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Home , Cell signaling, Signal transduction, Small molecule

617

β2-AR

Gγ

G 15 Gα β

Cell Signaling and : Between Cells

15.1 The Basic Elements of Signaling Systems The English poet John Donne expressed his belief in the 15.2 A Survey of Extracellular Messengers and Their interdependence of in the phrase “No man is an island.” Receptors The same can be said of the cells that make up a complex multi- cellular . Most cells in a plant or animal are specialized 15.3 G -Coupled Receptors and Their Second to carry out one or more specific functions. Many biological Messengers processes require various cells to work together and to 15.4 Protein- as a Mechanism coordinate their activities. To make this possible, cells have to for communicate with each other, which is accomplished by a 15.5 The Role of as an Intracellular Messenger process called . Cell signaling makes it possible 15.6 Convergence, Divergence, and Cross-Talk for cells to respond in an appropriate manner to a specific environmental . Among Different Signaling Pathways Cell signaling affects virtually every aspect of cell structure 15.7 The Role of NO as an Intercellular Messenger and , which is one of the primary reasons that this 15.8 () chapter appears near the end of the book. On one hand, an THE PER SP ECTIVE: Disorders Associated understanding of cell signaling requires knowledge about other with -Coupled Receptors types of cellular activity. On the other hand, insights into cell signaling can tie together a variety of seemingly independent THE HUMAN PER SP ECTIVE: Signaling Pathways and cellular processes. Cell signaling is also intimately involved in the Human Longevity

Three-dimensional, X-ray crystallographic structure of a signaling complex between a ␤2-adrenergic ( ␤2-AR), which is a represen- tative member of the G protein-coupled receptor (GPCR) superfamily, and a . The ␤2-AR is shown in green and the three subunits of the G protein are shown in orange, cyan, and purple. The plasma membrane is shown as a grey shadow. GPCRs are integral membrane characterized by their seven transmembrane helices. As a group, these proteins bind an astonishing array of biological messengers, which constitutes the first step in eliciting many of the body’s most basic responses. [CONTINUED ON NEXT PAGE ] Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 618 that are far away stimulat from potentially In the thesource. case of body, the throughout travel can that are in close proximity to the origin of the me stim and distance short a travel can messengerslar shown in the image) that bound to the to bound that image) the in shown otherthrough Cellsmost signaling usually communicate pathways. wit ar featuresthatgeneral the of few describinga by It may be helpful to begin the discussion of this c 15.1 cell signaling crucially important for understandin regulation of and division. This makes Figure 15.1 bind the to epinephrine) (e.g., an of Binding of Cell Signaling Systems OF rsalzto ehooy nti ae crystalliza case, this In technology. crystallization protei important these of structures high-resolution bloo high of treatment the for prescribed widely are n eaaino mohmsl el.Beta-adrenergi cells. muscle smooth of relaxation and protei G the bind to it causes that receptor the in n t srae ht a rsod o ht esne ( messenger that to 15.1 respond can that surface its on the cell thating, is producing the messenger expres during extracellularmatrix the to bind they or , by body because they are inherently or they trave a unstable, to ability their in limited usually are cules Paracrinecellthat isgeneratingmesse themessage. theextracellular space to cells that are in close get cells via passage through the bloodstream(Figu the throughpassage via cells get 15.1 (or inhibit) themselves. During During themselves. inhibit) (or Endocrinemessengerscalledalso are te cls n h bd (tp ,Fgr 1.) The 15.2). Figure 1, (step ranging body frominformationalsmall compound , the in extracellular environments of cells contain cells hundred other senger by a cell that is engaged in sendin CellFigure signaling15.2. is initiated with the re cally act on target cells located at distant sites in S a b An overview of cellular signaling pathways is depic issignalingcellularpathways of overview An ØREN ,messenger molecules), travel only short distances t .Cneunl,cells Consequently, releasing the message). will sti (a) endocrine | The Basic Elements .F R F. G. Autocrine (Autocrine extracellularmessenger molecules inln,messengersignaling, molecules reach their tar- ASMUSSEN a ), paracrine ( paracrine ), A, NDREW paracrine b ), and endocrine ( and endocrine ), ␤ 2 AR and the G protein to stabilize the complex durin complex the stabilize to protein G the and AR .KC. inln (Figuresignaling (b) ,wihtasistesga notecl,inducing cell, the into signal the transmits which n, tion of the signaling complex was accomplished using accomplished was complex signaling the of tion proximityto the autocrine ns had been lacking. This situation is now changing now is situation This lacking. been had ns , and they typi-they and , g how a cell can RUSE d and heart arrhythmias. GPCRs had been ver been had GPCRs arrhythmias. heart and pressure d the body. the study of omplex subject lease of a mes- n okti h xrclua oano the of domain extracellular the in pocket ing g messages to eetr r h agt fanme fimportant of number a of targets the are receptors c sg,or theyssage, aon the around l s of different Extracellu-. ses receptors re degraded e shared byshared e ngermole- c , lt cellsulate ) types of intercellular signaling.) types e 15.1re AND Finally,. n cellsing hrough mulate signal- Figure s (e.g.,s h each e inted B c RIAN ). .KK. malignant tumor. lose the ability to control and devel that it possesses at that particular time in its li intracellulthe receivesand it stimulithat the on Thus the type of activities in which a cell engages that become engaged in the response in these two ty differentinitialtracedstimulustobe intrace can differentThese mus outcomes following smoothinteraction with a in relaxation and cell liver a in down thisreceptor bycirculating leads togl shown in the chapter-opening image on Actipage 617. w mjr ots f inl rndcin u ke in keep but transduction, signal of routes major two focus will we discussion, following the In activated. recep of type the on depends taken route particular appropriate the whereelicits it interior, cell the transmi is signal the which by routes major two are membran plasma the of surface inner the reached has 15.2). Figure 3, (step domain cytoplasmic receptor’s that causes the signal to be relayed across the mem the in change conformational a induces interaction respondin the of surface outer the at receptor a to molecule that binds to the receptor is called a cally recognize and bind that messenger molecule (s ular extracellular message if they express to the surfaces Cellsof othercan onlycells. respo glucagonmones and(e.g., ) to huge glycoprot solublep steroidsneurotransmittersandsmall, to ) moh uce el bt pses the possess both cells muscle smooth Liver messenger. extracellular same the to ferently specificcellsEvensharethatreceptora respon may me extracellular different to respond to them allow types of cells possess different complements of rece OBILKA In most cases, the extracellular the messenger extracellular molecule In most cases, .) (c) g the crystallization process. crystallization the g ␤ 2 responses such as increased heart rate heart increased as such responses AR causes a conformational a causes AR two additional small proteins (notproteins small additional two as the result of recent advances inadvances recent of result the as rg,including , y difficult to crystallize so thatso crystallize to difficult y ␤ receptors 2 arnri receptor-adrenergic op into a ␤ fe. bokr,which-blockers, (C nd to a partic- epne Theresponse. ycogenbreak- depends both llularproteins ar machineryar OURTESY tr,whichptors, brane brane to the that specifi- el Thiscell. g pes of cells. roteinhor- e ) Thetep 2). eins bound d very dif-very d Different. tor that isthat tor the samethe on theseon el andcells tted intotted ssengers. vation of receptor Once itOnce l cell.cle ,theree, mind binds 619 there are other ways that extracellular can have an im- I Another type of receptor (Section 15.4) transmits a signal pact on a cell. For example, we saw on page 169 how neuro- by transforming its cytoplasmic domain into a recruiting transmitters act by opening plasma membrane channels station for cellular signaling proteins (step 4a). Proteins and on page 525 how hormones diffuse through the interact with one another, or with components of a cellular plasma membrane and bind to intracellular receptors. In the membrane, by means of specific types of interaction two major routes discussed in this chapter: domains, such as the SH3 domain discussed on page 62. I One type of receptor (Section 15.3) transmits a signal Whether the signal is transmitted by a second messenger from its cytoplasmic domain to a nearby (step 4), or by protein recruitment, the outcome is similar; a protein that which generates a second messenger (step 5). Because it is positioned at the top of an intracellular signaling pathway is brings about (effects) the cellular response by generating a activated (step 6, Figure 15.2). Signaling pathways are the in- second messenger, the enzyme responsible is referred to as formation superhighways of the cell. Each signaling pathway an . Second messengers are small substances that consists of a series of distinct proteins that operate in sequence typically activate (or inactivate) specific proteins. Depend- (step 7). Most “signaling proteins” are constructed of multiple ing on its chemical structure, a second messenger may dif- domains, which allows them to interact in a dynamic way with fuse through the or remain embedded in the a number of different partners, either simultaneously or se- bilayer of a membrane. quentially. This type of modular construction is illustrated by the Grb2 and IRS-1 proteins depicted in Figures 15.20 and 15.25 a, respectively. Unlike Grb2 and IRS-1, which function Signaling cell exclusively in mediating protein-protein interactions, many signaling proteins also contain catalytic and/or regulatory do- 1 mains that give them a more active role in a signaling pathway. Extracellular signaling molecule (first messenger) Each protein in a signaling pathway typically acts by altering the conformation of the subsequent (or downstream) protein in the series, an event that activates or inhibits that pro- Transmembrane tein (Figure 15.3). It should come as no surprise, after reading receptor 2 2 about other topics in cell , that alterations in the confor- mation of signaling proteins are often accomplished by protein

3 Effector 3 4a 4 P Protein 1 5 6

Second Active messenger Protein Protein 6 kinase 2 kinase 2

P 7 Inactive Active 7 Protein 3 kinase 3

P 15.1 Inactive Active Activated factor target SystemsSignaling Cell of Elements Basic The protein 8 8 P Inactive Active 9 9 Transcription Survival Protein synthesis DNA Movement P Cell death Metabolic change Figure 15.3 Signal transduction Figure 15.2 An overview of the major signaling pathways by which pathway consisting of protein extracellular messenger molecules can elicit intracellular responses. and protein whose catalytic mRNA Two different types of signal transduction pathways are depicted, one actions change the conformations, and thus in which a signaling pathway is activated by a diffusible second the activities, of the proteins they modify. In the example depicted messenger and another in which a signaling pathway is activated by here, protein kinase 2 is activated by protein kinase 1. Once activated, recruitment of proteins to the plasma membrane. Most signal protein kinase 2 phosphorylates protein kinase 3, activating the transduction pathways involve a combination of these mechanisms. It enzyme. Protein kinase 3 then phosphorylates a transcription factor, should also be noted that signaling pathways are not typically linear increasing its affinity for a site on the DNA. Binding of a transcription tracks as depicted here, but are branched and interconnected to form a factor to the DNA affects the transcription of the in question. Each complex web. The steps are described in the text. of these activation steps in the pathway is reversed by a . Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 620 s euae b sgas rgntn a te el sur cell the at originating signals by regulated is cel a which in activity every Virtually cell. the of th even or synthesis, DNA of activation permeability, chan a mobility, cell in decrease or increase an ton, cy the of reconfiguration a enzymes, metabolic of ity an alteration o involve a change in , pr target the by initiated response the message, and Depending on the basic cellular processes (step 9). s o nesad h rls f hs dvre posttra modifications in diverse the activities these of different of cell type roles the understand to is from two different The types primary of cells. certai inphosphorylation tyrosine of frequencythe diffemarkedthe shows which 15.4, Figure in seen is renceof and its presumed i nearly The36,000widespre sites ofphosphorylation. phosphoproteins6,000h that moreidentified tissues differecent9 Onestudyof varioustissues. latedin p are thatresidues specific the and kinasesprotein substratestheidentify to 70) employed(pagebeen that approache proteomic signal Large-scale a as degradation. protein act can it or another, to partment it can tions, induce a protein–proteinprotein decrease to move or increasefrom one can subc it enzyme, inac or activate can Phosphorylation ways. different in behavior protein change can phosphorylation tein plasmicproteins arephosphorylated moreoneorat is that at least 50 percent of transmembran transcription and variousfactors, types of regulato ch ion include also substrates the phosphatases—but arethemselves—mostenzymeszymes otheroftenkinas proteinsubstrates a ofthe of Many of proteinsubstrate. residue acid amino single a only phosphorylate phosphorylothers whereas substrates, their as teins numerous have phosphatases and kinases protein Some of all ignore and substrates specific its only nize each proteinphorylated, kinase or phosphatase is ab potentialresiduescontainb acidaminotheofwith i proteins of thousandsthough even that, remarkable membrane or lip smallmolecules, withotherproteins, activatedin the cell by covalent modification or by Depending state. on the particular these kinase, enzy Manykinases proteins. are present in the cell in a otherssolubleintegraarearecytoplasmic proteins, Someprotein kinaseslates tyrosineandph residues. but substrates, important protein group of their kinases (roughly 90 of in humous) p residues threonine mately reach mately phate groups from different protein substrates. subunit can form a host of different enzymesphosphatasingle that a r result, a As substratespecificity. phosphatasesregulatorykeycontainsubunit athat proteinkinases typically work as asingle subunit, and approximately 150 different human genome encodes more than 500 different protei movephosphate groups from other proteins (Figure 1 respectively,proteinphosphataseskinasesandthat, Most protein kinases transfer groups to s Signals transmitted along such signaling pathways u pathways signaling such along transmitted Signals target proteinstarget (step 8, Figure 15.2) involved in involved 15.2) Figure 8, (step ypoen.It ry proteins. manyprotein self-inhibited emove phos- s. interactions l is engaged is l l membranel ellular com- e and cyto- rentmouse le to recog- h others.the type of type cell f the activ- e catalyticse determines .Whereass. eing phos-eing mes can be mportance osphatases hosphory- hosphory- d o re- or add ae This face. proteinsn these en-these t o de- or ate otein mayotein ie.Pro-sites. challenge of variousof nslational adoccur- e n ion in ge .) The5.3). n kinases iae antivate arboring ec inrence ids. It is It ids. initiates toskele- interac- several a cell a n erine or e deathe annels, single a very very a haves s andes pro- lti- because because cells have to be responsive to additional m side a cell is referred to as as to referred is cell a side that changes into is translated molecules messenger ext by carried information which in process overall AABYDATA 4:3,21.R 2011. 144:639, Figure 15.4 eaiecnes (F . negative in many of t (PTPN12) phosphatase activity protein tyrosine This may correlate with t other breast cells. cancer phospho have a muchcells greater of tyrosine level that It is evident given along the top of the figure. The names of the ce at the bottom of the figure). key of the red shadi by the intensity line is indicated of pTyr The frequency residues in a given protein in a given cellright. express these three signature proteins is shown in of pTyr The frequency residues in breast cance HER2. and the gro receptor, receptor, express three major molecular signatures of many br Triple-neg lines. breast cell triple-negative cancer residues proteins si (named on the right in certain side of the figure showleft the of phosphfrequency cells. of breast cancer different types lation in two Finally, signaling has to be terminated. This is impo is This terminated. be to has signaling Finally, T ING A comparison in the frequency of tyrosine phosphory of tyrosine in the frequency A comparison - LEI PITDWT EMSINFROM PERMISSION WITH EPRINTED ROM G OFU .G A G. J. C ELL signal transduction signal BC ANDLBECK S IGNALING .S B S. J. T ng of the box (see the ECHNOLOGY ative cells do not ative cells the panels on the de of the figure) in the triple-negative he loss of a particular rylation than therylation otyrosine (pTyr)otyrosine RUGGE The panels on the east cancer cells–east cancer wth factor receptorwth ll lines tested arell r cells thatr cells . E LSEVIER essages that , FROM occur in-occur racellular he triple- C, ELL .) rtant - 621 they may receive. The first order of business is to eliminate the receptors translate the binding of extracellular signaling extracellular messenger molecule. To do this, certain cells pro- molecules into the activation of GTP-binding proteins. duce extracellular enzymes that destroy specific extracellular GTP-binding proteins (or G proteins ) were discussed in messengers. In other cases, activated receptors are internalized connection with vesicle budding and fusion in Chapter 8, (page 624). Once inside the cell, the receptor may be degraded dynamics in Chapter 9, protein synthesis in together with its ligand, which can leave the cell with de- Chapters 8 and 11, and nucleocytoplasmic transport in creased sensitivity to subsequent stimuli. Chapter 12. In the present chapter, we will explore their role in transmitting messages along “cellular information circuits.” R E V I E W I Receptor protein-tyrosine kinases (RTKs) represent a 1. What is meant by the term signal transduction ? What second class of receptors that have evolved to translate the are some of the steps by which signal transduction presence of extracellular messenger molecules into changes can occur? inside the cell. Binding of a specific extracellular ligand to 2. What is a second messenger? Why do you suppose it an RTK usually results in receptor dimerization followed is called this? by activation of the receptor’s protein-kinase domain, which is present within its cytoplasmic region. Upon activation, these protein kinases phosphorylate specific tyrosine residues of cytoplasmic substrate proteins, thereby 15.2 | A Survey of Extracellular altering their activity, their localization, or their ability to interact with other proteins within the cell. Messengers and Their Receptors I Ligand-gated channels represent a third class of cell- A large variety of molecules can function as extracellular car- surface receptors that bind to extracellular . The riers of information. These include ability of these proteins to conduct a flow of across the plasma membrane is regulated directly by ligand bind- I Amino acids and derivatives. Examples include ing. A flow of ions across the membrane can result in a glutamate, glycine, , epinephrine, , temporary change in , which will affect and . These molecules act as neurotrans- the activity of other membrane proteins, for instance, mitters and hormones. voltage-gated channels. This sequence of events is the I Gases, such as NO and CO. basis for formation of a nerve impulse (page 166). In I , which are derived from . Steroid addition, the influx of certain ions, such as Ca 2ϩ, can hormones regulate sexual differentiation, pregnancy, change the activity of particular cytoplasmic enzymes. As carbohydrate , and excretion of sodium and discussed in Section 4.8, one large group of ligand-gated potassium ions. channels functions as receptors for . I , which are nonpolar molecules containing I receptors function as ligand-regulated 20 carbons that are derived from a fatty acid named transcription factors. Steroid hormones diffuse across the 15.3 . Eicosanoids regulate a variety of plasma membrane and bind to their receptors, which are processes including pain, inflammation, blood pressure, present in the . Hormone binding results in a con- Messen Second Their and Receptors Protein-Coupled G and blood clotting. Several over-the-counter drugs that are formational change that causes the hormone–receptor com- used to treat headaches and inflammation inhibit plex to move into the nucleus and bind to elements present synthesis. in the promoters or enhancers of hormone-responsive I A wide variety of polypeptides and proteins. Some of (see Figure 12.47). This interaction gives rise to an increase these are present as transmembrane proteins on the surface or decrease in the rate of gene transcription. of an interacting cell (page 251). Others are part of, or I Finally, there are a number of other types of receptors that associate with, the . Finally, a large act by unique mechanisms. Some of these receptors, for ex- number of proteins are excreted into the extracellular ample, the B- and T-cell receptors that are involved in the environment where they are involved in regulating response to foreign antigens, associate with known signaling processes such as cell division, differentiation, the immune molecules such as cytoplasmic protein-tyrosine kinases. We response, or cell death and cell survival. will concentrate in this chapter on the GPCRs and RTKs. Extracellular signaling molecules are usually, but not always, recognized by specific receptors that are present on the surface of the responding cell. As illustrated in Figure 15.2, receptors 15.3 bind their signaling molecules with high affinity and translate | G Protein-Coupled Receptors this interaction at the outer surface of the cell into changes that and Their Second Messengers take place on the inside of the cell. The receptors that have G protein-coupled receptors (GPCRs ) evolved to mediate signal transduction are indicated below. are so named because they interact with I G protein-coupled receptors (GPCRs) are a huge family of G proteins, as discussed below. Members

receptors that contain seven transmembrane ␣ helices. These of the GPCR superfamily are also referred to as seven- gers Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 622 eooi eooi eetrAeyy yls Behavioral cyclase Adenylyl receptor Serotonin Serotonin yls.A niae ntefiue the in the figure, As indicated cyclase. activates an effect which G protein, with a trimeric the rec bound to their ligand, When spanning helices. me contain seven and , that bind epinephrine two or more receptor molecules.) ( (in the . protein are by lipid groups linked to the membrane G protein.heterotrimeric receptor transmembrane seven a of means by signals cGMP Visual excitati Visual cGMP phosphodiesterase Rhodopsin Light eiwo elBooyb nulRves n.Reprod Inc. Review of by Annual Reviews, tors eliciting andthe p of smell and taste), tastants (molecules and detected gusta by olfactory odoran system), immune neurotransmitters, the of cells phagocytic tract animal), molecules (e.g., and chemoattractants derivatives, opium plant (both ar diverse a hormones are GPCRs to bind that t ligands among natural Included superfamil genomes. largest animal by single encoded the proteins constitute GPCRs fact, proc cellular of spectrum extraordinary an toge regulate that mammals and plants flowering to from ra in containidentified Thousands been have GPCRs different they 15.5). (Figure because helices transmembrane receptors seven (7TM) transmembrane Figure 15.5 tmlsRcpo fetrPhysiologic response Sl Potassium channel Effector receptor Muscarinic reproduced wit R Bourne, and Stryer H. Adapted from L. Receptor Acetylcholine Epinephrine Stimulus Table 15.1 (from a it undergoes anmolecule) isomeriz absorbs a photon, theWhen retinal (shown in red on the left together with an unbound heterotrimeric G protein ( rhodopsin is shown in its inactive (dark-athe left, struc crystallographic of based the GPCR on X-ray rhodopsin -e etd hmtci eetrPopoiaeCChe C Chemotactic receptor Phospholipas IgE receptor Mast cell f-Met IgE–antigen complexes (a) Receptor cis to a Ligand The membrane-bound machinery for transducing transducing for machinery membrane-bound The trans Examples of Physiologic Processes Mediated by GPCRs GDP or GTP Note: om,which leads to the disruption of an ionicform), COOH NH Many GPCRs may be active as complexes of ( 2 a ) Receptors of this type, including those including ) Receptors of this type, α ␤ b G protein Arnri eetrAeyy yls Glycogen breakd cyclase Adenylyl -Adrenergic receptor ) A model depicting the activation ␣ γ β and ␥ subunits of the G Intracellular second dapted) conformation Effector cdwt emsino nulRves in the formauced with permission of Annual Reviews, rhodopsin or, such as adenylyl such or, h permission from the Annual Review of Cell Biology messengers that are embedded ation reaction called ). eptor interacts mbrane- and aand hotons. A A listhotons. tory recep-tory se.Inesses. ue.Ontures. that at-that ts and ts ray of ray nging of y ther of he (b) rn ei rdcre ro) which exposes a branebind helix (red arrow), curved including the outward tilt and rotationprotein, of This event in turn leadsthe to protein. a change in linkage between residues on the third and sixth tra S n te fetr truh hc te at s provide is act they which Table15.1. through effectors the and of thi by means operate that ligands of the of some 15.5). There are three loops present on the outside the on present loops threeare There 15.5). c the of inside the on present is carboxyl-terminus a length, varying of loops by connected are membrane usd ftecl,the seven outside of the cell, present is amino-terminus Their topology. following Receptors Receptorsby G Protein-Coupled Signal Transduction (in red) bound ( to the receptor’s cytoplasmic face. in the proposed active conformation with a portion subunit of The the rhodopsin G protein. molecule on R 11- retinal HAT ANDCHWARTZ e C PITDB PERMISSION FROMEPRINTED BY Rhodopsin (inactive) cis TM-VI and Heterotrimeric G Proteins G protein-coupled receptors normally have thehave normally receptors protein-coupled G W t Republish in a book via Copyright Clearance Cente o ,cprgt18,b nulRvesIc Annual by Annual Reviews Inc. copyright 1986, Vol 2, , A YNE Protein plug owing of pacemaker activity .HL. motaxis UBBE L L ␣ M sensitization and learning in and learning sensitization helices that traverse the plasma ACMILLAN on N, own A TURE TM-VI G protein ϩ G P ␣ UBLISHERS B the sixth transmem- nsmembrane helix of conformation of the 5,43 2008. 473, 455, peptide(active) F: of the G ing site for the G the right is shown ROM G ␣ T ell (Figure ell ␣ peptide s s pathway of the cellthe of HUE L subunit IMITED n theon nd thend exterior Interior Aplysia W. Cell Cell r . in d ␣ .) 623 that, together, form the ligand-binding pocket, whose struc- Ligand ture varies among different GPCRs.There are also three loops Receptor present on the cytoplasmic side of the plasma membrane that provide binding sites for intracellular signaling proteins. 1 It is very difficult, for a number of technical reasons, to pre- γ γ pare crystals of unmodified GPCRs that are suitable for α α G protein β β X-ray crystallographic analysis. For a number of years, GDP GDP rhodopsin was the only member of the superfamily to have its X-ray crystal structure determined. Rhodopsin has an unusually Effector stable structure for a GPCR, owing to the fact that its ligand (a retinal group) is permanently bound to the protein and the pro- 2 tein molecule can only exist in a single inactive conformation in γ the absence of a stimulus (i.e., in the dark). Beginning in 2007, α β as a result of years of effort by a number of research groups, a flurry of GPCR crystal structures appeared in the literature. For GTP GDP the most part, these structures revealed the GPCR in the inac- tive state, but more recent reports have described the structures of modified versions of several GPCRs in the active state.There is also a body of structural and spectroscopic data (of the type 3 described on page 135) that provide insights into some of the γ conformational changes that occur as a GPCR is activated and α becomes bound to a G protein. β The first X-ray crystal structure of an active GPCR with its GTP bound G protein was solved by Brian Kobilka and colleagues at Stanford University and is shown in the chapter-opening image on page 617. The inactive conformation of the GPCR is stabi- 4 lized by noncovalent interactions between specific residues in the γ α transmembrane ␣ helices. Ligand binding disturbs these interac- β tions, thereby causing the receptor to assume an active confor- GTP mation. This requires rotations and shifts of the transmembrane ATP cAMP ␣ helices relative to each other. Because they are attached to the cytoplasmic loops, rotation or movement of these transmem- brane ␣ helices causes changes in the conformation of the cyto- 5 plasmic loops. This in turn leads to an increase in the affinity of

γ 15.3 the receptor for a G protein that is present on the cytoplasmic α surface of the plasma membrane (Figure 15.5 b). As a conse- β GDP + P Messen Second Their and Receptors Protein-Coupled G quence, the ligand-bound receptor forms a receptor–G protein i complex (Figure 15.6, step 1). The interaction with the receptor induces a conformational change in the ␣ subunit of a G protein, causing the release of GDP, which is followed by binding of 6 GTP (step 2). While in the activated state, a single receptor can γ α β Figure 15.6 The mechanism of receptor-mediated activation (or GDP inhibition) of effectors by means of heterotrimeric G proteins. In step 1, the ligand binds to the receptor, altering its conformation and increasing its affinity for the G protein to which it binds. In step 2, the 7 G␣ subunit releases its GDP, which is replaced by GTP. In step 3, the G␣ subunit dissociates from the G ␤␥ complex and binds to an effector GRK P γ (in this case ), activating the effector. The G ␤␥ dimer α P β may also bind to an effector (not shown), such as an or an GDP enzyme. In step 4, activated adenylyl cyclase produces cAMP. In step 5, ATP the GTPase activity of G hydrolyzes the bound GTP, deactivating G . ␣ ␣ ADP In step 6, G ␣ reassociates with G ␤␥ , reforming the trimeric G protein, and the effector ceases its activity. In step 7, the receptor has been 8 phosphorylated by a GRK and in step 8 the phosphorylated receptor has been bound by an molecule, which inhibits the ligand- P γ α bound receptor from activating additional G proteins. The receptor P β bound to arrestin is likely to be taken up by . Arrestin GDP gers Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 624 pae rdcn ioio tipopae n diacylg Activated(page and 630). G trisphosphate producing sphate, G G activatedby iscyclase Adenylyl cyclase. adenylyl y yls.Glylcyclase. activate PLC than more trigger G and physiologic response. proteins G different with act althoughsomeGPCRs can teinwith which interacts, it r particular elicitedactivatedanby TheGPCR dependso typethe on couple. they which to effectors the unit has a low affinity for Gfor affinity low a has unit hyaeatv,G they are active, bound GTP amount a of certain time has after passed hy by off themselves turn and receptoractivated an interact the by on turned are They timers. molecular G heterotrimeric proteins In a , tein (step 6). un activate one or more cellular signalingturn, protei messengers Second below). (seephosphodiesterase GMP the reass and effector the from dissociate will subunit Other effectors include phospholipase(step 4). C- fectorleads to the production of the second messen activatio case, this In 3). step 15.6, (Figurecyclase suc effectorprotein, activatean to free tached)is the G with resultsan activatedin a GPCR, conformational followinginterac GTP, by GDP Replacementof subunit. G Proteins signal amplification (discussed further on page 632) activate a number of G protein providing molecules, uui,the subunit, rtiei bcue l o te cnit f he di three of called consist subunits, polypeptide them of all h as because described are erotrimeric They GTP. guanine or GDP bind either they because cleotides, proteins G as to referred protein These www.wiley.com/college/karp. at Web the fo be can which Pathways, Experimental the in cussed stud Their of Virginia. University the at colleagues Gil Alfred and Health of Institutes National the at and characterized by and his c rified, are covalently attached to the Heterotri proteins are held at the plasma membrane by lipid c chapter. this in later discussed are which such a monomeric G proteins, guishes them from small, oGP GGTP. to the the in increase an and effector the for affinity the d a causing change conformational a in results This GTP to GDP and inorganic phosphate (Pi) (Figure 15. at rnfrain olwn is iscain from dissociation its Following transformation. nant hasbeen associated with excessive cell proliferati other G protein families although their inappropria rmrccmlx Each dissociated Gtrimeric complex. andG s uuis Gsubunits. ␤␥ ␤␥ Theguanine - ispresent ont A G protein is said when to its be “on” Heterotrimeric G proteins come in Gfour flavors, ␣ 12/13 uui.In the itsG GTP-bound conformation, subunit. subunit to reform the inactive heterotrimeric G pro G heterotrimeric inactive the reform to subunit subunit. Thus, following hydrolysis of GTP, the G the GTP, of hydrolysis following Thus, subunit. Thisclassification is based . on the G ␣ ␤␥ subunits can turn themselves off by hydrolysis ofhydrolysis by off themselves turn can subunits ␤ 12/13 Heterotrimeric Heterotrimeric G pu-proteins were discovered, .PLC complex also has a signaling function and it can q ␣ aiy ebr cnan G contain members family membersare less well characterized than the subunits can turn on downstream effectors. ␤ hydrolyzes bispho- i s subunits function by inhibiting adeny- family members couple receptors toreceptorscouple members family ␣ ␤␥ ␣ , , leading to dissociation of theofdissociation to leading , ␤ and and , ␥ ␣ subunits (Figure 15.5 ␥ subunit (with GTP at- . This property This distin-property . ␣ subunit is bound ␣ ␣ onand malig- uuis that subunits h as adenylylas h subunitsand ns. te activation ␤ . affinity for affinity n of the ef-the of n ociate withociate function as ies areies dis- gercAMP TP-bound a means of drolysis of drolysis hains that and cyclic ecrease inecrease change in ,step 6, 5). olleagues a andman s ei G meric f G pro-G f ion withion h G the G, esponse While. und onund fferent lycerol inter- heG ␣ s Ras, q one nu- ares sub- G, tion in, et- a ). ␣ ␣ ␣ i - , ae called nase, the activated GPCR is phosphorylated by a specific type of ki- thecytoplasmic thefirst Instep, placetwosteps. in protein G additional on turning from receptors tive blindness resulting from the disease retinitis pigm of one be to thought is death cell retinal of type lead to the death of the photoreceptor incells the by rhodopsin of phosphorylation with interfere that desensitization is illustrated by the observation t importanc The environment. unchanging an endlessly of presence “fire” to continuing than rather vironment, on is Desensitization mechanisms that allows a cell to respond to cell. a chang the of surface outer the s cell while that stimulus is sti the sponding to the stimulus, because desensitization termed is action bi arrestin pro G additional of activation further the prevents consequence, het a with As binding proteins. for G compete erotrimeric and GPCRs to bind that called form proteins, a small family step 8). of (Figure 15.6, binding the is which step, ond -thr proteinof kinasesfamily smallthat specificallya recognizeform activate GRKs 7). step 15.6, olssmsweete r erdd(tp4.If to re where they are degraded (step 4). endo from traffic may receptors internalized outcome, In a from those that fromarise the plasma membrane. physiologic and properties different have endosomes transm signals the that appears it Now surface. cell resid they when transduction signal of capable only well) as RTKs (and GPCRs that assumption the under t surprise workingbeen had a who signaling cell of as field the in searchers came endosomes” “signaling of covery The 3). (step endosomes within localized GPCRs bound length later in the ischapter, thought to be activa discusseis which pathway, MAPK The nalingcomplexes. mole arrestin associated as serve a scaffold for the assembly of various cyt the where 312), (page somes receptors travel along the pathway endocytic cases, outcom alternative several in participate can tosis receptors that have been removed from the surface b Depending on the circu into the cell by endocytosis. phosphorylated of uptake the promotes 2) (step pits clathrin and arrestin bound between interaction The pits areclathrin-coated that in molecules situated 15 A to binding of arrestin aremolecules capable also Figure 1, (step GPCRs phosphorylated to bound are processes intracellular different in involved teins di of variety a to binding of capable are they that theirinactivestate. and thethe receptor, G effectorprotein, must all be to activate G Toproteins. regain sensitivity to fut receptorsblockedbefromhavec to overstimulation, turn on G and proteins, G proteins turn on effectors activate The activation. receptorin resultsbinding emnto o te Response the Termination of PLC i effectors, of types different of number a to couple Arrestins can be described as protein hubs (page 62 (page hubs protein as described be can Arrestins the for stage the sets GPCR the of Phosphorylation ␤ K, ϩ and Ca poenculd eetr kinase receptor protein-coupled G 2 Desensitization ϩ o hnes and adenylylion cyclase. channels, , the process theblocksthatac- , e ae en ht ligand that seen haveWe ( GRK ted by arrestin- ure the stimuli, hat mutations oplasmic sig- d GPCRs. the causes of causes the . While they While . rtn.This . (page 310).(page fferent pro-fferent entosa. es. In some In es. returned to To. prevent ll actingll on P2 adaptorP2 receptorsd domain of of proteins ceptors are into endo- en.Thisteins. e in its en- itted fromitted y endocy- mstances, ontinuing (Figure) ed at theat ed GPCRs ncluding o the of e a GRKa os re- tops arrestins -coated ,takes s, second l rolesal eonine n thein somes nding re- o cules were dis- ,in), sec- ofe d atd .7), - 625 degraded, the cells lose, at least temporarily, sensitivity for the ligand in question. Finally, according to a third scheme, the P AP2 6 Arrestin P arrestin-bound GPCRs may be dephosphorylated and re- 1 turned to the plasma membrane (step 5). If receptors are re- turned to the cell surface, the cells remain sensitive to the Recycling Clathrin endosome P 2 ligand (they are said to be resensitized ). P Signaling by the activated G ␣ subunit is terminated by a less complex mechanism: the bound GTP molecule is simply 5 hydrolyzed to GDP (step 5, Figure 15.6). Thus, the strength Endosome and duration of the signal are determined in part by the rate of 4 GTP hydrolysis by the G ␣ subunit. G ␣ subunits possess a 3 P weak GTPase activity, which allows them to slowly hydrolyze P the bound GTP and inactivate themselves. Termination of the response is accelerated by regulators of G protein signaling ERK Other pathways (RGSs ). The interaction with an RGS protein increases the rate of GTP hydrolysis by the G subunit. Once the GTP is Figure 15.7 ␣ Arrestin-mediated internalization of GPCRs . Arrestin- hydrolyzed, the G -GDP reassociates with the G subunits bound GPCRs (step 1) are internalized when they are trapped in ␣ ␤␥ clathrin-coated pits which bud into the cytoplasm (step 2). As to reform the inactive trimeric complex (step 6) as discussed discussed in Section 8.8, clathrin-coated buds are transformed into above. This returns the system to the resting state. clathrin-coated vesicles which deliver their contents, including the The mechanism for transmitting signals across the plasma GPCRs, to endosomes. When present in the endosomes, arrestins can membrane by G proteins is of ancient evolutionary origin and serve as scaffolds for the assembly of signaling complexes, including is highly conserved. This is illustrated by an experiment in those that activate the MAPK cascade and the transcription factor which yeast cells were genetically engineered to express a re- ERK (step 3). Alternatively, the GPCRs can be delivered to lysosomes, ceptor for the mammalian hormone . When these where they are degraded (step 4), or they can be returned to the plasma yeast cells were treated with somatostatin, the mammalian recep- membrane in a recycling endosome (step 5), where they can then tors at the cell surface interacted with the yeast heterotrimeric G interact with new extracellular ligands (step 6). (FROM S. L. R ITTER proteins at the inner surface of the membrane and triggered a re- AND R. A. H ALL , N ATURE REVIEWS MCB 10:820, 2009, B OX 1B. sponse leading to proliferation of the yeast cells. NATURE REVIEWS MOLECULAR CELL BIOLOGY BY NATURE The effects of certain mutations on the function of G PUBLISHING GROUP . R EPRODUCED WITH PERMISSION OF NATURE PUBLISHING GROUP IN THE FORMAT REUSE IN A BOOK /TEXTBOOK protein-coupled receptors are discussed in the accompanying VIA COPYRIGHT CLEARANCE CENTER .) Human Perspective. 15.3

THE HUMAN PERSPECTIVE Messen Second Their and Receptors Protein-Coupled G Disorders Associated with G Protein-Coupled Receptors GPCRs represent the largest family of genes encoded by the human gene that encodes rhodopsin, the visual pigment of the rods. Many of genome. Their importance in is reflected by the fact these mutations lead to premature termination or improper folding of that over one-third of all prescription drugs act as ligands that bind to the rhodopsin protein and its elimination from the cell before it this huge superfamily of receptors. A number of inherited disorders reaches the plasma membrane (page 288). Other mutations may lead have been traced to defects in both GPCRs (Figure 1) and het- erotrimeric G proteins (Table 1). Retinitis pigmentosa (RP) is an inherited disease characterized by progressive degeneration of the NH 2 retina and eventual blindness. RP can be caused by mutations in the Extracellular

Figure 1 Two-dimensional representation of a “composite” 2 8 transmembrane receptor showing the approximate sites of a number 7 of mutations responsible for causing human diseases. Most of the 3 1 4 mutations (numbers 1, 2, 5, 6, 7, and 8) result in constitutive stimula- 6 tion of the effector, but others (3 and 4) result in blockage of the recep- 5 tor’s ability to stimulate the effector. Mutations at sites 1 and 2 are Intracellular found in the MSH (melanocyte-stimulating hormone) receptor; 3 in the ACTH (adrenocorticotrophic hormone) receptor; 4 in the receptor; 5 and 6 in the TSH (thyroid-stimulating hormone) receptor; 7 in the LH (luteinizing hormone) receptor; and 8 in rhodopsin, the light-sensitive pigment of the retina. gers COOH Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 626 whereas a G protein with a G protein and thus cannot pass the signal downstream canno that molecule rhodopsin a of synthesis the to Human analogue coupled receptor Human analogue Neonatal severe G Familial hypocalciuric Disease G thyroid tumors Pituitary, osteodystrophy Albright’s hereditary Disease Pathway Table 1 A ecie ntetx,a G protein with a G in the text, *As described G syndrome McCune–Albright obndpeoiu uet G G Combined precocious puberty ovarian tumors Adrenocortical, aiilguootci Adrenocorticoptropic Rhodopsin receptor Cone opsin receptor hormone Luteinizing V2 vasopressin receptor Familial Thyrotropinreceptor spectral Color blindness, Retinitis pigmentosa X-linked nephrogenic Familial male Hyperthyroidism ing that the synthesis of the mutant protein and it and protein mutant the of synthesis the that ing recepto this lack normally that cells cultured into th by was introducing verified This conclusion tumor. proliferat cell but to the secretion mone excessive excessiv to only not co leads a that pathway sending the through surface, inner its on protein G a vates consti receptor TSH the mutation, the of result a As mutations 1, (Figure protein the of loop tracellular structu the affects that substitution acid amino an act to said (the TSH by stimulated be to having without hormone quant large secrete adenomas thyroid these of cells thyro ho pituitary the secrete by stimulation to response in that only cells thyroid normal Unlike noma. mu tumor, thyroid case, benign of type such a cause to one found been In function.” of “gain a as scribed lead the proteins signaling can have an alter effect, opposite that mutations Many receptor. encoded the via Copyright Clearance Center. Clearance via Copyright of Naturepermission Group Publishing in the format Nature by Nature Gro Publishing 1994. copyright 1994, Source: yeclei of BoPCAR1 receptor of BoPCAR1 receptor hyperparathyroidism hypercalcemia glucocorticoid deficiencyglucocorticoid and peudohypoparathyroidism ( ( and pseudohypoparathyroidisms deficiency and isolated hormone (ACTH) hormone receptor receptor and isolated deficiency variationssensitivity insipidus precocious puberty (thyroid adenomas) gip gsp RP results from a mutation that leads to a loss of of loss a to leads that mutation a from results RP .E lpa,rpitdwt emsinfo aue V reprinted with permission from Nature, Clapham, E. D. ) oncogene) Human Diseases Linked to the G Protein constitutively ). The TSH receptor in these cells contains cells these in receptor TSH The ). i ␣ inhibits the effector. Defective G protein- s ␣ (homozygous) acts to stimulate the effector, reuse in a book/textbook Defective G protein* p Reproduced withup. ion that causes the ion causes that r and demonstrat- and r re of the third in- third the re of ing ing to what is de- mn S,the TSH, rmone at sites 5 or 6). or 5 sites at to the effector. s incorporation s ities of thyroid of ities t activate its Gits activate t e thyroid hor- thyroid e e e mutant gene called an ade- an called ntinual signal ntinual tutively acti- tutively l31 .109, p. ol 371, tations have tations d hormone id tutr of structure function of function receptor is receptor s s s s i ␣ ␣ ␣ ␣ ␣ ular G tioned in a normal manner in these patients suggest The of facthypoparathyroidism. that most of the bod leadingt causethem tosecrete parathyroid hormone, stimulithat respondto notparathyroid could result a As inactive. remaintoprotein G the caused of the parathyroid , which function at a tempe a functionat which glands, parathyroidthe of no h pam mmrn ld o h cniuu prod continuous cells. engineered the genetically the in cAMP to led membrane plasma the into tumor. A mutation in a cell of the body, such as a th a as such a called body, the of cell a in mutation A tumor. to proliferated then which thyroid, the of cells the but inherited not was mutation the that indicating t in only but thyroid patient’s a of portion normal ways that control . cell control that ways stimulates which -8, for receptor active co a encodes genome virus The patients. AIDS in lent lesions skin purplish causes re which is constituti that sarcoma, virus a Kaposi’s herpes of as type a acts is virus The that GPCR. protein a encode to shown v cancer-causing one least At cancer. human of cause mutations somatic chapter, following the in evident individual the of all in present be would that tion gle amino acid substitution in one of the G Both patients were found to and hypoparathyroidism. prec disorders: endocrine of combination rare a pa from male two on report a by illustrated is This ders. to lead also can proteins G heterotrimeric of units ␤ certain alleles of Forthe ge example, case of GPCRs. thein documented well been has This individuals. other than ders susceptible less or more be to individuals certain morphisms may have considerable impact on human dis Yet it has in become recentclear years (page 416). variations within are thought “normal” of as common, polymorphisms Genetic gene. a of sequence nucleotide perature than the body’s visceral organs (33organs visceral body’s the than perature which are housed outside testes, of the body’s core, both in the presence and absence of bo was inactive, the mut at normal body , In contrast, and. in the even absence of a bound lig-tein remained in the active state, At temperatures the belowm normal body , in amino acid sequence two caused effects on the mu berty. In contrast, the mutation in this same Gsame this in mutation the contrast, In berty. and of synthesis premature to leading the of absence the in cAMP synthesize to stimulated prot G the bearing patients the of cells testicular h sex male the productionsubsequentof and cAMP of in receptors on the surface of the testicular cells, The circulating begins to be produced at that time. the time of in puberty response to the ho pituitary testoste initiate testes the of cells endocrine the focus of clinical research.focus of clinical asso identifying polymorphi genetic and susceptibility disease tween 417, page on discussed As dividuals. ( che a of alleles certain and schizophrenia; or abuse r increased with associated are receptor dopamine a ce pressure; blood high or asthma developing of hood CCR5 2 adrenergic receptor have been associated with an in The mutation that causes thyroid adenomas is The adenomas thyroid that mutation not causes fo As noted in Table 1, mutations in genes that encode that genes in mutations Table1, in noted As Mutations are thought of as rare and disabling chan disabling and rare as of thought are Mutations ␣ ) are associated with prolonged survival in HIV-infin survival prolonged with associated are ) isoform is not essential in the activities of most somatic mutation somatic to distinguish it from an inherited muta- inherited an from it distinguish to ␣ Њ sfrs The alterationisoforms. s 37vs. C ␣ ducing the synthesis rone production atproduction rone subunit in the cells the in subunit to particular disor- particular to ein mutation weremutation ein mn H which rmone LH, ’s cells. As will be will As ’scells. that genetic poly- have a lower tem- s that this partic- LH binds to LH inherited disor- inherited he tumor tissue, tumor he mokine receptormokine would normallywould give rise to the to rise give , the cells of theof cells the , n iad Theund ligand. isk of substanceof isk ne encoding the sms is a currenta is sms signaling path- signaling ily organs func- tients sufferingtients o theocondition arose in one of one in arose tant G protein. rccos pu-precocious rature of 37ratureof Њ ocious pubertyocious the population and is preva- is and rtain alleles of alleles rtain ) Normally,C). are a primary a are creased likeli- contain a sin- ant G protein irus has been has irus sponsible for for sponsible roe Theormone. utant G pro- other cells. ae causingease, ri el is cell, yroid , in contrast, in , LH ligand,LH itos be- ciations nstitutively ey active vely und und in the ges in thein ges cin of uction ected in-ected the sub-the Њ C, 627 Bacterial Because G proteins are so important to glycogen phosphorylase was present only in the supernatant the normal of multicellular organisms, they pro- fraction, the particulate material was required to obtain the vide excellent targets for bacterial pathogens. For example, hormone response. Subsequent experiments indicated that cholera (produced by the bacterium Vibrio cholerae ) ex- the response occurred in at least two distinct steps. If the erts its effect by modifying G ␣ subunits and inhibiting their particulate fraction of a liver homogenate was isolated and GTPase activity in the cells of the intestinal . As a incubated with the hormone, some substance was released result, adenylyl cyclase molecules remain in an activated that, when added to the supernatant fraction, activated the mode, churning out cAMP,which causes the epithelial cells to soluble glycogen phosphorylase molecules. Sutherland secrete large volumes of fluid into the intestinal lumen. The identified the substance released by the membranes of the loss of water associated with this inappropriate response often particulate fraction as cyclic monophosphate leads to death due to dehydration. (cyclic AMP , or simply cAMP ). This discovery is heralded as Pertussis toxin is one of several factors produced the beginning of the study of signal transduction. As will be by Bordetella pertussis , a microorganism that causes whooping discussed below, cAMP stimulates glucose mobilization by cough. Whooping cough is a debilitating respiratory tract in- activating a protein kinase that adds a phosphate group fection seen in 50 million people worldwide each year, causing onto a specific serine residue of the glycogen phosphorylase death in about 350,000 of these cases annually. Pertussis toxin polypeptide. also inactivates G ␣ subunits, thereby interfering with the sig- Cyclic AMP is a second messenger that is capable of dif- naling pathway that leads the host to mount a defensive fusing to other sites within the cell. The synthesis of cyclic response against the bacterial infection. AMP follows the binding of a first messenger—a hormone or other ligand—to a receptor at the outer surface of the cell. Figure 15.8 shows the diffusion of cyclic AMP within the cy- Second Messengers toplasm of a following stimulation by an extracellular The Discovery of Cyclic AMP, a Prototypical Second messenger molecule. Whereas the first messenger binds ex- Messenger How does the binding of a hormone to the clusively to a single receptor species, the second messenger of- plasma membrane change the activity of cytoplasmic en- ten stimulates a variety of cellular activities. As a result, second zymes, such as glycogen phosphorylase, an enzyme involved messengers enable cells to mount a large-scale, coordinated in glycogen metabolism? The answer to this question was response following stimulation by a single extracellular ligand. provided by studies that began in the mid-1950s in the labo- Other second messengers include Ca 2ϩ, phosphoinositides, ratories of Earl Sutherland and his colleagues at Case Western , diacylglycerol, cGMP, and . Reserve University. Sutherland’s goal was to develop an in vitro system to study the physiologic responses to hor- Phosphatidylinositol-Derived Second Messengers It mones. After considerable effort, he was able to activate wasn’t very long ago that the of cell membranes glycogen phosphorylase in a preparation of broken cells that were considered strictly as structural molecules that made had been incubated with glucagon or epinephrine. This membranes cohesive and impermeable to aqueous solutes. broken-cell preparation could be divided by centrifugation Our appreciation of phospholipids has increased with the re- 15.3 into a particulate fraction consisting primarily of cell mem- alization that these molecules form the precursors of a number branes and a soluble supernatant fraction. Even though of second messengers. Phospholipids of cell membranes are Messen Second Their and Receptors Protein-Coupled G

Figure 15.8 The localized formation of cAMP in a live cell in indicated. Notice that the cAMP levels drop by 109s despite the response to the addition of an extracellular messenger molecule. continued presence of the . (The cAMP level was This series of photographs shows a sensory nerve cell from the sea determined indirectly in this experiment by microinjection of a hare Aplysia . The concentration of free cAMP is indicated by the fluorescently labeled cAMP-dependent protein kinase labeled with color: blue represents a low cAMP concentration, yellow an interme- both fluorescein and rhodamine on different subunits. Energy transfer diate concentration, and red a high concentration. The left image between the subunits (see page 738) provides a measure of cAMP shows the intracellular cAMP level in the unstimulated neuron, and concentration.) (F ROM BRIAN J. B ACSKAI ET AL ., S CIENCE 260:223, the next three images show the effects of stimulation by the 1993. R EPRINTED WITH PERMISSION FROM AAAS.) neurotransmitter serotonin (5-hydroxytryptamine) at the times gers Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 628 Figure 4.10).Figure membran cellular most of component minor a inositol, phosphati compound the from derived substance a ger, second same the by triggered are secretion, to other contraction to leading one allergy responses, these an of of Both symptoms the trigger can that stance the cell of is a stimulated mast to cell, secrete hi a foreign When antigen binds to t lated to contract. cell muscle the stomach, the of wall the within cell smoo a of surface the to binds acetylcholine mitter Phosphorylation Phosphatidylinositol cussed. not is from derived messengers second group Another kinases. protein-tyrosine receptor and re protein-coupled G by signals of transmission the f generated are and phosphatidylinositol, from rived a which messengers, second lipid best-studied the on wi we section, this In messengers. second as function response to extracellular signals and the products enzyme classes depicted in Figure 15.9 Figure in depicted classes enzyme fo All . of classes main the by tacked th generalized a within sites cleavage ae p popoii mlcl.Fgr 15.9 Figure molecule. phospholipid a up blo make building different the connect that bonds ester sp hydrolyze that enzymes are Phospholipases zymes). (lipid-dephosphorylating phosphatases phospholipid enzym enzymes (lipid-splitting (lipid-phosphorylating kinases phospholipid phospholipases include zymes signals. extracellular to response in regulated are converted into second messengers by a variety of en Figure 15.9 ebaeadmyatrisezmtcatvt.( activity. and may altermembrane its enzymatic o to the inner surface holds the enzyme interaction of a phosphoinosi inositol ring binds to the phosphorylated domain that molecule containing a PH of a PLCportion enzyme seFgr 51) ( 15.10). (see Figure head group from the splits thewhich phosphorylated we wil Of these enzymes, sites. cule at the indicated of phospholipases t by four types subject to attack 2.22 phospholipidture (see Figure of a generalized (a) Phospholipid-based second messengers.Phospholipid-based b ) A model showing a between the interaction hn h neurotrans- the When a c ) Fluorescence ) Fluorescence can be activated inactivated be can hat cleave the mole-hat cleave ). Phospholipids are Phospholipids ). f the plasma l focus on PLC, diacylglycerol tmn,a sub-stamine, ( they they produce a a ) Thestruc- zymes zymes that hs en-These ie Thistide. hw theshows he surface th muscleth is stimu-is at are at-are at messen- ur of theof ur ollowing (b) k that cks of lipidof n theand ll focus ll ceptors attack. PH Domain es (seees e de-re ,and), ecific dis- dyl– es), en- EMSINFROMPERMISSION 3,4,5-trisphosphate (PIP s binds that an with stained been has cell the cell to which a chemical (i.e., chemoattractant micrograph that has been of a stimulatedcell to mo hs suis hy nuae sie o pgo pancr pigeon of o slices [ carry incubated To they pancreas. studies, the these in synthesis RNA on effect the study to out set had investigators These Unive McGill and Hospital MontrealGeneral of Hokin an Lowell by 1950s early the in out emer carried studies signals extracellular to responses cellular in deo h irtn el(ros.Brequals 15 Bar (arrows). cell edge of the migrating iae(IK eeae I4popae(I4P,whi (PI(4)P), 4-phosphate PI generates (PI4K) kinase PI of sugar inositol the of 4-position the to group transfer 7 of Fora single example, generate phosphoinositides. to cells in present kinases phoinositide or 5 carbons can be 4, phosphorylated by speci The 3, diacyl and inositol between linkage the in involved num Carbon atoms. carbon six has bilayer, the of face cytopl the at resides which ring, inositol the figure, As are indicated shownon the in left Figure 15.10. vated in response to a large variety of extracellul kinas lipid that established well now is It choline. such extracellularmolecules, messengertoresponse ac are that kinases lipid specific by phosphorylated tides which are collectivelyderivatives, referred to as which was rapidly converteditol to (PI), other phos theisotope was incorporated primarily into phospha i radioactivity of phospholipid Furtherfraction incorporation analysisof the cell. the to with tissue led the choline of treatment that found they ingly, RNA.of synthesisthe precursors during as used are triphosphates nucleoside into incorporated be would H J B T AMES 32 IOL RENDS ENRY ]rhpopae The idea was that [P]orthophosphate. The first indication that phospholipids might be inv Several of the reactions of phosphoinositide metabo phosphoinositide of reactions the of Several :5,1997; 7:559, . hs ugse ta ioio-otiig iis can inositol-containing that suggested This . .HH. .BR. C ELL RE ANDURLEY OURNE B IOL C E U, 040 00 B 2000. 10:470, . : LSEVIER FROM IEST OFNIVERSITY 3 J ), which is seen to be localized at the leading is seen to be localized which ), AY (c) P .GA. AULA .) ROBL E R R C T MGSRPITDWITH REPRINTED IMAGES OTH CETE AL ET ICKERT ALIFORNIA C, URR 32 ␮ pecifically to PIto pecifically O. P]orthophosphate satatd.Thisis attracted). S, .(m. ve toward a ., PIN AN phosphoinosi- OREYOFCOURTESY ar signals. B F: revealed that S. F s of acetyl-of s es are acti-are es RANCESCO side of this phorylated ROM asmic sur-asmic phosphate TRUCT by PI 4- PI by e from ged iae intivated tidylinos- as acetyl-as d Mabeld fic phos- distinct Interest- glycerol. t the nto which, ber 1 is1 ber acetyl- ch can ch a in eas olved rsity. . lism be ut ut , 629

Ligand

1 2 PI PI(4)P PI(4,5)P2 G protein PI(4,5)P2 DAG Kinase Kinase R 5

γ Inner leaflet 6 PKC PI-PLCβ α HO P HO P HO P β 3 HO P 6 2 1 OH OH OH OH 4 4 HO 3 OH HO OH HO P HO P 5 OH P P P 7 GTP GDP HO P OH HO P IP 3 8 P 9 IP 3 receptor

Ca 2+

Smooth ER

Figure 15.10 The generation of second messengers as a result of which PI(4, 5)P 2 is split into diacylglycerol (DAG) and inositol 1,4,5- ligand-induced breakdown of phosphoinositides (PI) in the lipid trisphosphate (IP 3) (step 5). DAG recruits the protein kinase PKC to bilayer. In steps 1 and 2, phosphate groups are added by lipid kinases the membrane and activates the enzyme (step 6). IP 3 diffuses into the 2ϩ to phosphatidylinositol (PI) to form PIP 2. When a stimulus is received cytosol (step 7), where it binds to an IP 3 receptor and Ca channel in by a receptor, the ligand-bound receptor activates a heterotrimeric G the membrane of the SER (step 8). Binding of IP 3 to its receptor protein containing a G␣q subunit (step 3), which activates the enzyme causes release of calcium ions into the cytosol (step 9). PI-specific - ␤(step 4), which catalyzes the reaction in

be phosphorylated by PIP 5-kinase (PIP5K) to form PI 4,5- or PIP 3 typically recruits the protein to the cytoplasmic face of 15.3 bisphosphate (PI(4,5)P 2; Figure 15.10, steps 1 and 2). the plasma membrane where it can interact with other mem- PI(4,5)P 2 can be phosphorylated by PI 3-kinase (PI3K) to brane-bound proteins, including activators, inhibitors, or G Protein-Coupled Receptors and Their Second Messen Second Their and Receptors Protein-Coupled G form PI(3,4,5)P 3 (PIP 3) (shown in Figure 15.25 c). The phos- substrates. Figure 15.9 c shows an example where PIP 3 is phorylation of PI(4,5)P 2 to form PIP 3 is of particular interest specifically localized to a particular portion of the plasma because the PI3K enzymes involved in this process can be membrane of a cell. PIP 3 is produced at the front of the cell by controlled by a large variety of extracellular molecules and a localized lipid kinase and is subsequently degraded at the PI3K overactivity has been associated with human cancers. rear and sides of the cell by a localized lipid phosphatase. The The formation of PIP 3 during the response to insulin is dis- cell shown in Figure 15.9 c is engaged in , which is cussed on page 645. to say that it is moving toward an increasing concentration of All of the phospholipid species discussed above remain in a particular chemical in the medium that serves as the the cytoplasmic leaflet of the plasma membrane; they are chemoattractant. This is the mechanism that causes phago- membrane-bound second messengers. Just as there are lipid cytic cells such as macrophages to move toward or kinases to add phosphate groups to phosphoinositides, there other targets that they engulf. Chemotaxis depends on the lo- are lipid phosphatases (e.g., PTEN) to remove them. The ac- calized production of phosphoinositide messengers, which tivity of these kinases and phosphatases are coordinated so bind to certain -binding proteins (page 372) to influence that specific phosphoinositides appear at specific membrane the formation of actin filaments and lamellipodia that are re- compartments at specific times after a signal has been received. quired to move the cell in the direction of the target. The role of specific phosphoinositides in membrane traffick- ing was discussed on page 311. Phospholipase C Not all inositol-containing second mes- The phosphorylated inositol rings of phosphoinositides sengers remain in the lipid bilayer of a membrane. When form binding sites for several lipid-binding domains (PH, PX, acetylcholine binds to a cell, or an antigen and FYVE) found in proteins. Best known is the PH domain binds to a mast cell, the bound receptor activates a het- (Figure 15.9 b), which has been identified in over 150 different erotrimeric G protein (Figure 15.10, step 3), which, in turn,

proteins. Binding of a protein by its PH domain to PI(3, 4)P 2 activates the effector phosphatidylinositol-specific phospholipase gers Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 630 oan n a hshioiie medd n h bilay the in PLC embedded phosphoinositide a and domain bound there by the (Figure 15.10), interaction betwe mediated bursts of Caof bursts mediated cytosol (step 7, Figure Figure 15.10) and bind to a specific (step cytosol 7, ecules, 15.9 C- iiy n h kde,pg 11.Vspesn id to binds ser a Vasopressin causes and surface 151). cell liver the page at receptor kidney, the in tivity mone vasopressin (the same hormone that has antidiu is the response of too, a liver ce So, calcium levels. secretory both granules are in triggere a mast histamine-cont cell, of and cell muscle smooth contractio above, used examples two the In responses. trigger cause they bind to various target molecules, messe second or intracellular as considered be also Calcium 9). (step cytoplasm the into diffuse to ions IP of Binding channel. phate (IP ( u (tp ) t a ntd n ae 8,ta te smoo IP The the cells. of that 280, page on storage noted calcium of site a is reticulumendoplasmic was It 8). (step lum endoplasmi smooth the of surface the at located tor ain y PLC by mation following membrane plasma the in remains that cule Diacylglycerol of these secondeach messengers separately. Weexaminewill signaling. cell in messengers second as roles el IPcell. ecule capable of rapid diffusion throughout the int Inositol 1,4,5-trisphosphate 1,4,5-trisphosphate Inositol an autoimmune skin disease.and to treat psoriasis, as trials clinical munosuppressant to rejectionprevent of transplante in tested being is which AEB071, PKCinhib specific a of studies in seen is responses susc in The tumors importance of PKC in mice. the development cause of im can and culture cell in notype malign permanent a exhibit C kinase proteinexpress cons to engineered genetically been have that cells In properties. growth normal their recover cells the me the from removed is ester phorbol the When cells. ma as temporarily behave and control growth lose to ca cells, cultured of variety a in isoforms C kinase ters called compounds, plant powerful of group a with seen is control growth in C kinase protein of tance apparenti The immuneresponses. and death, cell lism, cellul differentiation, and growth cellular in roles of variety wide a proteins. on residues threonine and serine iae Ckinase rtis ht er DGbnig 1 oan h best The proteins of family a is effectors these of studied domain. C1 DAG-binding a bear that proteins DAG ␤ , that resemble DAG. These compounds activate protein activate compounds These DAG. resemble that , isoforms have a number of importantof number a have isoforms C kinase Protein b ␤ (PLC ,PLC ) (step 5, Figure 15.10), both of which play importan play which of both 15.10), Figure 5, (step ) catalyzes 5)Pa catalyzes reaction that splits PI(4, 3 nstl 1,4,5-trisphosphate inositol molecules formed at the membrane diffuse into theinto diffuse membrane the at formed molecules ( 3 PKC sasgrpopaeasal water-soluble mol-) is a sugar phosphate—a small, ␤ ␤ (tp ) ie h poen eitd n Figure in depicted protein the Like 4). (step ) is situated at the innerof thesurface membrane ) (step 6, Figure 15.10), which phosphorylate which 15.10), Figure 6, (step ) 3 Diacylglycerol (Figure 15.10) is a lipid mole-lipid a is 15.10) (Figure Diacylglycerol ␤ receptor also functions as a tetrameric Ca tetrameric a as functions also receptor hr i rcut ad ciae effector activates and recruits it There . 2 ϩ 3 release, which appear as oscillationsas appear which release, pn te hne,alwn Ca allowing channel, the opens ( IP 3 nstl 1,4,5-trisphos- Inositol ) ( IP 3 ) and and ) 2 into two mol- called called d by elevated diacylglycerol ll ll to the hor- using themusing r metabo-ar erior erior of the ing specific in a varietya in e o IP of ies IP in studiesin phorbol es-phorbol itor calleditor gr be-ngers d kidneys en its PH contrast, ions canions titutively ant phe-ant c reticu-c retic ac- 3 n im- an lignant its for-its eptible protein mpor- aining recep- n of aof n target target dium, mune its 2 2 er. th 3 ϩ ϩ - - t frequency. (R increase the height (amplitude) of but the t spikes, calcium at periodic Higher intervals. concentrations cell to vasopressin leads to controlled spikes in t is proportional to the concentration of free calciu luminesces when it binds calcium The intensity ions. was injected awith protein aequorin, extracted from concentration in response to hormone stimulation. Figure 15.11 fectors. The human genome encodes at least 16 differ 16 least at encodesgenome human The fectors. effector can also as exist can ma in multiple forms, heterotrimeric G proteins that transmit signals fro targetofdifferent membranestypesof the in occur ceptor may coexist in or the t same plasma membrane, Differentisofor proteins. G differentofwith types forms can have different affinities for the ligand o mitter released by nerve cells in parts of the brai powerfuln a serotonin, receptorfortheisoforms of and15 whichadrenergicbinds epinephrine, receptor, isofordifferent 9 identified researchershave ample, for receptor the ligandcan exist in several different addition, versions (iso In ligands. of array verse GPCRs cellul are Thus capable as a group, of bindin of variety sponses. wide a triggering acro he membrane, and information plasma GPCRs transmit of to way proteins by G act erotrimeric stimuli, sensory and ters, neurotr includinghormones, agents, ofvarietywide A The Responses Specificity of G Protein-Coupled BOOK A IN REUSE RIGHT C IHPRISO OF PERMISSION WITH list of some of the responses mediated by IPby mediated responses the of some of list encode information that governs the cell’s oscillati specific such of intensity and frequency The in shown 15.11. recording the in calcium cytosolic free of Table 15.2. We will have more discussion about Ca Weabout discussion more have will Table15.2. Section 15.5.Section THBERTSONU 96 N1986. PITDWT EMSINFROM PERMISSION WITH EPRINTED TR BYATURE Experimental demonstration of changes in free calci 2+ , [Ca ] (nM) AND / ETOKVIATEXTBOOK 200 400 600 .H C H. P. N ATURE N ATURE OBBOLD 10 0.4 nMVasopressin Time (minutes) P UBLISHING C P UBLISHING OPYRIGHT N, 030 20 ATURE G he concentration of free os Exposurem ions. of the OPI H FORMAT THE IN ROUP C G hey do increase their 1:0,1986;319:601, .M W M. N. LEARANCE of hormone do not certain jellyfish that ROUP of the luminescence A single liver cell 3 .Different iso-n. om) Forex- forms). is indicated in indicated is R. r may interact m receptor to ny of the ef- epne Aresponse. EPRODUCED ms of a re-a of ms OODS cls Thecells. eurotrans- 2 s f the of ms ϩ C n mayons different given a hey may ansmit- Figure ent Gent ENTER COPY ions in ions g a di- s the ss .S. K. , r re- ar um - t- .) ␣ 631

Table 15.2 Summary of Cellular Responses Elicited Glycogen synthase by Adding IP 3 to Either Permeabilized or Intact Cells (Glucose)n – 1 + UDP-glucose Cell type Response

Vascular smooth muscle Contraction CH 2OH CH 2OH CH 2OH Stomach smooth muscle Contraction H O HHHO H O H Cyclic GMP formation, actin H H H OH H OH H OH H polymerization O O O PP Blood Shape change, aggregation HO i Salamander rods Modulation of light response H OH H OH H OH UTP oocytes Calcium mobilization, membrane n n – 1 n – 2 Glycogen Sea urchin eggs Membrane depolarization, cortical (glucose)n reaction CH 2OH P Lacrimal gland Increased potassium current i H O H O (Glucose) + n – 1 OH Adapted from M. J. Berridge, reproduced with permission from the Annual Review Glycogen OP O - of Biochemistry, Vol. 56, copyright 1987 Annual review of biochemistry. Volume 56, phosphorylase HO 1987 by Richardson, Charles C. Reproduced with permission of Annual Reviews, H OH O- Incorporated in the format Republish in a book via Copyright Clearance Center. Glucose 1-phosphate

subunits, 5 different G ␤ subunits, and 11 different G ␥ subunits, Glucose 6-phosphate along with 9 isoforms of the effector adenylyl cyclase. Different combinations of specific subunits construct G proteins having different capabilities of reacting with specific isoforms of both Fructose 6-phosphate Glucose + Pi receptors and effectors. As mentioned on page 624, some G proteins act by in- hibiting their effectors. The same stimulus can activate a stim- Blood ulatory G protein (one with a G ␣s subunit) in one cell and an Figure 15.12 The reactions that lead to glucose storage or inhibitory G protein (one with a G ␣i subunit) in a different mobilization. The activities of two of the key enzymes in these cell. For example, when epinephrine binds to a ␤-adrenergic reactions, glycogen phosphorylase and glycogen synthase, are controlled receptor on a cardiac muscle cell, a G protein with a G ␣s sub- by hormones that act through signal transduction pathways. Glycogen unit is activated, which stimulates cAMP production, leading phosphorylase is activated in response to glucagon and epinephrine, to an increase in the rate and force of contraction. In contrast, whereas glycogen synthase is activated in response to insulin (page 646). when epinephrine binds to an ␣-adrenergic receptor on a 15.3 smooth muscle cell in the intestine, a G protein with a G ␣i subunit is activated, which inhibits cAMP production, pro- ducing muscle relaxation. Finally, some adrenergic receptors which is sometimes called the “fight-or-flight” hormone—is Messen Second Their and Receptors Protein-Coupled G turn on G proteins with G ␣q subunits, leading to activation of produced by the in stressful situations. Epi- PLC ␤. Clearly, the same extracellular messenger can activate nephrine causes an increase in blood glucose levels to provide a variety of pathways in different cells. the body with the extra energy resources needed to deal with the stressful situation at hand. Insulin acts through a receptor protein- and Regulation of Blood Glucose Levels its signal transduction is discussed on page 644. In contrast, Glucose can be utilized as a source of energy by nearly all cell both glucagon and epinephrine act by binding to GPCRs. types present in the body. It is oxidized to CO 2 and H 2O by Glucagon is a small protein that is composed of 29 amino glycolysis and the TCA , providing cells with ATP that acids, whereas epinephrine is a that is derived can be used to drive energy-requiring reactions. The body from the amino acid tyrosine. Structurally speaking, these two maintains glucose levels in the bloodstream within a narrow molecules have nothing in common, yet both of them bind to range. As discussed in Chapter 3, excess glucose is stored in GPCRs and stimulate the breakdown of glycogen into glucose animal cells as glycogen, a large branched composed 1-phosphate (Figure 15.12). In addition, the binding of either of glucose monomers that are linked through glycosidic of these hormones leads to the inhibition of the enzyme glyco- bonds. The hormone glucagon is produced by the alpha cells gen synthase, which catalyzes the opposing reaction in which of the pancreas in response to low blood glucose levels. glucose units are added to growing glycogen molecules. Thus Glucagon stimulates breakdown of glycogen and release of two different stimuli (glucagon and epinephrine), recognized glucose into the bloodstream, thereby causing glucose levels to by different receptors, induce the same response in a single tar- rise. The hormone insulin is produced by the beta cells of the get cell. The two receptors differ from one another primarily in pancreas in response to high glucose levels and stimulates glu- the structure of the ligand-binding pocket on the extracellular

cose uptake and storage as glycogen. Finally, epinephrine— surface of the cell, which is specific for one or the other hormone. gers Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 632 duced by cAMPby duced In-Response a of Example An Mobilization: Glucose an increase in the levels of cAMP. heterotrimericactivateof samethetype proteinsG Following activation bo by their respective ligands, amplification of the signal (these steps are indicat are steps (these signal the of amplification 15.14 Figure in illustrated cascade reaction the in Many of signal generated from the original message. ampli greatly to mechanism a provides messenger ond production the Thus, time. of period short a in gers cyclase me cAMP of number large producea can which of each adenyl an activate can which of each teins, number a activate can surface cell the at molecule Amplification Signal (kinase protein cAMP-dependent a of unit plasm where they bind to an allosteric site on a re 15.14). Figure of cAMP (stepsthe 1 andformation 2, c enzyme activated The effector. cyclase adenylyl an the other tissues of the body (step 8).so and bloodstream the into diffuses which glucose, conve is reaction the in formed 1-phosphate glucose (ste glycogenbreakdown ofthe stimulating e this 6), (step activates polypeptide residue phosphorylase serine glycogen specific a to group phosphate single addi the 115), (page discoveredKrebsbyFischer and of phosphate groups to glycogen molec phosphorylase t catalyze to enzyme the activates kinase phorylase phosphorylation contrast, In glycogen. to glucose of t prevents thus and activity catalytic its inhibits glycogen of Phosphorylation phosphoryl 5). and and 4 (steps kinase synthase glycogen metabolism, glucose piv a play that enzymes two include cell liver a in active The catalytic subunits target of substra PKA. rel thereby subunits, regulatory the of dissociation cAMP binding c the activity catalytic of the enzyme. normall subunits regulatory The subunits. (C) alytic t and (R) regulatory two of composed heterotetramer PKA binds to its receptor, activating a Ga activating receptor, its to binds this in step first The F in illustrated as reactions, of chain a initiating cAMP evokes a response that leads to glucose mobili (Figure membrane plasma the of surface inner the at domain catalytic whose protein membrane integral an the response. helps which termi cAMP phosphodiesterase, the enzyme tion of cAMP molecules present in the cell is accom de The phosphorylase. glycogen and ki synthase, glycogen phosphorylase 15.14: Figure of enzymes phorylated the of all from remove can phosphatase-1, A member particular of this of family enzymes nases. contain cells by Liver added groups phosphate the remove that phatases indefinitely. state activated the would cell the otherwise above; discussed steps the ne omd AP oeue dfue no h cyto- the into diffuse molecules cAMP formed, Once As one might expect, a mechanism must exist to rever to exist must mechanism a expect, might one As ) (step 3, Figure 15.14). In its inactive form, PKA is ais PKA form, inactive its In 15.14). Figure 3, (step ) cAMP is synthesized by adenylyl cyclase, adenylyl by synthesized is cAMP reaction cascadereaction h bnig f snl hormone single a of binding The ␣ s subunit, which activates which subunit, occurs as the hormonethe as occurs protein kinase Akinase protein he conversionhe gulatory gulatory sub- igure 15.14.igure th receptors otal role inrole otal tes of PKA he transferhe thatcause easing theeasing plished by of G pro-G of d y the by ed remain inremain eut in result ) The7). p zation zation by auses the the steps y inhibity of a sec-a of synthase of phos-of tion of aof tion protein, effector, 15.13). atalyzes ls Asules. wo cat-wo resides reaches phos- td to rted the ki-the nzyme phos- n the in y thefy struc- ssen- nase, nate ase ase se , genes that play a role in the response to cAMP. In l In cAMP. to response the in role a play that genes re regulatory the in located are CREs ini of transcription. rate the increase and bind factors scription DNAwhe the in sites are elements response that 525 a transcription factor called called factor transcription a mos 15.14), Figure 9, (step proteins nuclear key late molecules translocate into the nucleus where they p activ the of fraction A response. the in participate g its and nucleus the cytoplasm, the in produced are of effects best-studied and rapid most the Although TransductionSignal Pathways cAMP of Aspects Other mobilization of glucose within the cell.mobilization stimulus at the cell surface transformed is rapidly per barely a as begins what Thus, phosphates. glucose nu larger much a of formation the catalyze can turn w larger number molecules, a of glycogen phosphorylase phosphorylate turn in which molecules, kinase lase pho of number large a phosphorylates subunit alytic PKA Each c cAMP molecules activate PKA. blue arrows). ann a atclr uloie eune (TGACGTCA) sequence knownthe as nucleotide particular a taining 1 step 15.14, (Figure DNA the on sites to dimer a as proteinbinding monophosphate. Figure 15.13 phosphodiesterase, which converts the t nucleotide the cyclic converts which phosphodiesterase, The breakdown of cAMP (not shown) is accomp domains. simila two situated between in a cleft the membrane The enzyme’s on is located dimensions). helices (sh containing six transmembrane each parts, cyclase,adenylyl – O O O P O O O – O O P – Formation of cyclic FormationAMP from ATP of cyclic by is catalyzed cAMP responsecAMP element ). The phosphorylated version of CREBbindsof version phosphorylated The ). an integral membrane an integral that consists of two O O P – H 1 C CH 11 9 2 CC HC C N OH C H 2 3 T cAMP ATP N NH C O 4 2 5 OH C H CREB N N C 6 CH H Active site ( ( CRE AP epne element- response cAMP 7 – O O 8 P ). Recall frompage Recall ). C H H O the inner surface ofthe inner surface 10 O r cytoplasmic r cytoplasmic own here in two C H CC HC C N C H 12 into a major o a 5 N C NH lished by a O hosphory- td PKAated ito of tiation 2 iver cells,iver t notablyt enes alsoenes Ј in ofgions mber ofmber OH COOH C H ceptible sphory- cAMP re tran-re 0) con-0) hich hich in n evenn N N C H + CH PP at- i , 633

1 Glucagon epinephrine, others Receptor

GTP Gαs G protein Adenylyl cyclase

2 Inactive Active Cytoplasm Nucleus CREB PKA AMP cAMP P Phosphodiesterase Glycogen 9 CREB Protein synthase Active kinase A 3 4

Phosphatase 10 P P Inactive P Active CREB CREB Phosphorylase DNA P 5 kinase CRE Inactive

mRNA Glycogen Active Phosphatase Inactive P phosphorylase 6

Inactive Phosphatase 2– Active CH 2OPO 3 O O O Figure 15.14 The response by a liver cell to glucagon or epinephrine. The steps in the response to hormonal stimulation that PO 2– 3 7 lead to glucose mobilization are described in the text. Many of the Glucose Glucose 6– Glucose 1– Glycogen phosphate phosphate steps in the reaction cascade are accompanied by a dramatic amplifica- 8 tion of the signal. Steps leading to amplification are indicated by clus- Plasma membrane ters of blue arrows. Activation of transcription by CREB occurs in conjunction with the usual array of coactivators (e.g., p300 and CBP) Bloodstream and chromatin-modifying complexes (not shown).

for example, several of the enzymes involved in gluconeogen- eral of the hormonal responses mediated by cAMP in mam- 15.3 esis, a pathway by which glucose is formed from the interme- malian cells are listed in Table 15.3. Cyclic AMP pathways diates of glycolysis (see Figure 3.31), are encoded by genes have also been implicated in processes occurring in the nerv- Messen Second Their and Receptors Protein-Coupled G that contain nearby CREs. Thus, epinephrine and glucagon ous system, including learning, memory, and . not only activate catabolic enzymes involved in glycogen Chronic use of opiates, for example, leads to elevated levels of breakdown, they promote the synthesis of anabolic enzymes adenylyl cyclase and PKA, which may be partially responsible required to synthesize glucose from smaller precursors. for the physiologic responses that occur during drug with- cAMP is produced in many different cells in response to drawal. Another cyclic nucleotide, cyclic GMP, also acts as a a wide variety of different ligands (i.e., first messengers). Sev- second messenger in certain cells as illustrated by the induced

Table 15.3 Examples of Hormone-Induced Responses Mediated by cAMP

Tissue Hormone Response

Liver Epinephrine and glucagon Glycogen breakdown, glucose synthesis (), inhibition of glycogen synthesis Skeletal muscle Epinephrine Glycogen breakdown, inhibition of glycogen synthesis Cardiac muscle Epinephrine Increased contractility Adipose Epinephrine, ACTH, and glucagon Triacylglycerol catabolism Kidney Vasopressin (ADH) Increased permeability of epithelial cells to water Thyroid TSH Secretion of Bone Parathyroid hormone Increased calcium resorption LH Increased secretion of steroid hormones Adrenal cortex ACTH Increased secretion of gers Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 634 provide a structural framework or or framework AK structural a figure, provide this in indicated As 15.16. Figure in shown are which of several since, discovered been have AKAPs ent At eredleast as proteins that co-purified with PKA. The first that AKAPsfunction as signaling were hubs. ciae a ope efco.Te fetr n hs c this in effector The effector. coupled a activates in part by the discovery of PKA-anchoring proteins sub PKA different express cells different that tion the by part in answered was question This type. cell in stimulus, particular a to response in substrates appro the phosphorylates PKA how to as question the b which functions, different out carry these of Most in in the vision. signaling pathway involved GMP cyclic also plays discussed in the next section, page on discussed cells muscle smooth of relaxation activation is depicted in 15.5Figure structu whose rhodopsin, that above mentioned was It and smell depends largely taste, Our ability to see, in Sensory PerceptionThe Role of GPCRs signal to a heterotrimeric G protein (called trans which molecule, rhodopsin the in change tional co a induces light of photon single a of Absorption provide us with color vision under conditions of br retin the of cones the in present are GPCRs related Several room. darkened a in or night at environment pictu black-and-white a with us provide and tensity which are the photoreceptor cells that respond to l rods the in present protein light-sensitive the is n AP ees nue b eiehie aorsi,a vasopressin, TSH to epinephrine, different responses. physiological by induced levels cAMP in su therebylinking different types, cell these of each in strates phosphorylate must PKA Clearly, hormone. secretion the to leads to TSH response in cell roid and the activation membrane of to the water, enzyme sponse to vasopressin causes an increase in the per ce tubule kidney a in enzyme same the of activation g of breakdown the to leads epinephrine to response live a in PKA of activation Although 15.15). (Figure thi by phosphorylated proteins specific the by mined typical is cAMP to cell given responsea the of PKA, similar role signaling. in cell t proteins of types different of variety a co-opted fu similar a with suggesting that AKAPsevol have proteins a diverse structure, most unlike that, note to diffe is It of levels. cAMP in increase an following strates presence the in PKA substrates and consequently of phosphorylation diff of localization in ing Different cells express different AK lar substrates. the subcellular localization of PKA in the presence Substrate selection thus is a partly con phorylated. beco to ones first the are they and subst by relevant close present the activated, is PKA and rise c levels When substrates. more or one to proximity close in PKA accu As a consequence, locations within the cell. to PKA sequestering by interactions protein–protein eas cM eet ms o is fet b activati by effects its of most exerts cAMP Because Over one hundred PKA substrates have been described been have substrates PKA hundred one Over b scaffold saGC.Rhodopsin is a GPCR. , transducin o coordinating for of our retina,our of o carry out aout carry o the increasethe meability meability of P,result-APs, on GPCRs. ighter light. ow light in- sequence of a particulara of particu- strates and strates of thyroidof interesting erent sub- a key role s i the is ase or me phos-me 50 differ- observa- ution has re of ourof re 66 As656. ae are rates ,whicha, ly deter- ly in a thy- rings up rings nforma- lycogen, specific ,which), s kinases ll in re-in ll r cell in cell r discov- mulates nction, AKAPs closely e andre is amits AMP priate AP s rent nd ng b- . are able to combine with a large variety of differe of variety large a with combine to able are exp taken humans that, receptors that odorant different 400 roughly estimated is It University. Columbia Ax Richard and Buck Linda by 1991 in identified first receptor odorant Mammalian nose. our enter that cals ceptors contain these epithelium, nasal the of in located are which tips distal The stem. brain our in located olfactory the to cavity nasal upper our lines that ep the from extend that olfactory along ted tion of action potentials along the optic nerve. the to lead may messenger, second a ofdecre concentration a by triggered is it that in unusual is which This channels. sodium the of closure the to leading l loweredcGMP in results phosphodiesterase cGMP of A current”). “dark(a current ionic inward continued leadin membrane,configuration, open an plasma in channels the the keeping in channels binding sodium of t capable cGMP-gated In thus and high retina. remain the levels of cGMP cells rod the in excitation sual in role important an plays cGMP 15.13). (Figure cAMP struc in similar messenger second a cGMP, nucleotide enzyme cGMP which phosphodiesterase, hydrolyzes the Figure 15.15 neatwt o hnes hshdetrss and G , with interact ion channels, pnprn,freape binds to a similar for example, Epinephrine, when it binds to even responses in different cells, elici can the same hormone In fact, . areeffects thought to be mediated by activation of in cAMP concentration. changes by be affected can el neg eaain nadto oPA cAMP is In addition to PKA, undergo cells relaxation. an are broken downtriacylglycerols in the fat cell, glycogen is broken down in the are quite different: The the production three of cAMP in all types. cell of th cells and smooth muscle fat cells, liver cells, Plasma membrane (glycogen formation) Our sense of smell depends on nerve impulses transm impulses nerve on depends smell of sense Our Triglyceride , which are GPCRs capable of binding various chemi- various binding of capable GPCRs are which , formation) (fatty acid (transport) Glycogen synthase

Kinase Schematic illustration of the variety of processes that of the variety illustration Schematic (glycogen breakdown) Phosphorylase kinase cAMP disassembly) (DNA synthesis, (assembly/ RNA synthesis) differentiation, Nucleus Kinase ␤

Kinase -adrenergic receptor in Kinase netn,causinge intestine, the same receptor. d the smooth muscle the same enzyme, liver cell, epne,however,responses, Endoplasmic t very different t very synthesis) reticulum All of theseAll EFs (page 641). (protein known to nt chemicalnt bulb that isthat bulb odorant re-odorant s i the in ase response, together, e dark, he ctivation neurons, ithelium genera- ture toture weres g to ato g cyclic evels, l ofel ress vi- to it- 635

L-type Ca2+ AMPA channel receptor NMDA receptor Plasma AKAP18 membrane AKAP79/150 PKA Microtubules PKC PKA PKA MAP2 PP2B

PP1 Centrosome PKA Yotiao PP1 PKN PP2A PDE PKA AKAP350 PDE mAKAP PKC PKA Gluco- GSK3␤ kinase PKA PP1 Pericentrin AKAP220 BAD D-AKAP1 PKC PKA Nucleus PP1 PKA WAVE1 PKA PP1 Rab32 Vesicles Abl PKA PKC PKA PKA PKA Gravin WAVE1 AKAP-Lbc Actin Rac Rho PKC PKD

Cytoskeleton

Figure 15.16 A schematic representation of AKAP signaling com- a number of different compartments, including the plasma membrane, plexes operating in different subcellular compartments. The AKAP mitochondrion, , centrosome, and nucleus. (R EPRINTED in each of these protein complexes is represented by the purple bar. In WITH PERMISSION FROM W. W ONG AND J. D. S COTT , N ATURE each case, the AKAP forms a scaffold that brings together a PKA REVIEWS MOL CELL BIOL 5:961, 2004; COPYRIGHT 2004. NATURE molecule with potential substrates and other proteins involved in the REVIEWS MOLECULAR CELL BIOLOGY BY NATURE PUBLISHING signaling pathway, including phosphatases (green triangles) that can GROUP . R EPRODUCED WITH PERMISSION OF NATURE PUBLISHING remove the added phosphate groups and phosphodiesterases that can GROUP IN THE FORMAT REUSE IN A BOOK /TEXTBOOK VIA terminate continued signaling. The AKAPs shown here target PKA to COPYRIGHT CLEARANCE CENTER .) structures (odorants). 1 Each olfactory receptor neuron ex- Our perception of taste is much less discriminating than presses only one allele of one of the hundreds of different our perception of smell. Each cell in the tongue 15.3 odorant receptor genes. Consequently, each of these sensory transmits a sense of one of only five basic taste qualities, neurons contains only one specific odorant receptor and is namely: salty, sour, sweet, bitter, or umami (from the Japanese Messen Second Their and Receptors Protein-Coupled G only capable of responding to one or a few related chemicals. word meaning “flavorful”). Taste receptor cells that elicit the As a result, activation of different neurons containing differ- taste of umami respond to the amino acids aspartate and glu- ent odorant receptors provides us with the perception of dif- tamate and to purine , generating a perception that ferent aromas. That does not mean that a single chemical a food is “savory.” This is the reason that monosodium gluta- cannot interact with more than one olfactory receptor, but mate and disodium guanylate are commonly added to rather that the specific combination of receptors that are acti- processed foods to enhance flavor. The pleasurable umami vated by that compound may play a key role in producing a taste is thought to have evolved as a mechanism to drive particular smell. Mutations in a specific gene encoding a par- mammals to seek high-protein foods. The perception that a ticular odorant receptor can leave a person with the inability food or beverage is salty or sour is elicited directly by sodium to detect a particular chemical in their environment that most ions or protons in the food. These ions pass through the ϩ ϩ other members of the population can perceive. When acti- plasma membrane of receptor cells via Na or H channels, vated by bound ligands, odorant receptors signal through het- respectively, eventually leading to a depolarization of the cell’s erotrimeric G proteins to adenylyl cyclase, resulting in the plasma membrane (page 166). In contrast, the perception that synthesis of cAMP and the opening of a cAMP-gated cation a food is bitter, sweet, or savory depends on a compound in- channel. This response leads to the generation of action po- teracting with a GPCR at the surface of the receptor cell. Hu- tentials that are transmitted to the brain. mans encode a family of about 30 bitter-taste receptors called T2Rs, which are coupled to the same heterotrimeric G pro- tein. As a group, these taste receptors bind a diverse array of 1 The human genome contains roughly 1000 genes that encode odorant recep- different compounds, including plant or cyanides, tors but the majority are present as nonfunctional pseudogenes (page 408). Mice, which depend more heavily than humans on their sense of smell, have that evoke a bitter taste in our mouths. For the most part, sub-

more than 1000 of these genes in their genome, and 95 percent of them encode stances that evoke this perception are toxic compounds that gers functional receptors. elicit a distasteful, protective response that causes us to expel Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 636 causes causes us to lose some of our appreciation for the c a have we when evident more becomes taste of tion our in neurons olfactory of importance The taste. of taste receptors (gustatory) that provides us with o It is this bymerged taste input receptors. from provided both o messages simple relatively the than eaten have allowing the brain to cosa, learn much more about th that travel via the throat to neurons olfactory in o releases het chewed is that TR1-TR3 food a Fortunately, of erodimer. consist receptors Umami a and sweeteners. monellin), (e.g., proteins and tasting certain sugars, to responds it and heterodimer) T1R3 receptor sweet-taste affinity high one only possess H carbohydrates. energy-rich contains that one be to t sweet a elicits that food a contrast, In agreeable. basic bitt is eaten have we food the that same simply is which the evoke substances diverse many result, substan noxious unrelated to respond that receptors dif of variety a contains sensation bitter a evokes taste-bu single a protein, receptor single a contain cel olfactory Unlike mouth. our from matter food the .Describe the relationship between phosphatidylino 8. .What ismeantbytheterm 3. Describe Sutherland’s experiment that led to the 2. What is the role of G proteins in a signaling pat 1. .Describe the steps that lead from the synthesis o 5. .How is it possible that the same first messenger, 4. .What isthemechanism offormationthesecond 7. Describe the steps between the binding of a ligand6. W E I V E R involving DAG? esters interferedo phorbol with signal pathways diacylglycerol, calcium ions, and protein kinase C. concept of the second messenger. formation ofIP stimuli? as glycogen breakdown, can be initiated by differen in different target cells? That the same response, ger, such as cAMP, can also evoke different respons different target cells? That the same second messen as epinephrine,canevokedifferent responsesin messenger IP cyclase. How is the response normally attenuated? and theactivationofaneffector, such asadenylyl such asglucagontoaseventransmembranereceptor phosphodiesterase? arrestin? By protein phosphatases? By cAMP and stream. How is this process controlled by GRKs a liver cell to the release of glucose into the blo attheinnersurfaceofplasmamembrane cAMP [Ca increase the possibilities for metabolic regulation increase the possibilities for metabolic cascade result in amplification of a signal? How doe signal transduction? How does the use of a reaction 2 ϩ ]? 3 ? What istherelationshipbetween 3 and an elevation of intracellular amplification in regardto our nasal mu- taste of food. ur rich sense aste is likelyis aste lfactory lfactory and ferent T2Rferent er and dis-and er hway? d cell that cell d od- (a T1R2-(a e food we ces. As a As ces. ? How f such percep- such old thatold dorants sitol, rtificial umans sweet ls thatls taste, t s it es - - Receptor protein-tyrosine kinases protein-tyrosine Receptor an unregulated version of the protein-tyrosine kina occurscellsinth forexample, leukemia, Oneoftype lead to uncontrolled cell division and the developm kinasesthatregulatedcannotbe continuallare and proteinmutant Expressionof cells. migrationof and attachmentextraceltheto survival, differentiation, These kinases are involved in the regulation g humanof gro the by encoded are protein-tyrosine kinases with the of multicellular Over organisms. phorylation is a mechanism for signal transduction ei n netaellrlgn idn oan an domain, binding ligand extracellular an and helix transm single a contain that proteins membrane gral receptor 15.4 for Signal Transduction Phosphorylation as a Mechanism r 15.17ure dimerizati receptor ligand-mediated causing thereby tion factor molecule to bind to two receptors at th orgrowth single a forpossible it made This sites. RTKsreceptorsuggestedcontainligandstwoofthat Early 15.17). (Figure dimerization receptor-mediated ligand-mediateddimerizat recognized: beenhave tion Twomechanisms pairaofreceptors. of for receptor ligand-bindinextracellular the of dimerization the techniquesotherligandbindinthatofbasis the on the solve to widely is it yet RTK, entire an ofdimensionalstructure have biologists structural though the inside changes biochemical into translated cell outsi the on factorgrowth a ofpresence the is How transsignalmechanicsconsideringofwhenthe mind Dimerization Receptor ofr t ti mdl oe rwh atr (.. EGF (e.g., factors growth Some model. this to fo conform were factors growth all not However, re site. binding a contains subunit each which in subunits, linked di identical or similar two of composed plat are (CSF-1) as such factors derivedgrowth factor (PDGF)colony-stimulatingor differentiation and growth that is focused on signal transduction by RTKs. th of section This migration. cell neuronal and sion, ce response, immune the as diverse as processes trol a signals extracellular by indirectly regulated are protein-tyrosin Non-receptor insulin. as such lators growth -derived or by metabolfactor (PDGF) ( factor growth epidermal as such factors entiation pag on RTKs are discussed activated directly by is extracellular growth TK non-receptor first the of ery i identified The was of VanderbiltCohen Stanley by University. 1978 EGFR, studied, be to RTK first The TKs. non-recept 32 and RTKs 60 nearly encodes genome man rti-yoie iae cn e iie i to grou two in divided be can kinases Protein-tyrosine | (or a Protein-Tyrosine .Ti mdl a spotd y h observation the by supported was model This ). cytoplasmic rti sbtae.Protein-tyrosine phos- substrates. protein phosphorylatespecific tyrosine residues on Protein-tyrosine kinases ) protein-tyrosine kinases protein-tyrosine n biu qeto cms to comes question obvious An ( RTKs ), which are inte-are which ), are enzymes that nd they con- they nd t,division,wth, that appeared ent of cancer. se ABL. e same time, lularmatrix, y activecany 90 different and differ- differentia- g resultsing domainsg EGF) andEGF) . The hu- The . at containat dimeriza- el Al- cell? accepted e chaptere de of theof de e kinasese -tyrosine -binding embrane on (Fig-on duction: l adhe- ll factor-1 ic regu- ion andion sulfide- d enome. ceptor- discov- n tound 696. e three- work non- elet- or ps: or n 637 Ligand Ligand

Inactive Inactive monomers monomers

Ligand induces dimerization interface

Inactive monomers Ligand-mediated dimerization

Receptor-mediated dimerization

Active dimers Active dimers 15.4 Protein-Tyrosine Phosphorylation as a Mechanism for Mechanism a as Protein-TyrosinePhosphorylation

Trans-autophos- Trans-autophos- phorylation phorylation

P P P P

Signal Signal transmission transmission

SH2 or PTB SH2 or PTB domain domain P P P P

(a) (b) Figure 15.17 Steps in the activation of a receptor protein-tyrosine are similar to those in part a, except that the ligand is monovalent and, kinase (RTK). (a) Ligand-mediated dimerization. In the nonactivated consequently, a separate ligand molecule binds to each of the inactive state, the receptors are present in the membrane as monomers. Binding monomers. Binding of each ligand induces a conformational change in of a bivalent ligand leads directly to dimerization of the receptor and the receptor that creates a dimerization interface (red arrows). The Transduction Signal activation of its kinase activity, causing it to add phosphate groups to ligand-bound monomers interact through this interface to become an the cytoplasmic domain of the other receptor subunit. The newly active dimer. (B ASED ON A DRAWING BY J. S CHLESSINGER AND formed phosphotyrosine residues of the receptor serve as binding sites A. U LLRICH , N EURON 9:384, 1992; BY PERMISSION OF CELL PRESS . for target proteins containing either SH2 or PTB domains. The target NEURON BY CELL PRESS . R EPRODUCED WITH PERMISSION OF proteins become activated as a result of their interaction with the CELL PRESS IN THE FORMAT REUSE IN A BOOK /TEXTBOOK VIA receptor. ( b) Receptor-mediated dimerization. The sequence of events COPYRIGHT CLEARANCE CENTER .) Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 638 ok o spot a eod ehns (iue 15.17 (Figure mechanism second a supports now work TGF sites that act as binding sites for cellular signaling proteins. It adjacent is to these the autophosp kinase domain. late each other on residuestyrosine that are prese pho to proceed subunits receptor the activated, been entering. activation Once of their the kinase kinase d domain. from ATP in a position away from the substrate-binding site, preventing thereby the activation Followingloop i its phosphorylation, site, binding sub the obstructs unphosphorylated, when loop, vation present in the in present residues tyrosine on by usua trolled is activity Kinase molecules. signaling plasmic the receptor’s kinase as or activity binding serve they can out two different RTKsfunctions: carry can close contact allows for side Bringing of twothe kinaseplasma dom membrane. ing of two protein-tyrosine kinase domains on the c receptordimerization mostRTKs, results in the juxt 15 Figure (see ligand of absence the in dimers tive including the areinsulin prese and IGF-1 receptors, ity of their receptors A to smallform numbedimers. ligands act as allosteric regulatorsanism, that tur exposure of a receptor With dimerization interface. leadingf theto extracellularreceptor, domaina of conformationalainducesbindingchang ligandwhich t i o n s Interac- Protein–Protein Phosphotyrosine-Dependent ActivationKinase Protein eetro h ie,and vicereceptor versa (Figure of the15.17 dimer, o domaincytoplasmictheresidues tyrosineinlates protein kinase activity of one receptor of the dime n dmis r the are domains ing sineresidues (as TheinFigure best-studied 15.17). containdomainsthatbind specifically phosphorylto suc because protein-tyrosinevated receptors, kinase Signalingproteins Figureare15.3). ableassociato teinsthat interact with one another in asequentia phosphotyrosine-binding nase signaling activity in these lower single-celle ing which protein, correlates with the overall lack budding-yeast genome encodes only interestingonenois to It SH2-domain-co residue). any be can Xwhich theSH2 domains ofPI 3-kinase bind topTyr-Met-X-M protein-tyrosinerecognizeskinasepTyr-Glu-Glu-Ile lated tyrosine For residues. the example, SH2 domain pho thesequenceimmediately acidadjacenttoamino Theresidues. specificity of the interactions is dete interactions occur following phosphorylation of spe phosphorylation-dependent protein–protein interacti Theymediatecoded largethehumanbyanumb genome. ardomainsSH2 110 than More 15.18). residue conserv (Figure a contain o and binding-pocketphosphorylatedaccommodatest thata composed acids amino are 100 They approximately viruses. (oncogenic) mor-causing tially identified as part of proteins encoded by the ␣ oti nyasnl eetrbnigst.Struc singlereceptor-bindinga contain only ) site. Signalingpathways consist chainaof signalingof activation loopactivation r-oooy 2Src-homology ( trans-autophosphorylation PTB ) of the kinase domain. The acti- The domain. kinase the of domain Autophosphorylation sites onsites Autophosphorylation . SH2 domains SH2 wereini- . ( SH2 ) domain d . genome of tu- of tyrosine-ki- rmined by the sites for cyto- in which the, n on the abil- lmanner (see r phosphory- nt in regions cific tyrosine te withteacti- ormationor a pTyr-bind- resulting in s stabilized f the otherthe f this mech- ytoplasmic , r of RTKs, te that thethatte horylation n.Theseons. nt as inac- proteinsh omain has aposition- b atedtyro- of the Src whereas, ). 2) For.24). ht are that regulate l con- lly n theand sphory- e in thein e sphory- yrosine strate- ains in ntain- en-e et(in b tural er ofer p r o - in) ed f (F residue when tyrosine the key but only interaction, creating a of the SH2 domain, on the surface pockets Figure 15.18 I E C tyrosine residue and the isoleucine residuetyrosine ( The phos The phosphate group is shown in light blue. arewhose side chains colored green is and backbone (Pro-Asn-pTyr-Glu-Glu-Ile-Pro) is shown as a space- heptap The phosphotyrosine-containing ribbon. purple areasurface represented by red dots and the polype domain of the protein is shown wi in a cutaway view residue. containingand a peptide a phosphotyrosine rtis rncito atr,and enzymes (Figure transcription factors, proteins, proteins,adaptor including RTKs, activatedwith act distinguishseveral groups signalingof proteins th lation sites present on the receptor (as in Figure P containingspecificautopsignalingtoproteins bind or SH2- which in complexes, signaling of formation thereforeactivation Receptor cytoplasm. the in ent domainsa signaling ofPTB proteins orety SH2 with moreresiduestyrosineautophosphorylatedorone on (R kinases protein-tyrosine receptor that Pathways seen have Signaling Downstream of Activation osre ad ifrn PB oan pses differe possess domains PTB residues that interact with their ligands. different and conserved are domains PTB motif. phosphorylated the to ically whereas Asn-Pro-X-Tyrphorylated others bind motif, u an to specifically bind to appear domains PTB some be however, complicated, more is story The Tyr)motif. (As asparagine--X-tyrosine an of part as ent bind to tyrosine phosphorylated residues that are u LSEVIER ELL ROM of a signaling complex (Figure 15.19of a signaling complex (Figure more signaling proteins to become joined together a Adaptor proteins function as linkers that enable tw PTB domains were discovered more recently. They can They recently. more discovered were domains PTB Arg 273 93 R 1993. 72:783, P–TYR Arg G α ABR IE L .) A2 β B5 -2 PRO The interaction between an SH2 domain of a proteinof domain SH2 an between interaction The W -1 ASN Trp KMNE AL ET AKSMAN PITDWT EMSINFROM PERMISSION WITH EPRINTED β β B1 β A B +1 GLU β C β D +2 GLU ., N + 3 I L E OREYOFCOURTESY α B ϩ BG 3) are seen to project into a .Adaptor proteins). +4 PRO ptide backbone as aptide backbone is phosphorylated. colored yellow. th the accessible tightly fitting tightly J phorylated phorylated OHN 51) We 15.17). can Tyr Leu BG4 The SH2 filling model eptide Tyr at can inter-canat β sually sually pres- D5 hosphory- K α 15.19). n-Pro-X- K) areTKs) eut inresults B9 URIY AN . A vari- A . o or re pres-re nphos- s part specif- poorly cause TB- We nt , 639 Figure 15.19 A diversity of signaling proteins. Cells contain numer- Ligand ous proteins with SH2 or PTB domains that bind to phosphorylated tyrosine residues. ( a) Adaptor proteins, such as Grb2, function as a link Active Ras between other proteins. As shown here, Grb2 can serve as a link between an activated RTK and Sos, an activator of a downstream protein named Ras. The function of Ras is discussed later. SH2 domain Ras (b) The docking protein IRS contains a PTB domain that allows it to GTP bind to the activated receptor. Once bound, tyrosine residues on the P P P Sos docking protein are phosphorylated by the receptor. These phosphory- lated residues function as binding sites for other signaling proteins. Grb2 (c) Certain transcription factors bind to activated RTKs, an event that (a) leads to the phosphorylation and activation of the transcription factor and its translocation to the nucleus. Members of the STAT family of transcription factors become activated in this manner. ( d) A wide array of signaling enzymes are activated following binding to an activated RTK. In the case depicted here, a phospholipase (PLC-␥), a lipid kinase (PI3K), and a protein-tyrosine phosphatase (Shp2) have all bound to phosphotyrosine sites on the receptor. PTB domain IRS P P P P P SH2 P P PI3K Shp2

(b)

Kinase activity 15.4 STAT family Protein-Tyrosine Phosphorylation as a Mechanism for Mechanism a as Protein-TyrosinePhosphorylation P P transcription P factor

P

P Active STAT dimer SH3-N Nucleus (c) SH3-C

Figure 15.20 Tertiary structure of an adaptor protein, Grb2. Grb2 consists of three parts: two SH3 domains and one SH2 domain. SH2 domains bind to a protein (e.g., the activated EGF receptor) containing a particular motif that includes a phosphotyrosine residue. SH3 domains bind to a protein (e.g., Sos) that contains a particular motif PI-PLCγ that is rich in proline residues. Dozens of proteins that bear these domains have been identified. Interactions involving SH3 and SH2 P P P domains are shown in Figures 2.40 and 15.18, respectively. Other PI3K Shp2 adaptor proteins include Nck, Shc, and Crk. (F ROM SÉBASTIEN

MAIGNAN ET AL . S CIENCE 268:291, 1995. REPRINTED WITH Transduction Signal (d) PERMISSION FROM AAAS. I MAGE PROVIDED COURTESY OF ARNAUD DUCRUIX .) contain an SH2 domain and one or more additional protein–protein interaction domains. For instance, the page 62, SH3 domains bind to proline-rich sequence adaptor protein Grb2 contains one SH2 and two SH3 motifs. The SH3 domains of Grb2 bind constitutively to (Src-homology 3) domains (Figure 15.20). As shown on other proteins, including Sos and Gab. The SH2 domain Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 640 I I I presence messenger molecules. of extracellular required changes to the biochemical to respond to t tha with activated of events RTKs initiate cascades signaling proteins that ass below, be described will be regulated can directly by phosphorylation. zymes Fi activity. resulting in catalytic in a change main, do- in the catalytic change a conformational that causes results in the SH2 domain in change a conformational binding to in phosphotyrosine which nism (page 115), m also be activated through can an allosteric zymes proximity to their substrates. places them in close as a result to the membrane simply of translocation be ac can Enzymes their association with a receptor. are activated foll these identifiedenzymes by which cruitment of STAT proteins (Figure 15.19 sites situated within a dimerized receptor leads to (Figure 15.19(Figure directly or indirectly as a consequence of this ass associate with activated RTKs and areenzymes turne thes equipped with SH2 domains, When vating proteins. and GTPase ac phospholipases, lipid kinases, phatases, protein p protein kinases, include enzymes Signaling 15.19 the SH2 domain of another STAT molecule (Figure rosine phosphorylation site that can act as a bindi STATssystem. contain an SH2 domain together with a play an important role in the function of the immun Transcription factors12. that belong to the STAT fa Transcription factors were discussed at length in C 15.19 with additional tyrosine phosphorylation sites (Fig supply certain recepto such as IRS, Docking proteins, 15. is presentwhich (Figure at the plasma membrane to a recep from or Grb2-Gab the cytosol of Grb2-Sos the Tyr-X-Asn motif on an RTK results in translocat o phosphorylation tyrosine Consequently, X-Asn motif. residues within a tyrosine binds to phosphorylated docking proteins that are expressed in a particular receptor to turn on signaling molecules canwi vary because the abilitytility to the signaling process, Docking proteins tionalprovide signaling molecules. phosphorylation sites then act as binding sites for tyrosine residues Th present on the docking protein. the receptor Once phosphorylbound together, protein. binding site for theor SH2PTB domain of the docki to whichautophosphorylation provid of the receptor, Binding of an extracellular ligand to sites. a recep an SH2 domain and a number of tyrosine phosphorylat where they stimulate the transcription of specific g move to the nucleus but not monomers, Dimers, dimers. these transcription factors and vice versa, protein, one STAT protein and the SH2 domain on a second STA interaction between the phosphorylated tyrosine res STAT As a result of the proteins are phosphorylated. tyrosine residuestion with in the receptor complex, naling an is discussed in Section 1 The role of STATs in an immunevolved response. in s c b .Tyrosine phosphorylation of). STAT SH2 binding .Docking proteins contain either domain a PTB or). d .Three have been mechanisms general ). c .Upon associa-). of the ociation tor leads the re- addi- ng site for cell. will form al,en-nally, enes in- En- ure hapter ociate th the these t lead T yr- which, echa- idue on owing he e ese 7.4. mil y tivated versa- es a

As hos- rs ng d on 19 ion tor, ig- ates ti- ion ty- a f ). T e in the context of the . A different cascade will b protein-tyrosine kinases. be the b on probably turned is that cascade signaling is characterized which pathway, kinase Ras-MAP the will we First RTKs. of downstream activated are that p important of couple a at closely more look can we nisms by which RTKs are able to activate signaling signaling.continued intracellular membrane become of part endosomal signaling plasma complexes and en the to returned lysosomes, in graded they can be de- RTKsnalized can have alternate several fates; As in the case of GPCRs (Figure ubiquitinat 15. internalization. receptor by followed is receptors vated 31 t complex Cbl (page the of Binding 542). (page internalization degradation for proteins those proteins marking other to a covalently is linked is that protein receptor. the to molecule ubiquitin a atta the catalyzes and receptor the Cbl with associates domain. SH2 an possesses which Cbl, for site ing as act can which residues, a tyrosine they tophosphorylate ligands, by activated are RTKs When Cbl. named One mechanism involves a receptor-binding research. are an remains internalization receptor causes what recept the of internalization by terminated usually nig h Response the Ending RAShum the of one in Mutations GDP. to GTP bound its of Ras is turned off downstreamby signaling proteins. tant versions of versions tant conta cancers human all of percent 30 approximately It was subsequently discove from its mammalian host. determined to only later, a retroviral oncogene and, Ras was into originally described tumor as cells. th norm transform to them enable that , called tion Some in of the these form viruses of conta RNA. Retroviruses are small viruses that carry their gen The Ras-MAP Kinase Pathway ure 15.19ure group that is embedded in the inner leaflet attache of the b covalently a by membrane plasma the of face many members G-protein of the small superfamily. The principles discussed in connectionport. with Ra nucleocytoplasmic and trafficking, vesicle ganization, cytos expression, gene differentiation, division, cell in processes, numerous of regulation the in involved These prote and (page 492). Sar1 (page 296), 302), Rabs the including proteins G (monomeric) small 150 of superfamily a of part are proteins Ras that note om:a atv GPbud om n a iatv GDP- 15.21 inactive (Figure an form and bound form GTP-bound active an d two in forms: present are proteins Ras subunit. small gle Ras consists of o however, heterotrimeric G proteins, time molecular a and switch a both as acts also Ras like G tho proteins that were discussed earlier and, Now that we have discussed some of the general mech general the of some discussed have we that Now Ras is a small GTPase that is anchored at the inner the at anchored is GTPase that small a is Ras genes that lead to tumor formation prevent the prot the prevent formation tumor to lead that genes a ). Ras is functionally similar to the heterotrimeric the to similar functionally is Ras ). RAS genes. At this point it is important to important is it point this At genes. a inl rndcin y Ts is RTKs by transduction Signal .RsGP id ad activates and binds Ras-GTP ). etic informa- e product of se proteins, ilayer (Fig- e described more thanmore r Exactlyor. y activatedy chment of chment keletal or-keletal be derived a of activeof a hydrolysis pathways, s apply to thereby, nly nly a sin- ) inter-7), .Unliker. in genes, ion and and ion athways red that discuss a bind- a cluding ifferent protein gage in n mu-in trans- lipidd al cellsal o acti-o ins are small (page ) or 2) sur- then or , ein an u- a- st 641

GEF Switch Switch II GEF GTP ON GEF Inactive Active G protein G protein 1b 2 GDP GDP Inactive GDP GTP G protein GDI 1a 3

GDI GEF Inactive Inactive G protein target 6 Active G protein protein GDP GAP GAP GTP 5 GAP Active 4 target GDP Switch I Clock GTP protein Switch Inactive OFF Signal target protein transmitted downstream (a) (b)

Figure 15.21 The structure of a G protein and the G protein cycle. bind to a downstream target protein (step 3). Binding to the (a) Comparison of the tertiary structure of the active GTP-bound state GTP-bound G protein activates the target protein, which is typically (red) and inactive GDP-bound state (green) of the small G protein an enzyme such as a protein kinase or a protein phosphatase. This has Ras. A bound guanine nucleotide is depicted in the ball-and-stick the effect of transmitting the signal farther downstream along the form. The differences in conformation occur in two flexible regions of signaling pathway. G proteins have a weak intrinsic GTPase activity the molecule known as switch I and switch II. The difference in that is stimulated by interaction with a GTPase-activating protein conformation shown here affects the molecule’s ability to bind to other (GAP) (step 4). The degree of GTPase stimulation by a GAP proteins. ( b) The G protein cycle. G proteins are in their inactive state determines the length of time that the G protein is active. when they are bound by a molecule of GDP. If the inactive G protein Consequently, the GAP serves as a type of clock that regulates the interacts with a guanine nucleotide dissociation inhibitor (GDI), duration of the response (step 5). Once the GTP has been hydrolyzed, release of the GDP is inhibited and the protein remains in the inactive the complex dissociates, and the inactive G protein is ready to begin a state (step 1a). If the inactive G protein interacts with a guanine new cycle (step 6). ( A: F ROM STEVEN J. G AMBLIN AND STEPHEN 15.4 nucleotide exchange factor (GEF; step 1b), the G protein exchanges its J. S MERDON , S TRUCT . 7:R200, 1999. R EPRINTED WITH PERMISSION GDP for a GTP (step 2), which activates the G protein so that it can FROM ELSEVIER .) Protein-Tyrosine Phosphorylation as a Mechanism for Mechanism a as Protein-TyrosinePhosphorylation

from hydrolyzing the bound GTP back to the GDP form. As leased, the G protein rapidly binds a GTP, which is pres- a result, the mutant version of Ras remains in the “on” posi- ent at relatively high concentration in the cell, thereby ac- tion, sending a continuous message downstream along the tivating the G protein. signaling pathway, keeping the cell in the proliferative mode. 3. Guanine nucleotide-dissociation inhibitors (GDIs ). GDIs The cycling of monomeric G proteins, such as Ras, be- are proteins that inhibit the release of a bound GDP from tween active and inactive states is aided by accessory proteins a monomeric G protein, thus maintaining the protein in that bind to the G protein and regulate its activity (Figure the inactive, GDP-bound state. 15.21b). These accessory proteins include The activity and localization of these various accessory pro- 1. GTPase-activating proteins (GAPs). Most monomeric G teins are tightly regulated by other proteins, which thus regu- proteins possess some capability to hydrolyze a bound late the state of the G protein. GTP, but this capability is greatly accelerated by interac- Ras-GTP can be thought of as a signaling hub because it tion with specific GAPs. Because they stimulate hydroly- can interact directly with several downstream targets. Here we sis of the bound GTP, which inactivates the G protein, will discuss Ras as an element of the Ras-MAP kinase cas- GAPs dramatically shorten the duration of a G protein- cade . The Ras-MAP kinase cascade is turned on in response mediated response. Mutations in one of the Ras-GAP to a wide variety of extracellular signals and plays a key role in genes ( NF1 ) cause neurofibromatosis 1, a disease in which regulating vital activities such as cell proliferation and differ- patients develop large numbers of benign tumors (neu- entiation. The pathway relays extracellular signals from the Transduction Signal rofibromas) along the sheaths that line the nerve trunks. plasma membrane through the cytoplasm and into the nu- 2. Guanine nucleotide-exchange factors (GEFs ). An inactive cleus. The overall outline of the pathway is depicted in Figure G protein is converted to the active form when the bound 15.22. This pathway is activated when a growth factor, such as GDP is replaced with a GTP. GEFs are proteins that EGF or PDGF, binds to the extracellular domain of its RTK. bind to an inactive monomeric G protein and stimulate Many activated RTKs possess phosphorylated tyrosine dissociation of the bound GDP. Once the GDP is re- residues that act as docking sites for the adaptor protein Grb2. Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 642 (as in Figure 15.19(as in Figure proximity close in Sos placing membrane, plasma the surf cytoplasmic the to cytoplasm the from Grb2-Sos transloc the promotes receptor activated an on site Grb2-bi a of Creation Ras. for GEF) (a factor change which is a guanine nucleo binds to Sos, in turn, Grb2, important signaling protein called Raf. Raf is then is Raf Raf. called protein signaling important inc proteins, of number a for interface binding a of site of Ras results in a conformational change and nucleotide-bindi nucleotid the in GTP for GDP of Exchange Ras GTP. rep is and released the is GDP opens result, a As Sos site. binding with Interaction notype. tion of Ras and transformation of the cell to a mal a constitutive in results mutant Sos membrane-bound Expression membrane. plasma the of surface inner the te permanently is that Sos of version mutant a with an by illustrated was This activation. Ras cause to Simply Simply bringing Sos to the plasma membrane is suffic Receptor PTK a-D Ras-GTP Ras-GDP Growth factor Soluble Raf E MEK MEK 3 MKP-1 R ERK ERK a ). Grb2 11 1 10 Receptor Membrane-bound FTF TF 9 2 Sos TF Raf 4 P Transcription P P 5 Gene 6 7 P P 8 P P P P MAPKKK MAPKK the creation ignant phe- experiment MAPK luding anluding thered tothered recruited laced bylaced to of ation tide ex- of thisof to Rasto c oface nding ctiva- ient ng e- IHPRISO OF PERMISSION WITH ) leading to an increase9), in the transcription of s scription factors increases their affinity for regul Pho suchtranscription as Elk-1. factors step 8), (TF, MAPK translocatesactivated, into the nucleus where residue thereby of this activating sequence, the enz MAPKKX-Tyr. phosphorylates MAPK on both the threoni MAPKs have a tripeptide near their catalytic site w they can phosphorylate tyrosine as well as serine a MAPKKs are(MAP dual-specificity kinase). a t kinases, MEK as a MAPKKkinase), and (MAP ERK kinase kinase), sequential kinase Raf is activity, known as a MAPKKK steps 5–7 is characteristic of all MAP kinase casca termed This ERK three-step (step 7). phosphorylation which in turn(step phosphorylates 6), and activates Raf phosphorylatesdepicted andhere, activates anot where it is phosphorylated and thus activated (step exchange which recruitsof Ras (step the 4), protein the Grb2-Sos proteins This complex (step causes3). tyrosine residues of the receptor (step 2) and the growth factor to its receptor (step 1) leads to the Figure 15.22 BOOK Jun further signaling S activity along (H. the pathway. threonine residues which of inactivaMAPK (step 11), the MKP family can remove phosphate groups from bot stimulated encodes a MAPK step phosphatase 1 (MKP-1; T CECSBYSCIENCES RENDS ) involved in the One growthof the genesresponse. os o bt EF n PG,hv as be identified been also have PDGF, and EGF both for tors the including of situated at the beginning of the pathway, several for Genes Jun). and Fos (e.g., pathway cancer-causing the at generated factors by transcriptional the of ber a and encoded Raf, Ras, for were genes the includes This oncogenes. they because covered pathway signaling Ras the in part a play that teins either become Many mutated of or are overexpressed. th genes cellular normal from derived are Oncogenes become to cells cause to ability their by identified key from a 14.8). cell G1 into S phasedriving (Figure a plays which D1, including proliferation, involv genes of activation the to leads pathway the Ev proteins. nuclear other and factors transcription sign the nucleus where other it and phosphorylates activates a and the MAP kinase receptors, is able Once to activated, m proteins. regulators, apoptotic cytoskele teins, kinases, protein factors, transcription ing be can that proteins 160 identified been have kinases these by phosphorylated Over ERK2. and ERK1 named k MAP two activate and phosphorylate to on goes phosphorylat Raf, of ME consequence a as 15.22). activated (Figure is MEK which kinase protein the is strates lation reactions. vated by a combination of andphosphorylation depho i where membrane plasma the of surface inner the to / ETOKVIATEXTBOOK s icse i te olwn catr noee are oncogenes chapter, following the in discussed As its of One kinase. protein serine-threonine a is Raf B IOCHEM I NTERNATIONAL The The steps of a generalized MAP kinase cascade. S CIENCE C E LSEVIER OPYRIGHT 944 94 T 1994. 19:484, U INOFNION L TD C LEARANCE . NTEFRA ES NA IN REUSE FORMAT THE IN B IOCHEMISTRY ED NBIOCHEMICAL IN RENDS atory sites on the DNA (step autophosphorylation of subsequent recruitment of pecific genes (e.g., NANDUN nd Allthreonine residues. e.Because of theirdes. m se ) Onceyme (step 7). 5.In the pathway 5). C ith the sequence Thr- Raf to the membrane, still another kinase her kinase named MEK sphorylation of the tran- the GTP-GDP ENTER it phosphorylates tes MAPK and stops scheme shown in (MAP kinase kinase whose expression is h tyrosine and erm denoting that ) Members of0). .K T K. N. .) ne and tyrosine R as a MAPK EPRODUCED end of theof end cancerous. number of were dis-were the recep- Binding of ed in cellin ed ONKS includ-, F o s entually, t is acti-is t the pro- a pro- tal ove into sphory- oe in role t haveat RTKs o by ion num- inases and sub- aling , K, 643 among the many known oncogenes. The fact that so many Mating Mating proteins in this pathway are encoded by genes that can cause factor High salt factor cancer when mutated emphasizes the importance of the path- way in the control of cell growth and proliferation. Ste2 Sho1 Ste2 Plasma membrane Adapting the MAP Kinase to Transmit Different Types G SH3 domain Chimeric G of Information The same basic pathway from RTKs βγ Ste5-Pbs2 βγ scaffold through Ras to the activation of transcription factors, as illus- Ste11 MAPKKK Ste11 Ste11 trated in Figure 15.22, is found in all eukaryotes investigated, Ste5 Pbs2 from yeast through flies and nematodes to mammals. Evolu- Ste7 MAPKK Pbs2 Ste7 kinase tion has adapted the pathway to meet many different ends. In Fus3 MAPK Hog1 yeast, for example, the MAP kinase cascade is required for Hog1 cells to respond to mating factors; in fruit flies, the pathway is utilized during the differentiation of the photoreceptors in the Transcription Transcription Transcription compound ; and in flowering plants, the pathway transmits factors activated factors activated factors activated signals that initiate a defense against pathogens. In each case, the core of the pathway contains a trio of enzymes that act se- Mating genes Osmoresponsive Osmoresponsive quentially: a MAP kinase kinase kinase (MAPKKK), a MAP activated genes activated genes activated kinase kinase (MAPKK), and a MAP kinase (MAPK) (Fig- ure 15.22). Each of these components is represented in a par- Mating response Osmoregulatory Osmoregulatory response response ticular organism by a small family of proteins. To date, 14 different MAPKKKs, 7 different MAPKKs, and 13 different (a) (b) (c) MAPKs have been identified in mammals. By utilizing differ- Figure 15.23 The roles of scaffolding proteins in mediating two ent members of these protein families, mammals are able to yeast MAPK pathways. (a) The MAPK pathway that regulates mating assemble a number of different MAP kinase pathways that in these cells is elicited by a mating factor that binds to a GPCR, Ste2, leading to the activation of a G which binds to the scaffolding transmit different types of extracellular signals. We have al- ␤␥ protein Ste5, which in turn binds the MAPKKK, MAPKK, and ready described how mitogenic stimuli are transmitted along MAPK proteins of the pathway, (b) The MAPK pathway that one type of MAP kinase pathway that leads to cell prolifera- regulates the yeast osmoregulatory response in cells exposed to high tion. In contrast, when cells are exposed to stressful stimuli, salt. The activated receptor (Sho1) binds to the Pbs2 scaffolding 15.4 such as X-rays or damaging chemicals, signals are transmitted protein by its SH3 domain. The MAPKKK Ste11 is shared in these along different MAP kinase pathways that cause the cell to two pathways but is recruited into one or the other response by virtue withdraw from the , rather than progressing through of its interaction with the appropriate protein scaffold. The scaffold for Mechanism a as Protein-TyrosinePhosphorylation it as indicated in Figure 15.22. Withdrawal from the cell cycle Pbs2 does not recruit a separate MAPKK, but has its own MAPKK gives the cell time to repair the damage resulting from the ad- enzymatic activity. (c) When cells are genetically engineered to express verse conditions. a chimeric Ste5-Pbs2 scaffold, they respond to a mating factor by Recent studies have focused on the signaling specificity of exhibiting the osmoregulatory response. (See Science 332:680, 2011, for a discussion of scaffold proteins and this experiment.) MAP kinase cascades in an attempt to understand how cells are able to utilize similar proteins as components of pathways that elicit different cellular responses. Studies of amino acid sequences and protein structures suggest that part of the an- swer lies in selective interactions between enzymes and sub- events. For example, they can induce a change in conforma- strates. For example, certain members of the MAPKKK tion of bound signaling proteins, leading to their activation or family phosphorylate specific members of the MAPKK fam- inhibition. A few scaffolding proteins are known to have an ily, which in turn phosphorylate specific members of the enzymatic role, as illustrated by the MAPKK activity of the MAPK family. But many members of these families can par- yeast Pbs2 scaffold shown in Figure 15.23 b. In addition to ticipate in more than one MAPK signaling pathway. facilitating a particular series of reactions, scaffolding proteins Specificity in MAP kinase pathways is also achieved by may prevent proteins involved in one signaling pathway from spatial localization of the component proteins. Spatial local- participating in other pathways. As a result, several pathways ization is accomplished by structural (i.e., nonenzymatic) pro- can share the same limited set of signaling proteins without teins referred to as scaffolding proteins , whose apparent compromising specificity. This is the case for the yeast MAP- function is to tether the appropriate members of a signaling KKK protein Ste11 shown in Figure 15.23 a,b , which partici- pathway in a specific spatial orientation that enhances their pates in both the mating and osmoregulatory response Transduction Signal mutual interactions. The AKAPs depicted in Figure 15.16 are depending upon which scaffolding protein it has interacted examples of scaffolding proteins involved in cAMP-driven with. The importance of scaffolding proteins is well illustrated pathways. Another group of scaffolding proteins, such as the by an experiment in which parts of two different yeast yeast proteins shown in Figure 15.23 a, b, play a role in routing MAPK-cascade scaffolding proteins (Ste5 and Pbs2) were signals through one of various MAP kinase pathways. In some genetically combined to form a chimeric protein (Ste5-Pbs2) cases, scaffolding proteins can take an active role in signaling (Figure 15.23 c). Normally these two scaffolds mediate two Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 644 otisteislnbnigst.The contains the insulin-binding site. olytic processing. The The processing. olytic b protein precursor single a from derived are which inactive state, is a tetramer consisting of two consisting of is two a tetramer inactive state, h Isln eetr s PoenTrsn Kinase an of Protein-Tyrosine composed is a receptor insulin Each Is Receptor Insulin The and/or decreasing gluconeogenesis. synthesis, eride increasingan glycogen increasingby uptake, glucose respond to rec such as cells in the liver, insulin their surface, express that Cells high. are levels cose cells informing molecule, messenger extracellular an Insulin f the pancreas respond by secreting insulin. b the levelsmeal, carbohydrate-rich a glucose after occurs as When rise, levels. glucose blood in increase an in resu glycogen of breakdown the stimulates and GPCRs acts glucagon earlier, discussed As glucagon. secrete o cells alpha the level, certain a below fall levels g blood When pancreas. the by monitored are culation i glucose of levels The problems. health serious and i electrolytes and fluids, glucose, of loss a in sults glucose blood in elevation persistent A metabolism. for glucose on largely depends system nervous tral a coma, and consciousness of loss to lead can levels A decrease in blo cose levels within a narrow range. bl maintaining effort considerable spend bodies Our Signaling by the Insulin Receptor the cells responded by displaying the osmoregulator r mating the stimulates normally that factor mating ex were protein chimeric the containing cells yeast differentMAPK signaling pathways (Figure 15.23 Figure 15.24 binding. ( causes a conformational change in the a conformational change causes yolsi rgo (iue 52) The 15.24). (Figure region cytoplasmic regi transmembrane single a region, extracellular an ikd oehr y iufie od (iue 52) Tw 15.24). these (Figure bonds disulfide by together linked b nti oe,binding of a single insulin molecul ) In this model, ␣␤ ( a h nui eetr shown here form in schematic ) The insulin receptor, heterodimers are held together by disulfide bondsdisulfide by together held are heterodimers The response of the insulin receptor to ligandThe α β chain (a) chain ␣ hi i etrl etaellr and extracellular entirely is chain ␤ subunits, which activates the which subunits, domain kinase Tyrosine ␤ ␣ and two two and chain is composed of ␣ Disulfide bond ␣ and e to the and a a and f the pancreasthe f ␤ this message ␤ subunits. n the urinethe n a,b unctions as y response. eta cells of cells eta od glucose posed to ato posed his are chains s the cen-the s its energyits pos oneptors n the cir-the n that glu-that on, and a and on, ␣ d triglyc-d levels re- levels through ␤ ood glu-ood esponse, .When). prote-y subunits in the lucose chain, lting of o (b) tyrosine kinase activity of the kinase activity tyrosine substrates (IRSs)substrates that are discussed below. residuesthe receptor on severa as tyrosine as well residues on the cyto located tyrosine phosphorylate come into close physical proximity (Figure 15.24 w cell, the of kinase domains onthe thetyrosine causes inside of o outside the repositioning on domains causes This ligand-binding molecule. insulin single dimer receptor insulin the that suggests work cent and receptor 15.24 activation (Figure tion of the kinase domains leads to trans-autophosp brane and in the carboxyl-terminal tail (Figure 15. t tail (Figure and in the carboxyl-terminal brane to adjacent present are that residues tyrosine on the vation receptorof the phosphoryl kinase domain, Fo open so that it bind can substrates. cleft alytic leaves now loop activation the addition, In together. ca for areresiduesessential that thereby bringing ea to respect with domain kinase the of lobes large sm the of rotation a requires conformation new This catalytic the from away conformation new a assumes acti the residues, tyrosine three the of phorylation U conformation in which it occupies the active site. a loop activation the state, unphosphorylated the In acti the in presentare sites phosphorylation these fied in the regioncytoplasmic of the insulin recept between between the tors are inactive in the absence of ligand (Figure (Figure ligand of absence the arein tors inactive insuli RTKs, other Like dimers. stable as present are insulin monomers, as surface cell the on present be Insulin Receptor Substrates 1 and 2and 1 Substrates Receptor Insulin ancnann sgaig rtis a i Fgr 15 Figure in (as proteins signaling main-containing recrui directly that sites autophosphorylation sess strates n poen (iue 15.19 (Figure proteins ing it because associates instead with a small fam rule, H dmi-otiig inln poen.Sm o th of Some events that occur during insulin signaling are proteins. show signaling domain-containing SH2 and Insulin Several tyrosine phosphorylation sites have been id been have sites phosphorylation tyrosine Several d ). The insulin receptor is an exception to this gene this to exception an is receptor insulin The ). ( IRSs ␣ .TeIS,i un provide the binding in sites turn, for The IRSs, ). his hs while most RTKs are thought to Thus, chains. P P P (c) ␤ uuis (subunits. b ), called called ), c ) Theactivated c ). insulin-receptor sub- insulin-receptor l insulin receptor P P P plasmic domain of Most RTKs pos-RTKs Most b 15.24 llowing acti- talysis closer talysis .Juxtaposi-). vation loop.vation r Three ofor. ily of ily - vation loopvation n in Figure ␤ 24 S2 do- SH2 t the tocell horylation pon phos- e mem-he ates itself subunits receptors ch other, ch ssumes assumes the cat-the c n recep-n ). id a binds .19 a l and all .Re-). f the the f cleft. enti- hich a , ral c e , 645

PI3K Grb2 Shp2 PI 3-kinase PI-5-P'tase

YY Y YY Y Y Y YY YY YY YYY Y Y N PH PTB C PI(4,5)P2 PI(3,4,5)P3 PI(3,4)P2 PTEN (a)

Plasma membrane Insulin receptor

PTB domain 5' 3'

PIP 4 5 P Protein synthesis 3 3 P Ras P P P P P P GTP P P PH domain P P P Grb2 Sos IRS-1 Glucose uptake P P Activated PDK1 P PKB Other P P signaling proteins mTOR mTORC2 PI3K Glycogen synthesis complex (2 subunits) (b) (c) Figure 15.25 The role of tyrosine-phosphorylated IRS in activating illustration.) ( c) Activation of PI3K leads to the formation of a variety of signaling pathways. (a) Schematic representation of an membrane-bound phosphoinositides, including PIP 3. One of the key IRS polypeptide. The N-terminal portion of the molecule contains a kinases in numerous signaling pathways is PKB (AKT), which interacts PH domain that allows it to bind to phosphoinositides of the at the plasma membrane with PIP 3 by means of a PH domain. This in- membrane and a PTB domain that allows it to bind to a specific phos- teraction changes the conformation of PKB, making it a substrate for phorylated tyrosine residue (#960) on the cytoplasmic domain of an ac- another PIP 3-bound kinase (PDK1), which phosphorylates PKB. The tivated insulin receptor. Once bound to the insulin receptor, a number second phosphate shown linked to PKB is added by a second kinase, of tyrosine residues in the IRS may be phosphorylated (indicated as Y). mostly likely mTOR. Once activated, PKB dissociates from the plasma 15.4 These phosphorylated can serve as binding sites for other membrane and moves into the cytosol and nucleus. PKB is a major proteins, including a lipid kinase (PI3K), an adaptor protein (Grb2), component of a number of separate signaling pathways that mediate the for Mechanism a as Protein-TyrosinePhosphorylation and a protein-tyrosine phosphatase (Shp2). ( b) Phosphorylation of insulin response. These pathways lead to translocation of glucose trans- IRSs by the activated insulin receptor is known to activate PI3K and porters to the plasma membrane, synthesis of glycogen, and the synthe- Ras pathways, both of which are discussed in the chapter. Other path- sis of new proteins in the cell. PKB also plays a key role in promoting ways that are less well defined are also activated by IRSs. (The IRS is cell survival by inhibiting the proapoptotic protein Bad (page 659) drawn as an extended, two-dimensional molecule for purposes of and/or activating the transcription factor NF- ␬B (page 660).

15.25. Following ligand binding and kinase activation, the in- ent on these docking proteins (Figure 15.25 b). Both IRS-1 and sulin receptor autophosphorylates tyrosine #960, which then IRS-2 contain a large number of potential tyrosine phospho- forms a binding site for the phosphotyrosine binding (PTB) rylation sites that include binding sites for the SH2 domains domains of insulin receptor substrates. As indicated in Figure of PI 3-kinase, Grb2, and Shp2 (Figure 15.25 a,b). These pro- 15.25 a, IRSs are characterized by the presence of an N-terminal teins associate with the receptor-bound IRS-1 or IRS-2 and PH domain, a PTB domain, and a long tail containing tyro- activate downstream signaling pathways. sine phosphorylation sites. The PH domain may interact with PI 3-kinase (PI3K ) is composed of two subunits, one phospholipids present at the inside leaflet of the plasma mem- containing two SH2 domains and the other containing the brane, the PTB domain binds to tyrosine phosphorylation catalytic domain (Figure 15.25 b). PI 3-kinase, which is acti- sites on the activated receptor, and the tyrosine phosphoryla- vated directly as a consequence of binding of its two SH2 tion sites provide docking sites for SH2 domain-containing domains to tyrosine phosphorylation sites, phosphorylates signaling proteins. At least four members of the IRS family phosphoinositides at the 3 position of the inositol ring (Figure have been identified. Based on the results obtained in knock- 15.25c). The products of this enzyme, which include PI 3, Transduction Signal out experiments in mice, it is thought that IRS-1 and IRS-2 4-bisphosphate PI(3,4)P 2 and PI 3,4,5-trisphosphate (PIP 3), are most relevant to insulin-receptor signaling. remain in the cytosolic leaflet of the plasma membrane where Autophosphorylation of the activated insulin receptor at they provide binding sites for PH domain-containing signal- Tyr960 provides a binding site for IRS-1 or IRS-2. Only after ing proteins such as the serine-threonine kinases PKB and stable association with either IRS-1 or IRS-2 is the activated PDK1. As indicated in Figure 15.25 c, PKB (more commonly insulin receptor able to phosphorylate tyrosine residues pres- known as AKT) plays a role in mediating the response to Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 646 lcs TransportGlucose (Figure PKB of activity kinase phosphorylate Ser/Thr the can activate PDK1 which in setting a provides P to proximity close in membrane, plasma the to PDK1 as well R as to other insulin, extracellular signals. R insulin.by Figure 15.26 (Figure 15.25(Figure phosphat lipid the by ring inositol the on position phosphate the removalof by terminated is signaling activi cellular numerous regulating in role crucial ha which mTOR, kinase, second a by phosphorylation on also PKB of Activation PKB. of activation for cient no is it essential, is PDK1 by phosphorylation While synthase kinase-3 (GSK-3) has been identified as a negativea as identified been has (GSK-3) kinase-3 synthase Gl o residues. turned threonine and is serine on that phosphorylation enzyme an synthase, glycogen by out i synthesis Glycogen glycogen. of form the in stored GLUT4 translocation is still lacking. pathwayss togetherworktwotothe howstanding of Detailed occur. translocationto GLUT4 foressential secondais pathway downstream theofinsulin recep thatstimulates Thissuggests GLUT4 translocation. t insuthe whereasonly is it receptorsactivate PI3K, tha known or well is It PI3K translocation. of stimulatesGLUT4 overexpression addition, In translocation. on experiments showing that inhibitors of PI3K bloc on activation of PI 3-kinase This andconclusio PKB. translocation GLUT4 d glucoseuptake(Figure 15.26). glucose transporters in the plasma membrane leads t ferred Thetoincrease as GLUT4 translocation. in nu w thprocess a fuse insulin, response vesicles to membraneinplasma These 15.26). (Figure cells sponsive ins of cytoplasm the in vesiclesmembrane in present from the blood In (pagethe 157). absence of insulin porter GLUT4 carries out insulin-dependent glucose glucos The synthesis. glycogen and transportglucose rnsBohm Sci.Trends Biochem. is from translocation the insulin receptor to GLUT4 A second path mediatewhere glucose uptake. can they t the transporters delivering (exocytosis), membrane fus The vesicles periphery. to the cell mic vesicles the translocati triggers which IRS-PI3K-PKB pathway, a signal is transm increases, the insulin level When vesicles that form by budding from the plasma membr B IOCHEMISTRY PITDB EMSINOF PERMISSION BY EPRINTED Excess glucose that is taken up by muscle and liver cells is Glucose transporters are storedGlucose transporters of cyt in the walls Regulation of glucose uptake in muscle and fat cell of glucose uptake in muscle Regulation c 2, ). E 125 06.(.V (D. 2006). 31:215, ; COPYRIGHT PKB is directly involved in regulatingin involveddirectly is PKB J OHN 95 J 1995, W E ANDOET OHN ILEY W & S e with the plasma ILEY itted through the .G V G. J. ONS o the cell surfaceo the cell not shown (see ane (endocytosis). way leading & S on of cytoplas- I, ecruitment ecruitment of NC OET GLUT4 is, lin receptorlin o increased s PTEN P ase ONS is PI3Kties. k GLUT4 torthatis n is based at the 3-the at oplasmic .) transport hatthere 15.25 mbersof s carrieds depends timulate , at is re-is at trans-e under- ln re-ulin t theith manyt I, ycogen suffi-t epends PKB NC f by ff KB, and and s s as . c ). Human Perspective.Human accom the in discussed is lifespan and pathways ing The relationshipthe subject between of insu debate. condi this to lead effects metabolic consequent its insulin resist by which but the molecular mechanism vessels,blood body’s the to damage to due be to thought are di kidney and reduced circulation in the limbs leading to amp resul blindness, that risks disease, health the diabetes—cardiovascular of Most pancreas. the of insulin-secreti the of death the to lead ultimately cyc viscious a up setting insulin, more even secrete p the stimulates which levels, glucose blood in tion This is presence turn of leads the to hormone. a chr resistance activation (Figure 15.14).activation (Figure s glycogen to further contributes dephosphoryla to synthase, known glycogen enzyme an 1, phosphatase tein Diabetes MellitusDiabetes in the body, which leads to a condition referred to referred condition a to leads which body, the in liver the in cells target overstimulate insulin of Ele secretion. insulin in increase chronic a to lead is lifestyle sedentary a with combined diet calorie i disease the ha eating of and lifestyle changing of result a likely incidence rising The rate. alarming wo the around increasing is incidence whose disease Typemorea is diabetes 2 Perspective17. Chapter of th in discussed is and insulin produce to inability Typecaus is diabetes 1 percent. remaining90–95 the acco which 2, type and cases, a the of which percent 5–10 for 1, type varieties: two in occurs Diabetes ing. ins in defects by caused is mellitus, diabetes eases, rae n S- kns atvt,rslig n n inc 15.25 (Figure an activity synthase glycogen in resulting activity, kinase GSK-3 in crease t leads insulin to response in pathway 3-kinase-PKB activation of Thus, following by phosphorylation PKB. ina is turn, in GSK-3, synthase. glycogen of regulator membrane Plasma , in which these target cells stop responding to the to responding stop cells target these which in , GLUT4 One of the most common human dis-human common most the of One Glucose vesicle Cytoplasmic

Fusion GLUT4 c ). Activation of pro-of Activation ). PKB and elsewhereand bits. A high- A bits. PDKI ng beta cellsbeta ng tion remaintion IR thought tothought vated levelsvated ulin signal-ulin PI3K onic eleva- le that canthat le utations— ancreas toancreas lin signal- Humane as complex ance and IRS-1 rld at anat rld ed by anby ed panying es inrease unts forunts ctivated ynthase ccounts o a de-a o most s the PI t from from t insulin sease, te 647 THE HUMAN PERSPECTIVE Signaling Pathways and Human Longevity Many factors are known to contribute to the aging process, some ge- reduced, which suggests a decrease in production of reactive oxygen netic and others nongenetic. Discussions of aging have appeared in species (page 35). several places in the text: in the Human Perspective on free radicals A number of early studies demonstrated that the lifespan of a (page 35), in the Human Perspective on mitochondrial diseases worm or fruit fly can also be dramatically increased by reducing the (page 208); in the Human Perspective on DNA repair deficiencies (page activity of insulin-like growth factors and their receptors. Studies 569); in the section on the nuclear lamina (page 490); and in the dis- of humans support this relationship; humans that live exceptionally cussion of (page 508). In recent years, a new contributor to long often exhibit unusually high insulin sensitivity—that is, the aging process has received attention: the activity of a signaling their tissues respond fully to relatively low circulating insulin levels. pathway involving insulin and a related protein IGF-1, which is the Low insulin levels are also linked to reduced incidence of cancer. focus of the present Human Perspective. Thus, just as high insulin levels and increased insulin resistance are The lifespans of animals can be increased by restricting the calo- associated with poor health, low insulin levels and increased insulin ries present in the diet. As first shown in the 1930s, mice that are sensitivity appear to be associated with good health. It is interest- maintained on very strict diets typically live 30 to 40 percent longer ing to note that calorie restriction in laboratory animals leads to than their littermates who are fed diets of normal caloric content. decreased insulin levels and increased insulin sensitivity, so that Two separate long-term studies are currently in progress on rhesus these two paths to increased longevity may be acting by the same monkeys to see if they too live longer and healthier lives when main- mechanism. tained on calorie-restricted diets. Significant differences in the pub- Although the medical has typically focused on in- lished data between these two groups have made it difficult to draw sulin as the primary metabolic hormone, many basic researchers in firm conclusions on the value of calorie restriction (CR) in primates. the field of aging have focused on the related hormone insulin- One team of researchers at the Wisconsin National Primate Research growth factor 1 (IGF-1). In one study, it was found that mutations in Center reported in 2009 that animals in their CR group have lower the gene encoding the IGF-1 receptor are especially frequent in a blood levels of glucose, insulin, and triglycerides and were less prone group of centenarians (humans living over the age of 100). Both to age-related disorders such as diabetes and coronary artery disease. insulin and IGF-1 share the downstream effector, mTOR. mTOR The effect of calorie restriction on the external appearance of one of became a central focus of the field of mammalian aging when it was these animals is seen in the photographs of Figure 1. The Wisconsin group also reported that 37 percent of the control group (i.e., animals that had enjoyed unrestricted diets) had died during the 20 years of 15.4 the study compared to only 13 percent of the CR group. In contrast, the other team of researchers at the National Institute of Aging re- for Mechanism a as Protein-TyrosinePhosphorylation ported in late 2012 that calorie restriction did not improve survival outcome. In fact, individuals in their CR group did not exhibit the re- duced cardiovascular disease that characterized the CR individuals in the Wisconsin study. One important difference between the CR and control groups in the NIA study was noted: none of the animals in the CR group had died from cancer as compared to five deaths from cancer from those in the control group. It has been suggested that dif- ferences in animal survival between the two studies may be explained by the fact that individuals of the control group in the Wisconsin study were allowed to eat unlimited amounts of high-sucrose-con- taining foods, which may have made them less healthy than the control animals in the NIA study. Neither of these studies has been conducted long enough to determine if the animals’ maximum span (more than 40 years) is increased as a result of CR. As reported in numerous television news shows, a growing num- ber of humans are hoping to extend their life span by practicing calo- rie restriction, which in essence means that they are willing to subject themselves to an extremely limited, but balanced, diet. The National (a) (b) Institutes of Aging has also begun a study (named CALERIE) on Figure 1 The effects of calorie restriction on Rhesus macaques. human subjects who are overweight (but not obese) that are kept on Photographs of (a) a typical control animal at 27.6 years of age (about diets containing about 25 percent fewer calories than would be re- the average life span) and (b) an age-matched animal on a calorie- quired to maintain their initial body weight. After a period of six restricted diet. (Contasting results from the NIA study can be found in Transduction Signal months of calorie restriction, these individuals show remarkable an advanced online publication in the 8/30/2012 issue of Nature.) metabolic changes; they have a lower body temperature, their blood (F ROM R. J. C OLMAN , ET AL ., S CIENCE 325:201, 2009. REPRINTED insulin and LDL-cholesterol levels are lower, they have lost weight as WITH PERMISSION FROM AAAS. C OURTESY RICKI COLMAN , would be expected, and their energy expenditure is reduced beyond WISCONSIN NATIONAL PRIMATE RESEARCH CENTER , U NIVERSITY that expected due simply to their lower body mass. In addition, the OF WISCONSIN .) level of DNA damage experienced by the cells of these individuals is Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 648 Etr1enco is proteins plant these of studied best the of cytopl the to signal the transmits that domain nase his cytoplasmic, a and stimuli external for receptor are enzymes the membrane proteins with an extracellular domain that eukaryotes, of types both In flo and plants. yeast both in discovered then were but cells signa these were enzymes thought to be restricted to environmental 1993, of variety a to response cell’s kinase that phosphorylates residues and m tein kinase that is absent from animal cells. plants contain a t On the other hand, in plant cells. any if arekinases tyrosine Receptor little, signaling. cell plant play to appear messengers, cell animal ubiq most the be may which nucleotides, cyclic ample, but other pathways are unique to each major kingdom for the gas ethylene (Cethylene gas the for including the use of Caof use the including mechanisms,signaling basic certain share animals and Plants Signaling Pathways in Plants especially aminothe acidavailability of nutrients, i whichespeciallyis sensitive rapamycin, to mTORC1, as a component of two distinct complexes mTORC1 and regulator of mTOR cellular is metabolism. a protein sensornutrient a is mTOR process. aging the in role an plays mTOR that notion the support findings These mTO the through signaling reduce to shown been also ha also has itself considered to be a Ca it viable anti-aging drug. and Unfor pamycin is also a potent suppressor mammals, offlies. the immune and s worms, in yeast, to given span when capability life increase to age-related This is disorders. the first compound tha decreasedandmice lifespan ofextended theicantly aninhibitor ofthe mTOR discoveredthat rapamycin, become more apparent. should pathways signaling animal ferencesand plant between seque Arabidopsis from obtained data of amount massive the lyze research As response. hormone the for required teins factors that activate expression of specific genes transc e are plants in pathway kinase MAP the of gets the downstr As in other eukaryotes, and animal cells. yeastin found cascade kinase MAP the to similar very is that along signals of transmission to leads receptor its Binding of and eth fruit ripening. flowering, mination, including processes, developmental of array diverse .Describe the steps between the binding of an insu 1. .What is the role of Ras in signaling pathways? Ho 2. W E I V E R It has long been known that bacterial cells have a have cells bacterial knownthat been long has It receptor tyrosine kinases? insulin differ from otherligandsthatactbymeansof Howdoestheactionof activation oftheeffector PI3K. molecule atthesurfaceofatargetcelland gene. The product of the of product The gene. and other plant genomes, the similarities and dif-and similarities the genomes, plant other and 2 2 H ϩ 4 ), a that regulatesthata hormone plant a ), and phosphoinositide messengers,phosphoinositide and Etr1 gene encodes a receptora encodes gene ,and can stimulates, lorie restriction has t has been shown the incidenceofthe ystem so it is not kinase that exists and a primarya and ncoding pro- iae signif-kinase, s activateds by also lackingalso a pathwaya ype ype of pro- uaey ra-tunately, ded by theby ded mTORC2. ediates the pathway.R important s.Oneasm. tidine ki-tidine seed ger-seed bacterial s Until ls. acts as a eam tar- oe in role , For ex-. ers ana-ers w is ylene ylene to protein lin wering ription uitous trans- s this this s ncing is maintained at very low levels, typically about 10about typically levels, low very at maintained is and/or Cachangers, debate as to how mTOR inhibition increases lifespan theredifficultand very componentsbeproven tohas defense against Untanglingoxidative the stress). ro proteins include molecular chaperones and proteins wh genes of expression activates (which FOXO factor Atg proteins teins), (which and regulate nonh autophagy), and histones from groups acetyl removes (which dea protein the protei ), mRNAenhancingthereby in involved proteins numerous phosphorylates includi lifespan, regulatingin implicated been have A number of proteinsing. both upstream and downstre reduce as occurs during calorie ability, restriction, p and autophagy, Studiesgrowth inhibit suggest and thatproliferation. reduc synthesis, protein and lipid calcium level is kept very low because (1) Ca(1) because low very kept is level calcium The cytosol. the than higher times 10,000 typically extra the in ion cell plant a ERor the of lumen the within or this space of concentration the contrast, o,ad 2 eeg-rvn Caplasma and ER membranes pump calcium out of the cyt energy-driven (2) and ion, to impermeable highly ke membranes these making normally are closed, membranes ER and plasma the both in os but their role was considered in Chapter 5 and ions, 2 Ca cytosolic of elevation Abnormal Caof regulatedactivity the by controlled co cellular particular a in ions calcium of tration concentration Th of calcium ions within the cytosol. dramatic a to leads and surface cell the at messa ceived extracellular an cases, these of and each In movement, death. cell transcription, synap metabolism, fertilization, mission, secretion, division, cell sponses, im contraction, muscle including activities, cellular remarkablea in role significant a play ions Calcium 15.5 Intracellular Messenger The concentration of Ca Figure in (as compartment the surround that branes death. lead can to occur in brain cells following a , Mitochondria also play an important role also in sequest play an important Mitochondria .What domain, and what role does it play is an SH2 3. .What istherelationshipbetweentype2diabetesand5. HowdoestheMAPkinasecascadealter 4. Ras differ from a heterotrimeric G protein? How doe this affected by the activity of a Ras-GAP? insulin sensitivity might help treat this disease? insulin production? How is it that a drug that incr transcriptional activity of a cell? signaling pathways? | The Role of Calcium as an 2 ϩ ion channels located ionwithin channels located the mem- 2 ϩ ions in the of cytosol a resting cell 2 ϩ rnpr sses f the of systems transport 2 ϩ will not be discussed will here. concentration, as can as concentration, ering and releasingering Ca 2 ϩ s mTORC1 signal- up,Capumps, les of these various 2 ed nutrient avail- that play a role in ϩ ng S6K1 (whichS6K1 ng the transcription is considerableis . mpartment is mpartment ion channelsion am of mTOR massive cell cetylase Sir2cetylase s encodedose synthesis, n nrae inincrease ooe cell romote vacuole isvacuole soe pro-istone variety of variety e concen- Ϫ ue re-mune i trans-tic eases cytosolic ge is re-is ge 7 cellular in 15.28). 2 .InM. osol. ϩ s this 2 cell ϩ ex- pt 2 649 2؉ IP 3 and Voltage-Gated Ca Channels We have de- scribed in previous pages two major types of signaling recep- tors, GPCRs and RTKs. It was noted on page 630 that interaction of an extracellular messenger molecule with a GPCR can lead to the activation of the enzyme phospholi- pase C- ␤, which splits the phosphoinositide PIP 2, to release the molecule IP 3, which opens calcium channels in the ER membrane, leading to a rise in cytosolic [Ca 2ϩ]. Extracellular messengers that signal through RTKs can trigger a similar re- sponse. The primary difference is that RTKs activate mem- bers of the phospholipase C- ␥ subfamily, which possess an SH2 domain that allows them to bind to the activated, phos- phorylated RTK. There are numerous other PLC isoforms. For example, PLC ␦ is actived by Ca 2ϩ ions, and PLC ⑀ is ac- tivated by Ras-GTP.All PLC isoforms carry out the same re- action, producing IP 3 and linking a multitude of cell surface receptors to an increase in cytoplasmic Ca 2ϩ. There is another major route leading to elevation of cytosolic [Ca 2ϩ], which was encountered in our discussion of synaptic transmission on page 169. In this case, a nerve impulse leads to a depolariza- tion of the plasma membrane, which triggers the opening of voltage-gated calcium channels in the plasma membrane, al- lowing the influx of Ca 2ϩ ions from the extracellular medium.

؉ Visualizing Cytoplasmic Ca 2 Concentration in Real Time Our understanding of the role of Ca 2ϩ ions in cellular responses has been greatly advanced by the development of in- dicator molecules that emit light in the presence of free cal- cium. In the mid-1980s, new types of highly sensitive, fluorescent, calcium-binding compounds (e.g., fura -2) were Figure 15.27 Experimental demonstration of localized release of ؉ developed in the laboratory of Roger Tsien at the University of intracellular Ca 2 within a single dendrite of a neuron. The mecha- 2ϩ California, San Diego. These compounds are synthesized in a nism of IP 3-mediated release of Ca from intracellular stores was de- form that can enter a cell by diffusing across its plasma mem- scribed on page 630. In the micrograph shown here, which pictures an brane. Once inside a cell, the compound is modified to a form enormously complex Purkinje cell (neuron) of the cerebellum, calcium that is unable to leave the cell. Using these probes, the concen- ions have been released locally within a small portion of the complex tration of free calcium ions in different parts of a living cell can “dendritic tree.” Calcium release from the ER (shown in red) was be determined over time by monitoring the light emitted using induced in the dendrite following the local production of IP 3, which a fluorescence microscope and computerized imaging tech- followed repetitive activation of a nearby . The sites of release of 2ϩ 15.5 niques. Use of calcium-sensitive, light-emitting molecules has cytosolic Ca ions are revealed by fluorescence from a fluorescent calcium indicator that was loaded into the cell prior to stimulation of provided dramatic portraits of the complex spatial and tempo- the cell. (F ROM ELIZABETH A. F INCH AND GEORGE J. A UGUSTINE , MessengerIntracellular an as Calcium of Role The ral changes in free cytosolic calcium concentration that occur NATURE , VOL . 396, COVER OF 12/24/98. REPRINTED BY PERMISSION in a single cell in response to various types of stimuli. This is FROM MACMILLAN PUBLISHERS LIMITED .) one of the advantages of studying calcium-mediated responses compared to responses mediated by other types of messengers whose location in a cell cannot be readily visualized. Depending on the type of responding cell, a particular IP 3 receptors described earlier are one of two main types stimulus may induce repetitive oscillations in the concentra- of Ca 2ϩ ion channels present in the ER membrane; the other tion of free calcium ions, as seen in Figure 15.11; cause a wave type are called receptors (RyRs ) because they bind of Ca 2ϩ release that spreads from one end of the cell to the the toxic plant ryanodine. Ryanodine receptors are other (see Figure 15.29); or trigger a localized and transient found primarily in excitable cells and are best studied in car- release of Ca 2ϩ in one part of the cell. Figure 15.27 shows a diac and skeletal muscle cells, where they mediate the rise in Purkinje cell, a type of neuron in the mammalian cerebellum Ca 2ϩ levels following the arrival of an . Muta- that maintains synaptic contact with thousands of other cells tions in the cardiac RyR isoform have been linked to occur- through an elaborate network of postsynaptic dendrites. The rences of sudden death during periods of . Depending micrograph in Figure 15.27 shows the release of free calcium on the type of cell in which they are found, RyRs can be in a localized region of the “dendritic tree” of the cell following opened by a variety of agents, including calcium itself. The in- synaptic activation. The burst of calcium release remains re- flux of a limited amount of calcium through open channels stricted to this region of the cell. in the plasma membrane induces the opening of ryanodine Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 650 of Ca The calcium ions are subsequently removed from the Na stored Ca ryanodine receptors in the SER le membrane (step 2), nr arw hogotteetr g.The blue co (arrow) throughout the entireentry egg. a small amount of Ca voltage-gated calcium channels in the plasma membra A depolarizationmuscle in cell. membrane voltage ca Figure 15.28 Figure 15.29 BOOK A IN REUSE OF PERMISSION WITH DUCED (R sperm. rise in Carise at 10-secon and photographed fertilized, rescent dye, 1993; which leads This to relaxation. cycle is repeated af PITDWT EMSINAFTER PERMISSION WITH EPRINTED ϩ /Ca 2 COPYRIGHT ϩ [Ca [Ca [Ca The unfertilized egg was injected with a calcium-se The unfertilized High Low Low 2 pumps located in the membrane of the SER (step 4) an ϩ 2 2 2+ 2+ 2+ ϩ ϩ secondary transport system in the plasma membrane (s ] ] ] into which the triggerscytosol (step the cell’s3), c concentration is seen to spread from the point of s (RyR) Voltage-gated Ca Calcium-induced calcium release, Calcium wave in a starfish egg induced by a fertilizing induced by egg Calcium wave in a starfish / membrane ETOKVIATEXTBOOK 93 N1993. Ca SER 2 ϩ [Ca 2+ High into the The cytosol calcium (step ions 1). bind to 1 TR BYATURE 2+ ] N + ATURE channel membrane Plasma C Ca OPYRIGHT N 2+ ATURE P 4 .J B J. M. UBLISHING Calcium ion Ca of theSER (SERCA) 3Na 2+ P E R R I D G E 2 C UBLISHING Na pump LEARANCE + ter each heart beat. + /Ca as it occurs in a cardiac G 3 5 uses the opening of cytosol by the action OPI H FORMAT THE IN ROUP lor indicates lowlor indicates ading to release of e allowing entry of ne, nevl.Thed intervals. 2+ N, exchanger G A TURE C 1Ca ROUP nsitive fluo- ENTER ontraction. 2+ d a perm R. 361:317, tep 5), EPRO .) - h yoo.I oersoss the elevation In of some Caresponses, the cytosol. to open and release additional calcium into adjacen act by opening a small typically number of Ca free [Ca (C a phospholipase C that is brought into the egg by t at the cell surface at the As site of the stimulus. the most dramatic Ca dramatic most the compartment. calcium cytoplasmic entire the throughspreads of wave propagated a cases, other In 15.27). (a cytosol the of region small a to localized mains hog tee hnes n etr h ctsl hy a they Ca cytosol, nearby the enter and channels these through receptors in causingthe theER, release of Ca hnes n h pam mmrn a dpce i Figu in depicted Ca as Once these channels have opened, 15.30. membrane plasma ca the of in opening the channels to leading response a triggers ER SOCE During the depletion of calcium level depleted. cellula repeated of the periods stockpile of intracellular calcium sponses, ions During ER. the in stored try try focused on a phenomenon known as into the ERback space. and/or the extracellular cyto the of out pumped rapidly are ions the because are waves Calcium division. mitotic first its toward t drive that 575) (page kinases cyclin-dependent of including events, of number a triggers fertilization fo concentration calcium cytoplasmic in rise sudden tact with the plasma membrane of the egg (Figure 15 or so following fertilization and is induced by the Ca releasecalcium called is phenomenon This 15.28). (Figure brane of a Caa of brane ment in the ER membrane, the STIM1 clusters act to r to act clusters STIM1 the ERthe in ment membrane, rea Followingtheir another. one to nm) (25–50 imity p close into come membranes plasma and ER the where ER and plasma membrane. In this system, the depletion the Casystem, this In membrane. plasma and ER be operating system signaling a by orchestrated are f th mystery that recently discovered was unsolved it until years an many been had SOCE for responsible The me thereby replenishing the ER’s stores. calcium the cytosol from where they can be pumped back into UTS OFOURTESY 2 2 ϩ (or ϩ xrclua sgas ht r tasitd y Ca by transmitted are that signals Extracellular Recent research in the field of has has signaling calcium of field the in research Recent wave in mammalian eggs is triggered by the formatio in the ER leads to the clustering within the ER mem ER the within clustering the to leads ER the in 2 SOCE ϩ ,whereas the red high free color indicates [Ca], 2 ϩ ), in which the “store” refers to the calcium ions calcium the to refers the “store” which in ), ion channels in the ER, causing these channelsthese causing ER, the in channels ion S 2 ( CICR ϩ TEPHEN -sensing protein called STIM1 into regionsinto STIM1 called protein -sensing ). 2 ϩ .SA. waves occurs within the first minutefirst the within occurs waves TRICKER .) store-operated calcium en- 2 he fertilizing sperm.he fertilizing 2 ϩ ϩ 2 calcium-induced Ca into the cytosol ϩ ions can enter sperm’s con- ion channels can become 2 2 2 ϩ t regions of ϩ s in Figurein s ϩ n of IP activation ese eventsese .A similar]. tween the tween he zygotehe 2) The.29). ions rush levels levels re- transient the ER, chanism 2 llowing rrange- o andsol s in the One ofOne ϩ release lcium ecruit t on ct re- r 3 rox- ions by of or re - 651

Plasma Orai1 (closed) membrane

Cytosol

STIM1 ER membrane Ca 2+ Depletion of Figure 15.30 A model for store-operated calcium entry. When Ca 2+ stores the ER lumen contains abundant Ca 2ϩ ions, the STIM1 proteins of the ER membrane and the Orai1 proteins of the plasma mem- Orai1 (open) brane are situated diffusely in their respective membranes, and the Orai1 is closed. If the ER stores are depleted, a signaling system operates between the two membranes, causing the two proteins to become clustered within their respective membranes in close proximity to one another. Apparent interac- Cytosol tion between the two membrane proteins leads to opening of the Orai1 channel and the influx of Ca 2ϩ ions into the cytosol from where they can be pumped into the ER lumen.

subunits of a plasma membrane protein called Orail into nucleotide phosphodiesterase, ion channels, or even to the adjacent regions of the plasma membrane (Figure 15.30). calcium-transport system of the plasma membrane. In the lat- Orai1 is a tetrameric Ca 2ϩ ion channel that had been identi- ter instance, rising levels of calcium activate the system re- fied as being involved in a particular type of inherited human immune deficiency that results from a lack of Ca 2ϩ stores in T lymphocytes. Contact between the cytosolic surfaces of the Table 15.4 Examples of Mammalian Proteins 2؉ STIM1 and Orai1 proteins in these ER-plasma membrane Activated by Ca junctions leads to the opening of the Orai1 channels, the in- Protein Protein function flux of Ca 2ϩ into microdomains of the cytosol near the STIM1 clusters, and the refilling of the cell’s ER stores. Modulator of muscle contraction Ubiquitous modulator of protein kinases ؉ Ca 2 -Binding Proteins Unlike cAMP, whose action is and other enzymes (MLCK, CaM kinase II, adenylyl cyclase I)

usually mediated by stimulation of a protein kinase, calcium 15.5 , retinin Activator of guanylyl cylase can affect a number of different types of cellular effectors, in- B Phosphatase cluding protein kinases (Table 15.4). Depending on the cell MessengerIntracellular an as Calcium of Role The type, calcium ions can activate or inhibit various enzyme and PI-specific PLC Generator of IP 3 and diacylglycerol transport systems, change the ionic permeability of mem- ␣-Actinin Actin-bundling protein branes, induce membrane fusion, or alter cytoskeletal struc- Implicated in endo- and exocytosis, ture and function. Calcium does not bring about these inhibition of PLA 2 responses by itself but acts in conjunction with a number of Phospholipase A 2 Producer of arachidonic acid calcium-binding proteins (examples are discussed on pages Protein kinase C Ubiquitous protein kinase Actin-severing protein 303 and 370). The best-studied calcium-binding protein is 2ϩ IP 3 receptor Effector of intracellular Ca release calmodulin , which participates in many signaling pathways. 2ϩ Calmodulin is found universally in plants, animals, and Ryanodine receptor Effector of intracellular Ca release Na ϩ/Ca 2ϩ exchanger Effector of the exchange of Ca 2ϩ for Na ϩ eukaryotic microorganisms, and it has virtually the same across the plasma membrane amino acid sequence from one end of the eukaryotic spectrum Ca 2ϩ-ATPase Pumps Ca 2ϩ across membranes to the other. Each molecule of calmodulin (Figure 15.31) con- Ca 2ϩ antiporters Exchanger of Ca 2ϩ for monovalent ions tains four binding sites for calcium. Calmodulin does not have Caldesmon Regulator of muscle contraction 2ϩ sufficient affinity for Ca to bind the ion in a nonstimulated Villin Actin organizer ϩ cell. If, however, the Ca 2 concentration rises in response to a Arrestin Terminator of photoreceptor response stimulus, the ions bind to calmodulin, changing the confor- Ca 2ϩ buffer mation of the protein and increasing its affinity for a variety of Adapted from D. E. Clapham, Cell 80:260, 1995, by copyright permission of effectors. Depending on the cell type, the calcium–calmodulin Cell Press. Cell by Cell Press. Reproduced with permission of Cell Press in the 2ϩ (Ca –CaM) complex may bind to a protein kinase, a cyclic format reuse in a book/textbook via Copyright Clearance Center. Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 652 ihfu on acu os(ht pee) Bindin ions (white spheres). with four bound calcium guard cells that regulate the diameter of the micro ftre rtis (Cof target proteins. uolar membrane (tonoplast).uolar membrane membrane plasma the in situated proteins transport lowby very kept is cell plant resting a of cytosol ser same residue as PKA 15.14). (Figure the on CREB phosphorylates one kinases protein case, best-studied the In factors. transcription phosph that (CaMKs) kinases protein various of tion complex also can stimulate gene throutranscription o itaellr acu cnetain.Te Ca The concentrations. calcium intracellular low thus constituting a self-regulatory mechanism for m quantities excess of cell the ridding for sponsible A that promotessurface of interaction Ca exposin ions the changes conformation of calmodulin, Figure 15.31 ih,pesr,gaiy n te ocnrto o pla of concentration The concentration the of Ca mones such as abscisic acid. and gravity, pressure, light, including stimuli, of variety a to response in certai cells within dramatically change calcium cytosolic The cells. plant in messengers intracellular portant Calcium ions (acting in conjunction with calmodulin Regulating Calcium Concentrations in Plant Cells AAAATLABAMA The role of Caof role The B Calmodulin. IRMINGHAM OURTESY 2 ϩ in plant cell signaling is illustrated by illustrated is signaling cell plant in A ribbon diagram of calmodulin (CaM) of calmodulin diagram A ribbon .) M ICHAE L 2 ϩ –CaM with a large number C ARSON U, g of these Ca g a hydrophobic IEST OFNIVERSITY the action ofaction the scopic pores of the ion,the of changes in changes aintaining gh activa- levels of levels 2 2 ) are im- and vac-and ϩ f these of ϩ t hor-nt plant n orylate –CaM in the 2 ϩ ine h ur eldcess The drop in turgor pressur the guard cell decreases. of the stomatal pore decreases as the fluid (turgor) aper their d The of dehydration. prevents which diameter the controlled, tightly and plants, in loss water (stomata) of a leaf (Figure 15.32 which triggers the release triggers which of Ca R (step 3a) and opens K Figure 15.32 plays a role in these events.) ( a role in theseplays events.) by (Phosphorylation osmotic loss of water (step 4). movements lead to a drop solute concent in internal subsequent elevation of intracellular [Ca of intracellular subsequent elevation ( them to bulge outwar causing within the guard cells, The stomata are kept open as turgor pre guard cells. h lsammrn r pnd allowing the influx o arethe plasma membrane opened, io calcium ABA rise, levels When abscisic acid (ABA). closure. b (b) ESEARCHERS ) One of the factors controlling stomatal pore size H H 2 2 H O O (closed) K channel + Guard cell Influx ( a ) Photograph of stomatal pores, each flanked by each a pai of stomatal pores, ) Photograph (a) Tonoplast K A simplified model of the role of Ca I, 3a + NC H 2 2 O O .) H ϩ 2 O 4 n no fu hnes(tp3) These ionand anion (step efflux 3b). channels Vacuole Ca Ca stomatal pore 2+ 2+ A Closure of D: 2 2 ϩ H 1 2 R rmitra trs(tp2.Thefrom internal stores (step 2). O J. 3b a EREMY ). Stomata Stomata are a major site of). K K channel + 2 (open) + ϩ membrane Efflux ] closes K] closes Plasma B URGESS H H 2 2 O H O protein kinases also Cl ssure is kept high is the hormone ration and the ration 2 d as seen here. ϩ channel ؉ − (open) Efflux Anion , NO influx channelsinflux n channels inn channels cell Guard pore Stomatal /P in guard cell f Ca HOTO pressure in 3 − e is caused 2 ϩ (step 1), iameter H 2 ue is ture 2 r of O O 653 in turn by a decrease in the ionic concentration (osmolarity) of target. In actual fact, signaling pathways in the cell are much the guard cell. Adverse conditions, such as high temperatures more complex. For example: and low humidity, stimulate the release of the plant stress hor- I Signals from a variety of unrelated receptors, each binding mone abscisic acid. Studies suggest that abscisic acid binds to to its own ligand, can converge to activate a common effec- a GPCR in the plasma membrane of guard cells, triggering ϩ tor, such as Ras or Raf. the opening of Ca 2 ion channels in the same membrane ϩ (Figure 15.32 b). The resulting influx of Ca 2 into the cytosol I Signals from the same ligand, such as EGF or insulin, can ϩ triggers the release of additional Ca 2 from intracellular diverge to activate a variety of different effectors and path- ϩ stores. The elevated cytosolic Ca 2 concentration leads to clo- ways, leading to diverse cellular responses. ϩ sure of K influx channels in the plasma membrane and open- I Signals can be passed back and forth between different ϩ ing of both K and anion efflux channels. These changes pathways, a phenomenon known as cross-talk . ϩ Ϫ Ϫ produce a net outflow of K ions and anions (NO and Cl ) 3 These characteristics of cell-signaling pathways are illustrated and a resulting decrease in turgor pressure. schematically in Figure 15.33. Signaling pathways provide a mechanism for routing R E V I E W information through a cell, not unlike the way the central ϩ 1. How is the [Ca 2 ] of the cytosol maintained at such a routes information to and from the various or- low level? How does the concentration change in gans of the body. Just as the collects in- response to stimuli? formation about the environment from various sense organs, 2. What is the role of calcium-binding proteins such as the cell receives information about its environment through calmodulin in eliciting a response? the activation of various surface receptors, which act like sen- 3. Describe the role of calcium in mediating the diameter sors to detect extracellular stimuli. Like sense organs that are of stomata in guard cells. sensitive to specific forms of stimuli (e.g., light, pressure, or sound waves), cell-surface receptors can bind only to specific ligands and are unaffected by the presence of a large variety of unrelated molecules. A single cell may have dozens of differ- 15.6 | Convergence, Divergence, ent receptors sending signals to the cell interior simultane-

ously. Once they have been transmitted into the cell, signals 15.6 and Cross-Talk Among Different from these receptors can be selectively routed along a number

of different signaling pathways that may cause a cell to divide, Diffe Cross-TalkAmong and Divergence, Convergence, Signaling Pathways change shape, activate a particular , or even The signaling pathways described above and illustrated commit suicide (discussed in a following section). In this way, schematically in the various figures depict linear pathways the cell integrates information arriving from different sources leading directly from a receptor at the cell surface to an end and mounts an appropriate and comprehensive response.

Figure 15.33 Examples of convergence, diver- G Protein-coupled receptors gence, and cross-talk among various signal- transduction pathways. This drawing shows the Acetylcholine, histamine outlines of signal-transduction pathways NA, 5-HT, ATP, PAF, TXA 2, Glutamate, Angiotensin II, R initiated by receptors that act by means of both Vasopressin, Bradykinin, heterotrimeric G proteins and receptor protein- , Bombesin, III Y, Thrombin, G tyrosine kinases. The two are seen to converge by Cholecystokinin, Endothelin, II the activation of different phospholipase C iso- Neuromedin, TRH, GnRH, I forms, both of which lead to the production of PTH 2+ Odorants, Light PLCβ IP 3 IP 3R Ca the same second messengers (IP 3 and DAG). PIP2 Activation of the RTK by either PDGF or EGF leads to the transmission of signals along three Tyrosine kinase-linked receptors PLCγ DAG PKC different pathways, an example of divergence. P Cellular activity Cross-talk between the two types of pathways is & illustrated by calcium ions, which are released PDGF, mitogenesis from the SER by action of IP and can then act EGF, etc. PI3K PIP3 3 on various proteins, including protein kinase P rent Signaling PathwaysSignaling rent MAP C (PKC), whose activity is also stimulated by GAP Ras Raf kinase DAG. (M. J. B ERRIDGE , REPRINTED WITH PERMISSION FROM NATURE VOL . 361, P. 315, 1993, COPYRIGHT 1993. N ATURE BY NATURE PUBLISHING GROUP . R EPRODUCED WITH PERMISSION OF NATURE PUBLISHING GROUP IN THE FORMAT REUSE IN A BOOK /TEXTBOOK VIA COPYRIGHT CLEARANCE CENTER .) Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 654 2 . 1. and Cross-Talk Among Signaling Pathways Examples of Convergence, Divergence, 3. tor, an , and a all and a receptor tyrosine an integrin, tor, and are then transmitted along the MAP kinase casca and are then transmitted Figure 15.34 ure 15.15 or 15.25 look at A quick in this chapter. have been described of the examples of signal all transduc in virtually Divergence. promoting target genes cell. in each of and a similar translation set of g transcription signals from diverse receptors lead can convergence, As a result of signals down the MAP kinase pathway. plex results and transmiss in the activation of Ras com- The recruitment of the Grb2-Sos 15.34). (Figure proximity to the plasma membr in close protein Grb2 sites for the SH2 domain of the ad docking tyrosine lead to theof ph formation of them can all ligands, dif of receptorsthese may three bind to very types Althoug . namely, was discussed in Chapter 7, receptor of signal tra cell-surface that is capable Another typ receptors kinases. and receptor tyrosine G protein-co receptors in thiscell-surface chapter: Convergence. pathway can participate in events occurring in othe occurring in events participate pathway can connected web in which components produced in one are in cells that operate more to resemble likely a circ the information In fact, of events. linear chain as an indepe operated as if each signaling pathways Cross-talk. signals out along a variety of different pathways.signals out along a variety ligand binding to a GPCR or an insulin receptor—sen Extracellular matrix Grb2 Integrin Signals transmitted from a G protein-coupled transmitted recep- Signals npeiu etos we examined a number ofIn previous sections, Evidence of signal divergence has been evident Sos We of distinct types have discussed two G protein- receptor coupled b MAP kinasecascade , c Neurotransmitters, illustrates howillustrates a single stimulus—a growth factors Grb2 hormones, Ras Sos Receptor tyrosine kinase P Grb2 Growth factors, e.g.,EGF, PDGF converge on Ras Sos de. P nsduction rowth- tion that n inter- ion of ferent uits upled ospho- ndent, r path- Fig- to the aptor e of ane this ds h to specific sites on the DNA. Figure 15.35 serine residue, activating the transcription factor activating the transcription residue, serine factor CREB the transcription phosphorylate cascade both PKA and the kinases of the In addition, cascade. inhibits the which activation of th to Raf, from Ras the transmission to block cAMP-dependent kinase PKA, pathways. paetta CREB is a substrate ofapparent that a much wider ra It has since become PKA. cAMP-dependent kinase, by the years that CREB be phosphorylated could only It was assumed f effector of cAMP-mediated pathways. CREB on page 632 as a terminal was described CREB. fact the transcription signaling effector, important ure These 15.35). two pathways also intersect at ano the protein thatRaf, heads the MAP kinase cascade ( whichdependent can phosphorylate kinase, and inhibi thought to accomplish this the by cA activating PKA, mitted through Cyclicthe MAP AMP kinase is cascade. cluding fibroblasts by blocking and signalfat cells, cAMP can also inhibit the growth of a variety of ce reaction cascade leading to Hoglucose mobilization. of cro of this type the importance that illustrates look at an example we will involving within a cell, and fo be passed back can that information the ways than attempting to c Rather that is discovered. ways the more cross-talk signaling between ing in cells, The more aboutsig that information is learned ways. Cyclic AMP was depicted earlier as an initiator of receptor ylcAPat nsm el,by means of the AMP acts in some cells, Cyclic P EGF An example of cross-talk between two major signaling two An example of cross-talk between MAPKK MAPK kinase Rsk-2 CREB Growth factor CRE P CREB SH2 P PKA β Grb2 P -adrenergic receptor a r b2 drener CREB transcription SosS Sos g ic receptor s factor + GGTGTP GTP RRas Ras TP as and allowing it to bind Raf e MAP kinase Epinephrine on the same MAP kinase cAM cAMP Gene activity PKA – of signals ss-talk. + s trans- or ther l,in-lls, path- cAMP wever, atalog Fig- rth MP- nge nal- t a or 655 of kinases. For example, one of the kinases that phosphory- accidental observation. It had been known for many years that lates CREB is Rsk-2, which is activated as a result of phos- acetylcholine acts in the body to relax the smooth muscle cells phorylation by MAPK (Figure 15.35). In fact, both PKA of blood vessels, but the response could not be duplicated in and Rsk-2 phosphorylate CREB on precisely the same vitro. When portions of a major blood vessel such as the aorta amino acid residue, Ser133, which should endow the tran- were incubated in physiologic concentrations of acetylcholine scription factor with the same potential in both pathways. in vitro, the preparation usually showed little or no response. In the late 1970s, Robert Furchgott, a pharmacologist at a A major unanswered question is raised by these examples New York State medical center, was studying the in vitro re- of convergence, divergence, and cross-talk: How are different sponse of pieces of rabbit aorta to various agents. In his earlier stimuli able to evoke distinct responses, even though they uti- studies, Furchgott used strips of aorta that had been dissected lize similar pathways? PI3K, for example, is an enzyme that is from the . For technical reasons, Furchgott switched activated by a remarkable variety of stimuli, including cell adhe- from strips of aortic tissue to aortic rings and discovered that sion to the ECM, insulin, and EGF.How is it that activation of the new preparations responded to acetylcholine by undergo- PI3K in an insulin-stimulated liver cell promotes GLUT4 ing relaxation. Further investigation revealed that the strips translocation and protein synthesis, whereas activation of PI3K had failed to display the relaxation response because the deli- in an adherent epithelial cell promotes cell survival? Ultimately, cate endothelial layer that lines the aorta had been rubbed these contrasting cellular responses must be due to differences away during the dissection. This surprising finding suggested in the protein composition of different cell types. Part of the an- that the endothelial cells were somehow involved in the re- swer probably lies in the fact that different cells have different sponse by the adjacent muscle cells. In subsequent studies, it isoforms of these various proteins, including PI3K. Some of was found that acetylcholine binds to receptors on the surface these isoforms are encoded by different, but related genes, of endothelial cells, leading to the production and release of an whereas others are generated by alternative splicing (page 534), agent that diffuses through the cell’s plasma membrane and or other mechanisms. Different isoforms of PI3K, PKB, or causes the muscle cells to relax. The diffusible relaxing agent PLC, for example, may bind to different sets of upstream and was identified in 1986 as nitric oxide by Louis Ignarro at downstream components, which could allow similar pathways UCLA and Salvador Moncada at the Wellcome Research to evoke distinct responses. The variation in responses elicited Labs in England. The steps in the acetylcholine-induced re- by different cells possessing similar signaling proteins may also laxation response are illustrated in Figure 15.36. be partly explained by the presence of different protein scaffolds in each of the cell types. As shown in Figure 15.23, the speci- ficity of a response can be orchestrated by the scaffolds with which the signaling proteins can interact. But it isn’t likely that Endothelial variations in isoforms and scaffolds can fully explain the ex- cell traordinary diversity of cellular responses any more than differ- ences in the structures of neurons can explain the range of ACh 1 responses evoked by the nervous system. Hopefully, as the sig- naling pathways of more and more cells are described, we will gain a better understanding of the specificity that can be 2+ achieved through the use of similar signaling molecules. Ca

Nitric oxide 2 Guanylyl synthase cyclase GTP 15.7 Ca 2+ 5

15.7 | The Role of NO as an Lumen MessengerIntercellular an as NO of Role The Intercellular Messenger Arg 3 cGMP Relaxation NO 6 During the 1980s, a new type of messen- 4 ger was discovered that was neither an or- ganic compound, such as cAMP, nor an GTP Smooth muscle cells ion, such as Ca 2ϩ; it was an inorganic gas—nitric oxide (NO). NO is unusual because it acts both as an extracellular Guanylyl messenger, mediating intercellular communication, and as a cyclase second messenger, acting within the cell in which it is gener- Relaxation cGMP ated. NO is formed from the amino acid L- in a reac- tion that requires oxygen and NADPH and that is catalyzed Figure 15.36 A signal transduction pathway that operates by means of by the enzyme (NOS). Since its discov- NO and cyclic GMP that leads to the dilation of blood vessels. The ery, it has become evident that NO is involved in a myriad of steps illustrated in the figure are described in the text. (F ROM R. G. biological processes including anticoagulation, neurotrans- KNOWLES AND S. M ONCADA , T RENDS BIOCHEM SCIENCE 17:401, 1992. mission, smooth muscle relaxation, and . TRENDS IN BIOCHEMICAL SCIENCES BY INTERNATIONAL UNION OF BIOCHEMISTRY REPRODUCED WITH PERMISSION OF ELSEVIER LTD . IN THE As with many other biological phenomena, the discovery FORMAT REUSE IN A BOOK /TEXTBOOK VIA COPYRIGHT CLEARANCE CENTER .) that NO functions as a messenger molecule began with an Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 656 of actions within the body that do not involve prod involve not do that body the within actions of tion had unexpected side effects. discoveredwas angina potential a when Viagra inhibited not drug. is h fortunately of but regulation the in contraction, role muscle key a plays PDE3, enzyme, isofo Another penis. the in acts that version the is w particular PDE5, specific isoform of cGMP quite phosphodiesterase, is Viagra erection. an of maintenance destroysdevelopment the promotes which cGMP, that of levels vated maintained, to enzyme leads enzyme this the of Inhibition cGMP. phosphodiesterase, cGMP of as acts instead but cyclase, guanylyl of activation of release the on effect no has drugs) related (and andsubsequent production cGMP.of cyclase the guanylyl of activation by re- cells this muscle mediates smooth NO in sponse above, described As blood. with gan engorgement and vessels blood penile of lining the which causes relaxation of smooth muscle release NO, in endings nerve arousal, sexual During (sildenafil). of development the to led also has messenger second Inhibiting Phosphodiesterase awarde was of NO as a signaling agent.1998 for the discovery estate, Nobel’s Alfred from donation a by i which Prize, Nobel the that fitting only is It work. to suffer more from the pain of angina on days they w factory Nobel’sAlfred dynamite in nitroglycerine work who disease Personsheart with observation. ing benefits of nitroglycerine were discovered through a The the increasing blood flow to the organ. the heart, stimulat v blood the which lining muscles smooth the of oxide, relaxation nitric to metabolized is erine sults from an inadequate flow of blood to the heart. angi of pain the treat to 1860s the since used been nitroglycerine of action the explained also studies activat cyclase guanylyl actual the as nitric served which into enzymatically converted being was demonstrated azide ultimately colleagues and Murad cellular tracts. in production cGMP stimulated molecule the disc to chanced and transport, electron of inhibitor tion of the blood vessel. (step cell muscle the of relaxation causing strates specific phosphorylates which PKG), (a kinase protein structure to Cyclic cAMP. GMP binds to a cGMP-depend cyclic simil messenger second important an synthesizes is which that (cGMP), enzyme the 5), (step cyclase stimulates and binds it where 4), (step cells muscle smooth adjacent the into and membrane plasma the across The NO formed in the endothelial cel thase (step 3). NO as an Activator of Guanylyl CyclaseGuanylyl of Activator an as NO sity of Virginia. Murad Murad was working with azide (N sity of Virginia. the at colleagues and Murad Ferid by 1970s late the wa cyclase guanylyl of activator an as acts NO that lic Ca ri a signals 15.36) Figure 1, (step cell endothelial Recent investigations have revealed that NO has a v The binding of acetylcholine to the outer surface o surface outer the to acetylcholine of binding The 2 ϩ concentration (step 2) that activates nitric oxide The discovery of NO The as discovery a The discovery The se in cytoso-in se 6) and dila-and 6) an inhibitoran , which had which , NO or theor NO 3 na that re-that na Nitroglyc- n interest- weren’t at ,a potent), s made inmade s rm of theof rm of the or-the of ere foundere r Theseor. guanylyl the penisthe uction ofuction over thatover l diffuses s fundeds Univer- essels ofessels medica- rapeutic enzyme y the by ed withed cells in Viagra o onefor GMP oxide, Viagra s the es ariety sub- that r inar and hich in d syn- ex- anf eart ele- ent alters the activity, turnover, or interactions of the or interactions turnover, alters the activity, posttranslational modification, which which is called posttranslational modification, . and channels, ryanodine Ras, hemoglobin, ing hundredoveraprote residueswell in cysteine tain ce of group —SH the to added is NO Forexample, cGMP. h crs”b paoyoi.Aotss s fe contrasted often is with a different of type cell death Apoptosis called phagocytosis. by “corpse” the engulf rapid the and dissecti the fragments, small into chromatin surface, cell the at blebs of formation neighboring to adhesion of loss the nucleus, its and of volume in shrinkage overall the by characterized (Figur process orderly neat, a o is apoptosis death by Death the to leads that events of sequence trated Apoptosis occurs through is unique to animal cells. Apoptosis 15.8 Cell Death) binding tightly to proteins present on the surfaces the on present proteins to tightly binding cap receptors possess that produced are lymphocytes development embryonic During lymphocytes. T of faces are recognized by specific receptors that are presen ta These pathogen-infectedcells. targetor abnormal phocytes are cells of the that recogn processas it occurs in mice are shown in Figure 15 elimination of the excess cells Threeby apoptosis. The fingers gers. are essentially carved out of the p without any space between the tissues that will bec the earliest form of the human hand resemblesment, almost sweranywhere During is: embryonicyou d look. cellsfindthatbecome targeted elimination?for The debris. for what happensout to concern the flying plosives as compared to simply blowing up the struc carefully using building a of implosion controlled compare be might apoptosis process, orderly and safe Because inflammation. of induction resulting the and m the into contents cell of leakage breakdown, brane membranous internal its and cell the sw both the by characterized is Necrosis nature. in derly less much although process, programmed and regulated considere generally is necrosis apoptosis, Like sult. bioche or trauma physical of type some follows ally ment. It has been estimated that 10that estimated been has It ment. Figure d embryonic of end the (see with stop not doesApoptosis apoptosis by eliminated are capability thathavelymphocytesthis T cellswithinbody. the genetic blueprint can result in unregulated cell di This is bleimportant genomic because damage. damage eliminationtheinvolved cells thatofhavesustai apoptos Forexample, apoptosis. byday every diebody .Describe the steps in the signaling pathway by wh 1. W E I V E R Why Why do andour wherebodies havedo unwanted cells, nitric oxide mediates dilation of blood vessels. | Apoptosis (Programmed rporme eldah is or a programmed normal cell process death, that, 10 –10 necrosis, 11 cells in the adultthe in cells S- vision and the which which gener- protein. ned irrepara-ned stages of this addle via the ome the fin- t on the sur- n,includ-ins, 3.Tlym- .38. ize and kill of placed ex-placed an orches- dangerous ture with- short an-short n f the of on cls thecells, d to be abe to d mical in- mical rget cellsrget lig of elling the cellthe ,mem-s, e 15.37)e a paddle ment of ment d to theto d a cell. a f ich is is in-is is evelop- evelop- 17.25). be ofable edium, normal to the t s a is it This or- T, we r- , 657 development of cancer. Apoptosis is also responsible for the death of cells that are no longer required, such as activated T cells that have responded to an infectious agent that has been eliminated. Finally, apoptosis appears to be involved in neu- rodegenerative diseases such as Alzheimer’s disease, Parkin- son’s disease, and Huntington’s disease. Elimination of essential neurons during disease progression gives rise to loss of memory or decrease in motor coordination. These examples show that apoptosis is important in shaping tissues and organs during embryonic development and in maintaining homeosta- sis within the bodies of adult animals. Serious diseases can re- sult from both the failure to carry out apoptosis when the elimination of cells (e.g., cancer cells) is appropriate and from the overactive induction of apoptosis when the elimination of (a) cells is not appropriate (e.g., type 1 diabetes). The term apoptosis was coined in 1972 by John Kerr, Andrew Wylie, and A. R. Currie of the University of Ab- erdeen, Scotland, in a landmark paper that described for the first time the coordinated events that occurred during the pro- grammed death of a wide range of cells. Insight into the mo- lecular basis of apoptosis was first revealed in studies on the nematode worm C. elegans , whose cells can be followed with absolute precision during embryonic development. Of the 1090 cells produced during the development of this worm, 131 cells are normally destined to die by apoptosis. In 1986, Robert Horvitz and his colleagues at the Massachusetts Insti- tute of Technology discovered that worms carrying a mutation in the CED-3 gene proceed through development without losing any of their cells to apoptosis. This finding suggested (b) that the product of the CED-3 gene played a crucial role in the process of apoptosis in this organism. Once a gene has been identified in one organism, such as a nematode, re- searchers can search for homologous genes in other organ- isms, such as humans or other mammals. The identification of the CED-3 gene in nematodes led to the discovery of a ho- mologous family of proteins in mammals, which are now called caspases . Caspases are a distinctive group of cysteine (i.e., proteases with a key cysteine residue in their 15.8 Apoptosis (Programmed Cell Death)Cell (Programmed Apoptosis

(c)

Figure 15.37 A comparison of normal and apoptotic cells. (a,b) Figure 15.38 Apoptosis carves out the structure of the mammalian Scanning electron micrographs of a normal ( a) and apoptotic ( b) T-cell digits. Three stages in this process in a mouse . In this particu- hybridoma. The apoptotic cell exhibits many surface blebs that are lar mouse, which is called a MacBlue mouse, all of the embryonic budded off in the cell. Bar equals 4 mm. ( c) Transmission electron macrophages express the cyan fluorescent protein. Fluorescent micrograph of an apoptotic cell treated with an inhibitor that arrests macrophages have infiltrated the regions of the footpad where apoptosis at the membrane blebbing stage. (A,B: F ROM S. J. M ARTIN apoptosis has occurred and are clearing the space between the digits. ET AL ., T RENDS BIOCHEM . S CI . 19:28, 1994. R EPRINTED WITH (F ROM DAVID A. H UME , N ATURE IMMUNOL . 9:13, 2008; © 2008, PERMISSION FROM ELSEVIER ; C: COURTESY OF NICOLA J. REPRINTED BY PERMISSION FROM MACMILLAN PUBLISHERS MCCARTHY .) LIMITED .) Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 658 I I I I arethe targets of caspases the following: protei essential of group select a cleaving by feat all, not if accompl Caspases most, death. cell during observed changes triggering for responsible are and stag early an at activated are that site) catalytic y idn t a rnmmrn rcpo,TF1 The TNFR1. r of family receptor, a of member a is TNFR1 trimeric a to binding by messengers TNF evokes discussed its in this chapter, types other Like . cancer in used those agent chemical toxic or e infection, viral radiation, temperature, ionizing to exposure as such conditions, t response in system immune the of cells certain by prote trimeric a is TNF cells. tumor kill to ability named was messenger which (TNF), factor extracellular necrosis tumor called an stimulu the by carried figure, is the in apoptosis depicted case the In 15.39. illustrated are pathway extrinsic the in steps The The Extrinsic Pathway of Apoptosis rs-ak ewe tee ahas n ta extracel that and apoptotic signals can cause pathways activation of the intri these the that between noted be cross-talk should it However, separately. ways pathway that utilized by internal stimuli, which is called t called is which stimuli, internal by utilized that the called pathway, ing ies indicate that external stimuli activate apoptos product testosterone with interfere that drugs with reaso the prostate cancer that has spread to is other tissues is This testosterone. hormone sex male the de when apoptotic become prostate the of cells lial For example secreted by cells of the immune system). such as certain and external stimuli, DNA, abnormalitie as such stimuli, internal both by gered ca Apoptosis program. suicide cell’s a of activation the NF- signals by inact of survival the generation disrupt Caspases signaling pathways. prosurvival to disrupt s as PKB, such other kinases, Inactivation of certain of the fromto apoptotic detachment cell its neighb le adhesion, is presumed cell to disrupt for example, An endonuclease called activatedAn DNase shape. quent inactivation of these proteins lead to change Cleavage and co and gelsolin. tubulin, actin, filaments, the cytoskeleton of Proteins of the nucleus. lamina and shrinkage the nuclear Cleavage of lamins leads to the disassembl envelope. Lamins kinase including focal a More than a dozen protein kinases, severing it into fragments. into it severing where DNA, it attacks to the nucleus the cytoplasm CAD translocates Once activated, protein. inhibitory of an is activated following cleavage which caspase Recent studies have focused on the events that lead Here we will discuss the extrinsic and . intrinsic path- ( FAK , which make up the inner lining of the nuclear make up the inner lining of the nuclear which , ␬ B pathway (page 660). ), K,PC and Raf1. PKC, PKB, extrinsic pathway extrinsic , such as those of intermediate such , Inactivation of FAK, , that is distinct fromdistinctis that , e of apoptosisof e ( is by a signal- often treated nsic pathway. CAD in producedin s Amongns. he o.Stud-ion. dhesion ivating n be trig-be n n Figurein o adverseo response prived of prived s such as such s (proteins s in cell

epithe-, i the in s intrinsic ors. ), protein ading ish thisish also erves levated of firstof y of f the of whyn to the elated for itsfor from for s lular nse- e is re inner surface of the plasma membrane are two procas two are membrane plasma the of surface inner 15.39. in Figure as indicated proteins, mediatesnu a of recruitment the to leads which domain, death that 15.39) r the of conformation in change a produces receptor Figure in the to of TNF Binding interactions. protein–protein segment green dom “death a (each called acids amino 70 about of segment conta subunit receptor TNF each of domain toplasmic procesapoptotic the on turns that “deathreceptors” of which leads to cell survival rather than self-de rather survival leads to cell of which Figure 15.39 between between and TNF also activates other signalin TNFR1 be noted t It can out the death sentence. that carry an initiator complex that activates downstream (exe molecule containing segme four polypeptide caspase-8 the twoprocaspase molecules cleave one another to Once assembled i as browndomains (indicated boxes). FADD by means interact of homologous regions called are present as protein green in each (indicated box act with one another by homologous regions d called and TR FADD, domains of the cytoplasmic receptor, TNF protein of the plasma complex at the inner surface tor proteins (TRADD and FADD) to f and procaspase-8 the activated receptor different binds two (TNFR1), mediated) pathway of apoptosis. membrane Plasma Executioner procaspases The last proteins to join the complex that assemble Procaspase-8 Executioner caspase Initiator caspase-8 A simplified model of the extrinsic (receptor- extrinsic the of model A simplified FADD When When binds to a TNF receptorTNF TRADD TNF Death domains TNFR1 struction (page 660).struction s.Procaspase-8 andes). hat the interaction ebae Themembrane. cutioner) caspases generate an active eath domains that cytoplasmic adap-cytoplasmic t.Caspase-8 isnts. n the complex, ahas oneg pathways, death effector orm a multi-orm ADD inter- s. The cy- The s. eceptor’s trimeric mber ofmber s at the pase-8 n ains ain” 659 molecules (Figure 15.39). These proteins are called “procas- types of stress that the BH3-only proteins are expressed or ac- pases” because each is a precursor of a caspase; it contains an tivated, thereby shifting the balance in the direction of apop- extra portion that must be removed by proteolytic processing tosis. In these circumstances, the restraining effects of the to activate the enzyme. The synthesis of caspases as proen- antiapoptotic Bcl-2 proteins are overridden, and the proapop- zymes protects the cell from accidental proteolytic damage. Unlike most proenzymes, procaspases exhibit a low level of Internal cellular damage proteolytic activity. According to one model, when two or more procaspases are held in close association with one an- other, as they are in Figure 15.39, they are capable of cleaving Activation of proapoptotic BH3-only protein one another’s polypeptide chain and converting the other and subsequent activation of Bak or Bax molecule to the fully active caspase. The final mature enzyme (i.e., caspase-8) contains four polypeptide chains, derived from two procaspase precursors as illustrated in the figure. Activation of caspase-8 is similar in principle to the acti- Bax vation of effectors by a hormone or growth factor. In all of these signaling pathways, the binding of an extracellular lig- and causes a change in conformation of a receptor that leads to the binding and activation of proteins situated downstream in the pathway. Caspase-8 is described as an initiator caspase because it initiates apoptosis by cleaving and activating down- stream, or executioner , caspases, that carry out the controlled + Cytoplasmic factors self-destruction of the cell as described above. (e.g. Apaf-1)

+ Procaspase-9 The Intrinsic Pathway of Apoptosis Internal stimuli, such as irreparable genetic damage, lack of oxygen (hypoxia), extremely high concentrations of cytosolic Ca 2ϩ, viral infection, ER stress, or severe (i.e., the production of large numbers of destructive free radicals), trigger apoptosis by the intrinsic pathway illustrated in Figure 15.40. Activation of the intrinsic pathway is regulated by Activated initiator members of the Bcl-2 family of proteins, which are character- caspase-9 complex ized by the presence of one or more small BH domains. Bcl-2 (Apoptosome) family members can be subdivided into three groups: (1) proapoptotic members (containing several BH domains) that promote apoptosis (Bax and Bak), (2) antiapoptotic members Executioner procaspases (containing several BH domains) that protect cells from 3 apoptosis (e.g., Bcl-x L, Bcl-w, and Bcl-2), and (3) BH3-only proteins (so-named because they contain only one BH do- Executioner caspase main), which promote apoptosis by an indirect mechanism. According to the prevailing view, BH3-only proteins (e.g., Bid, Bad, Puma, and Bim) can exert their proapoptotic effect in two different ways, depending on the particular proteins in- Figure 15.40 volved. In some cases they promote apoptosis by inhibiting The intrinsic (mitochondria-mediated) pathway of 15.8 antiapoptotic Bcl-2 members, whereas in other cases they apoptosis. Various types of cellular stress cause proapoptotic members of the Bcl-2 family of proteins–either Bax or Bak–to oligomerize within promote apoptosis by activating proapoptotic Bax or Bak. In the outer mitochondrial membrane, forming channels that facilitate the Death)Cell (Programmed Apoptosis either case, the BH3-only proteins are the likely determinants release of cytochrome c molecules from the intermembrane space. Once as to whether a cell follows a pathway of survival or death. In in the cytosol, the cytochrome c molecules form a multisubunit complex a healthy cell, the BH3-only proteins are either absent or with a cytosolic protein called Apaf-1 and procaspase-9 molecules. strongly inhibited, and the antiapoptotic Bcl-2 proteins are Procaspase-9 molecules are apparently activated to their full proteolytic able to restrain proapoptotic members. The mechanism by capacity as the result of a conformational change induced by association which this occurs is debated. It is only in the face of certain with Apaf-1. Caspase-9 molecules cleave and activate executioner caspases, which carry out the apoptotic response. The intrinsic pathway can be triggered in some cells (e.g., hepatocytes) by extracellular signals. 3The first member of the family, Bcl-2 itself, was originally identified in 1985 as This occurs as the initiator caspase of the extrinsic pathway, , a cancer-causing oncogene in human lymphomas. The gene encoding Bcl-2 was cleaves a BH3-only protein called Bid, generating a protein fragment overexpressed in these malignant cells as the result of a translocation. We now (tBid) that binds to Bax, inducing insertion of Bax into the OMM and understand that Bcl-2 acts as an oncogene by promoting survival of potential release of cytochrome c from mitochondria. cancer cells that would otherwise die by apoptosis. Chapter 15 Cell Signaling and Signal Transduction: Communication Between Cells 660 protein complex called the called complex protein the cytosol, cytochrome cytochrome the cytosol, apoptos to cell the commits irreversibly that event sential roles of these proapoptotic proteins in thi revealing apoptosis, from protected are Bak and Bax cytochrome cytochrome bring bring about apoptosis. caspases executioner downstream activates that pase init an is caspase-9 above, described pathway diated which is activated by Like the caspase-8, rec 15.40). cleavag proteolytic require not do and complex tein m the joining simply by activated become to thought molecul Procaspase-9 procaspase-9. of molecules eral totic cell in a Cell period as short as five minutes. fro released be can mitochondria cell’s a of all in possibly independent of cytochrome independent of cytochrome possibly es o cran iohnra poen,ms notabl most tochrome proteins, mitochondrial certain of lease dramaticall channel this increases the of permeability the OMM and promotes formed, Once OMM. the within protein-lined multisubunit, a into assemble to them c that conformation in change a undergo OMM) the of res permanent are which themolecules, Bak (and/or cules Although that thought is it (OMM). clear, entirely not is mechanism membrane mitochondrial outer c the from translocate to free is Bax protein totic 4 Figure 5.17). Nearly Nearly all of the cytochrome Figure 5.17). eg b atvtn te ae xctoe csae,w caspases, executioner targets. the same cellular cleave same the activating by verge ultimate pathways (mitochondria-mediated) intrinsic bionts and that do not undergo apoptosi bionts and that prokaryotes that these have evolved from prokaryotic one when perplexing more even is apoptosis in dria m of role key The time. present the at question this obvious no is There apoptosis. initiating in volved wo plant, power cell’s the as functions that ganelle and the mitochondri of the electron transport chain, arpae.Aottc el et tu ocr withou the releasbecause This is important en occurs 15.41). (Figure extracellular the thus into special content death cellular by spilling cell signal Apoptotic me” “eat an macrophages. as recognized are they phosphatidylse moves membran plasma the of leaflet outer the to molecules “scramblase” phospholipid a sis, Pho a During surface. on the inner leaflet of the plasma membrane. their on p normally is that phospholipid a is phatidylserine phosphatidylserine by of recognized are presence bodies apoptotic The hour. an than apoptot executed be can program apoptotic membrane-enclosed entire This body. condensed, a into tegrates th with Finally, their neighbors and start to shrink. Other intrinsic pathways that are pathways independent intrinsic of Ap Other ees o poppoi mtcodil rtis such proteins mitochondrial proapoptotic of Release You might be wondering why cytochrome As cells execute the apoptotic program, they lose co lose they program, apoptotic the execute cells As c hc rsds n h itrebae pc (see space intermembrane the in resides which , c saprnl h pito ortr, hti,an that is, is apparently the “point of no return,” 4 The extrinsic (receptor-mediated) and c forms forms part of a wheel-shaped multi- apoptosome c have also been described., , that also includes sev- includes also that , f1adcsae9 andaf-1 and caspase-9, c molecules present c a component, s lacking s process. ytosol to theto ytosol m an apop-an m e cell disin- is. Once in Once is. resent onlyresent Bax mole-Bax vironment endosym- eptor-me- uld be in-be uld e of cellu- answer toanswer n an or-on, iator cas-iator considers e (Figuree s. itochon- channel e wheree ultipro- which, y con- ly h es-the popto- the re- n lessin idents s are es cy- y auses ntact hich the ized both rine as s- ic y t B apoptotic signals. de or survival cell—whether a depends on a delicate balance between proapoptotic of fate the that pear I pression of genes encoding proteins. cell-survival .HM. Figure 15.41 key key transcription factor called NF- act the through mediated typically is survival Cell orig was hoped that it could be used asTNF an agent tu to kill because finding disappointing a was This wit treated when apoptosis undergo not do receptors p that cells most result, a As survival. cell ulating ano and apoptosis stimulating one interior: cell the si opposing and distinct two transmits often ceptor with of TNF interaction fact, In survival. cell tain a cell to self-destruction,signa opposing are there Signaling Cell Survival E of the engulfed cell and the dense state of its chr cell of the engulfed Note th of a . within the cytoplasm “corpse” phagocytosis. cant amount of tissue damage.cant which can cause lar debris can trigger inflammation, IOL .What is the role of the formation of caspase-cont 2. What aresomeofthefunctionsapoptosisinverte- 1. LSEVIER W E I V E R chondrial membraneandthedeathofcell. proapoptotic Bcl-2 member binds to the outer mito- and theeventualdeathofcell(b)timea molecule binds to its receptor(a) the time that a TNF brate biology? Describe the steps that occur betwee complexes in the process of apoptosis? 1R9,20,F 2001, 11:R796, . ENSON .) D, This electron micrograph shows an apoptotic cell Clearance of apoptotic cells is accomplished by cells of apoptotic Clearance ONNA of m Cytopl .BL. a IG croph a sm 1. RATTON A a ©2001,, ge Just as there are signals that commit , AND ␬ IHPRISO FROM PERMISSION WITH B, which which activates the ex-B, V AL E RIE cell Apoptotic mtn (Fomatin. .FA. e compact nature chrom fr Condensed, ls that main-that ls ADOK a ossessTNF t would ap- ivation of aof ivation gmented ROM a TNF re-a TNF ther stim-ther gnals intognals mor cells. and anti- a signifi- aining a h TNF.h C, tin P ath— inally n ETER URR .