13 SIGNALING AT THE CELL SURFACE
Three-dimensional structure of the G protein (blue) and (purple) complex as obtained by x-ray crystallography.
o cell lives in isolation. In eukaryotic microorgan- plasma membrane and bind to intracellular receptors. Sig- isms such as yeast, slime molds, and protozoans, se- naling from such intracellular receptors is discussed in Ncreted molecules called pheromones coordinate the Chapter 11. aggregation of free-living cells for sexual mating or differ- In this and the next two chapters, we focus on signaling entiation under certain environmental conditions. Yeast from a diverse group of receptor proteins located in the mating-type factors are a well-understood example of plasma membrane (Figure 13-1). The signaling molecule pheromone-mediated cell-to-cell signaling (Chapter 22). acts as a ligand, which binds to a structurally complemen- More important in plants and animals are extracellular sig- tary site on the extracellular or membrane-spanning do- naling molecules that function within an organism to control mains of the receptor. Binding of a ligand to its receptor metabolic processes within cells, the growth and differentia- causes a conformational change in the cytosolic domain or tion of tissues, the synthesis and secretion of proteins, and domains of the receptor that ultimately induces specific cel- the composition of intracellular and extracellular fluids. Ad- lular responses. The overall process of converting signals jacent cells often communicate by direct cell-cell contact. For into cellular responses, as well as the individual steps in this example, gap junctions in the plasma membranes of adjacent cells permit them to exchange small molecules and to coor- dinate metabolic responses. Other junctions between adja- OUTLINE cent cells determine the shape and rigidity of many tissues; other interactions adhere cells to the extracellular matrix. 13.1 Signaling Molecules and Cell-Surface Such cell-cell and cell-matrix interactions, which are covered Receptors in Chapter 6, may also initiate intracellular signaling via 13.2 Intracellular Signal Transduction pathways similar to those discussed in this and subsequent chapters. 13.3 G Protein–Coupled Receptors That Activate Extracellular signaling molecules are synthesized and re- or Inhibit Adenylyl Cyclase leased by signaling cells and produce a specific response only in target cells that have receptors for the signaling molecules. 13.4 G Protein–Coupled Receptors That Regulate In multicellular organisms, an enormous variety of chemi- Ion Channels cals, including small molecules (e.g., amino acid or lipid de- 13.5 G Protein–Coupled Receptors That Activate rivatives, acetylcholine), peptides, and proteins, are used in Phospholipase C this type of cell-to-cell communication. Some signaling mol- ecules, especially hydrophobic molecules such as steroids, 13.6 Activation of Gene Transcription retinoids, and thyroxine, spontaneously diffuse through the by G Protein–Coupled Receptors
533 534 CHAPTER 13 • Signaling at the Cell Surface
Exterior
P P Cytosol P
P Nucleus
P P P P P
G protein-coupled Cytokine receptors Receptor tyrosine TGFβ receptors Hedgehog (Hh) Wnt receptors Notch receptor receptors kinases receptors
Linked to a trimeric G Associated with Cytosolic domain Cytosolic domain Hh ligand tethered to Palmitoylated Wnt Ligand, Delta, is a protein that controls cytosolic JAK kinases with tyrosine kinase with serine/threonine membrane of ligand binds seven transmembrane the activity of an activity kinase activity signaling cell by transmembrane protein on signaling effector protein (here cholesterol anchor protein receptor cell adenylyl cyclase) complex
Activate cytosolic or Activate cytosolic Activate cytosolic Activate Smad Control processing Release an activated Cytosolic domain of nuclear transcription STAT transcription kinases (here MAP transcription factors of transcription factor transcription factor Notch released by factors via several factors by kinase) that trans- in the cytosol by by proteolysis; from a multiprotein proteolysis acts in pathways (here one phosphorylation locate to the nucleus phosphorylation Hh binding causes complex in the association with involving protein and activate nuclear release from cytosol nuclear transcription kinase A) transcription factors cytosolic complex factors by phosphorylation
▲ FIGURE 13-1 Overview of seven major classes of cell- stimulation may lead to activation of cytosolic protein kinases surface receptors discussed in this book. In many signaling that then translocate into the nucleus and regulate the activity of pathways, ligand binding to a receptor leads to activation of nuclear transcription factors. Some activated receptors, transcription factors in the cytosol, permitting them to translocate particularly certain G protein–coupled receptors, also can induce into the nucleus and stimulate (or occasionally repress) changes in the activity of preexisting proteins. [After A. H. Brivanlou transcription of their target genes. Alternatively, receptor and J. Darnell, 2002, Science 295:813.] process, is termed signal transduction. As we will see, signal- transduction pathways may involve relatively few or many 13.1 Signaling Molecules components. and Cell-Surface Receptors We begin this chapter with two sections that describe general principles and techniques that are relevant to most Communication by extracellular signals usually involves the signaling systems. In the remainder of the chapter, we con- following steps: (1) synthesis and (2) release of the signaling centrate on the huge class of cell-surface receptors that ac- molecule by the signaling cell; (3) transport of the signal to tivate trimeric G proteins. Receptors of this type, the target cell; (4) binding of the signal by a specific recep- commonly called G protein–coupled receptors (GPCRs), tor protein leading to its activation; (5) initiation of one or are found in all eukaryotic cells from yeast to man. The more intracellular signal-transduction pathways by the acti- human genome, for instance, encodes several thousand vated receptor; (6) specific changes in cellular function, G protein–coupled receptors. These include receptors in the metabolism, or development; and (7) removal of the signal, visual, olfactory (smell), and gustatory (taste) systems, which often terminates the cellular response (see Figure many neurotransmitter receptors, and most of the receptors 13-1). The vast majority of receptors are activated by bind- for hormones that control carbohydrate, amino acid, and ing of secreted or membrane-bound molecules (e.g., hor- fat metabolism. mones, growth factors, neurotransmitters, and pheromones). 13.1 • Signaling Molecules and Cell-Surface Receptors 535
Some receptors, however, are activated by changes in the signal an adjacent cell by direct contact. Cleavage by an ex- concentration of a metabolite (e.g., oxygen or nutrients) or tracellular protease releases a soluble form of EGF, which by physical stimuli (e.g., light, touch, heat). In E. coli, for can signal in either an autocrine or a paracrine manner. instance, receptors in the cell-surface membrane trigger sig- naling pathways that help the cell respond to changes Receptors Activate a Limited Number in the external level of phosphate and other nutrients (see Figure 4-18). of Signaling Pathways The number of receptors and signaling pathways that we dis- Signaling Molecules in Animals Operate cuss throughout this book initially may seem overwhelming. Moreover, the terminology for designating pathways can be over Various Distances confusing. Pathways commonly are named based on the In animals, signaling by soluble extracellular molecules can general class of receptor involved (e.g., GPCRs, receptor ty- be classified into three types—endocrine, paracrine, or au- rosine kinases), the type of ligand (e.g., TGF , Wnt, Hedge- tocrine—based on the distance over which the signal acts. hog), or a key intracellular signal transduction component In addition, certain membrane-bound proteins act as signals. (e.g., NF- B). In some cases, the same pathway may be In endocrine signaling, the signaling molecules, called referred to by different names. Fortunately, as researchers hormones, act on target cells distant from their site of syn- have discovered the molecular details of more and more re- thesis by cells of the various endocrine organs. In animals, an ceptors and pathways, some principles and mechanisms are endocrine hormone usually is carried by the blood or by beginning to emerge. These shared features can help us make other extracellular fluids from its site of release to its target. sense of the wealth of new information concerning cell-to- In paracrine signaling, the signaling molecules released cell signaling. by a cell affect target cells only in close proximity. The con- First, external signals induce two major types of cellular duction by a neurotransmitter of a signal from one nerve cell responses: (1) changes in the activity or function of specific to another or from a nerve cell to a muscle cell (inducing or pre-existing proteins and (2) changes in the amounts of spe- inhibiting muscle contraction) occurs via paracrine signal- cific proteins produced by a cell, most commonly as the result ing (Chapter 7). Many growth factors regulating develop- of modification of transcription factors leading to activation ment in multicellular organisms also act at short range. Some or repression of gene transcription. In general, the first type of of these molecules bind tightly to the extracellular matrix, response occurs more rapidly than the second type. Signaling unable to signal, but subsequently can be released in an ac- from G protein–coupled receptors, described in later sections, tive form. Many developmentally important signals diffuse often results in changes in the activity of preexisting proteins, away from the signaling cell, forming a concentration gradi- although activation of these receptors on some cells also can ent and inducing various cellular responses depending on induce changes in gene expression. their concentration at a particular target cell (Chapter 15). The other classes of receptors depicted in Figure 13-1 In autocrine signaling, cells respond to substances that operate primarily to modulate gene expression. In some they themselves release. Some growth factors act in this fash- cases, the activated receptor directly activates a transcription ion, and cultured cells often secrete growth factors that stim- factor in the cytosol (e.g., TGF and cytokine receptor path- ulate their own growth and proliferation. This type of ways) or assembles an intracellular signaling complex that signaling is particularly common in tumor cells, many of activates a cytosolic transcription factor (e.g., Wnt path- which overproduce and release growth factors that stimulate ways). In yet other pathways, specific proteolytic cleavage inappropriate, unregulated proliferation of themselves as of an activated cell-surface receptor or cytosolic protein well as adjacent nontumor cells; this process may lead to for- releases a transcription factor (e.g., Hedgehog, Notch, and mation of a tumor mass. NF- B pathways). Transcription factors activated in the cy- Signaling molecules that are integral membrane proteins tosol by these pathways move into the nucleus, where they located on the cell surface also play an important role in de- stimulate (or occasionally inhibit) transcription of specific velopment. In some cases, such membrane-bound signals on target genes. Signaling from receptor tyrosine kinases leads one cell bind receptors on the surface of an adjacent target to activation of several cytosolic protein kinases that translo- cell to trigger its differentiation. In other cases, proteolytic cate into the nucleus and regulate the activity of nuclear tran- cleavage of a membrane-bound signaling protein releases the scription factors. We consider these signaling pathways, exoplasmic region, which functions as a soluble signaling which regulate transcription of many genes essential for cell protein. division and for many cell differentiation processes, in the Some signaling molecules can act both short range and following two chapters. long range. Epinephrine, for example, functions as a neuro- Second, some classes of receptors can initiate signaling transmitter (paracrine signaling) and as a systemic hormone via more than one intracellular signal-transduction pathway, (endocrine signaling). Another example is epidermal growth leading to different cellular responses. This complication is factor (EGF), which is synthesized as an integral plasma- typical of G protein–coupled receptors, receptor tyrosine membrane protein. Membrane-bound EGF can bind to and kinases, and cytokine receptors. 540 CHAPTER 13 • Signaling at the Cell Surface that are used to solubilize receptor proteins from the cell membrane. The labeled ligand provides a means for detect- Receptor for ing the receptor during purification procedures. ligand other Another technique often used in purifying cell-surface than X receptors that retain their ligand-binding ability when solu- bilized by detergents is similar to affinity chromatography using antibodies (see Figure 3-34). To purify a receptor by this technique, a ligand for the receptor of interest, rather than an antibody, is chemically linked to the beads used to Ligand X form a column. A crude, detergent-solubilized preparation of membrane proteins is passed through the column; only No binding of X; no cellular response the receptor binds, and other proteins are washed away. Passage of an excess of the soluble ligand through the col- Transfection with cDNA umn causes the bound receptor to be displaced from the expression vector and selection of transformed cells beads and eluted from the column. In some cases, a receptor can be purified as much as 100,000-fold in a single affinity chromatographic step. Ligand X Once a receptor is purified, its properties can be studied and its gene cloned. A functional expression assay of the cloned cDNA in a mammalian cell that normally lacks the encoded receptor can provide definitive proof that the proper protein indeed has been obtained (Figure 13-6). Such ex- mRNA Ligand X pression assays also permit investigators to study the effects receptor of mutating specific amino acids on ligand binding or on “downstream” signal transduction, thereby pinpointing the receptor amino acids responsible for interacting with the lig- cDNA for receptor and or with critical signal-transduction proteins. for ligand X The cell-surface receptors for many signaling molecules are present in such small amounts that they cannot be puri- fied by affinity chromatography and other conventional bio- Binding of X; normal cellular response chemical techniques. These low-abundance receptor proteins ▲ EXPERIMENTAL FIGURE 13-6 Functional expression can now be identified and cloned by various recombinant assay can identify a cDNA encoding a cell-surface receptor. DNA techniques, eliminating the need to isolate and purify Target cells lacking receptors for a particular ligand (X) are them from cell extracts. In one technique, cloned cDNAs pre- stably transfected with a cDNA expression vector encoding the pared from the entire mRNA extracted from cells that pro- receptor. The design of the expression vector permits selection duce the receptor are inserted into expression vectors by of transformed cells from those that do not incorporate the techniques described in Chapter 9. The recombinant vectors vector into their genome (see Figure 9-29b). Providing that these then are transfected into cells that normally do not synthesize cells already express all the relevant signal-transduction proteins, the receptor of interest, as in Figure 13-6. Only the very few the transfected cells exhibit the normal cellular response to X if transfected cells that contain the cDNA encoding the desired the cDNA in fact encodes the functional receptor. receptor synthesize it; other transfected cells produce irrele- vant proteins. The rare cells expressing the desired receptor can be detected and purified by various techniques such as KEY CONCEPTS OF SECTION 13.1 fluorescence-activated cell sorting using a fluorescent-labeled ligand for the receptor of interest (see Figure 5-34). Once a Signaling Molecules and Cell-Surface Receptors cDNA clone encoding the receptor is identified, the sequence ■ Extracellular signaling molecules regulate interactions of the cDNA can be determined and that of the receptor pro- between unicellular organisms and are critical regulators tein deduced from the cDNA sequence. of physiology and development in multicellular organisms. Genomics studies coupled with functional expression as- says are now being used to identify genes for previously un- ■ Binding of extracellular signaling molecules to cell-surface known receptors. In this approach, stored DNA sequences receptors triggers intracellular signal-transduction pathways are analyzed for similarities with sequences known to encode that ultimately modulate cellular metabolism, function, or receptor proteins (Chapter 9). Any putative receptor genes gene expression (Figure 13-1). that are identified in such a search then can be tested for their ■ External signals include membrane-anchored and se- ability to bind a signaling molecule or induce a response in creted proteins and peptides, small lipophilic molecules cultured cells by a functional expression assay. (e.g., steroid hormones, thyroxine), small hydrophilic mol- 13.2 • Intracellular Signal Transduction 541
ecules derived from amino acids (e.g., epinephrine), gases more enzymes or nonenzymatic proteins. In muscle, a signal- (e.g., nitric oxide), and physical stimuli (e.g., light). induced rise in cytosolic Ca2 triggers contraction (see 2 ■ Signals from one cell can act on nearby cells (paracrine), Figure 19-28); a similar increase in Ca induces exocytosis on distant cells (endocrine), or on the signaling cell itself of secretory vesicles in endocrine cells and of neurotransmit- (autocrine). ter-containing vesicles in nerve cells (see Figure 7-43). Simi- larly, a rise in cAMP induces various changes in cell ■ Receptors bind ligands with considerable specificity, metabolism that differ in different types of human cells. The which is determined by noncovalent interactions between a ligand and specific amino acids in the receptor protein
(see Figure 13-2). NH2
■ N The maximal response of a cell to a particular ligand N Signaling Pathways F MEDIA CONNECTIONS generally occurs at ligand concentrations at which most of ocus Animation: Second Messengers in 5 N N its receptors are still not occupied (see Figure 13-3). O CH2 O ■ The concentration of ligand at which half its receptors 4 1 are occupied, the Kd, can be determined experimentally and
is a measure of the affinity of the receptor for the ligand 3 2 (see Figure 13-4). O POOH ■ Because the amount of a particular receptor expressed O is generally quite low (ranging from ≈2000 to 20,000 mol- 3 ,5 -Cyclic AMP (cAMP) ecules per cell), biochemical purification may not be feasi- ble. Genes encoding low-abundance receptors for specific Activates protein kinase A (PKA) ligands often can be isolated from cDNA libraries trans- fected into cultured cells. O
■ Functional expression assays can determine if a cDNA N NH encodes a particular receptor and are useful in studying 5 N N NH2 the effects on receptor function of specific mutations in its O CH2 sequence (see Figure 13-6). O 4 1
3 2 13.2 Intracellular Signal Transduction O POOH The various intracellular pathways that transduce signals O downstream from activated cell-surface receptors differ in 3 ,5 -Cyclic GMP (cGMP) their complexity and in the way they transduce signals. We describe the components and operation of many individual Activates protein kinase G (PKG) and opens cation channels in pathways later in this chapter and in other chapters. Some rod cells general principles of signal transduction, applicable to dif-
ferent pathways, are covered in this section. 2 1 PO3 OPO 2 CH3 (CH2)n C O CH2 3 O 6 5 Second Messengers Carry Signals O 2 OH 1 4 from Many Receptors CH3 (CH2)n C O CH OH HO 2 OPO3 The binding of ligands (“first messengers”) to many cell- O 2 3 Fatty acyl groups 3 surface receptors leads to a short-lived increase (or decrease) CH2OH in the concentration of certain low-molecular-weight Glycerol Inositol intracellular signaling molecules termed second messengers. 1,2-Diacylglycerol 1,4,5-trisphosphate (IP ) These molecules include 3 ,5 -cyclic AMP (cAMP), 3 ,5 - (DAG) 3 cyclic GMP (cGMP), 1,2-diacylglycerol (DAG), and inositol Activates protein kinase C Opens Ca2+ channels in (PKC) the endoplasmic reticulum 1,4,5-trisphosphate (IP3), whose structures are shown in Fig- 2 ure 13-7. Other important second messengers are Ca and ▲ FIGURE 13-7 Four common intracellular second various inositol phospholipids, also called phosphoinosi- messengers. The major direct effect or effects of each tides, which are embedded in cellular membranes. compound are indicated below its structural formula. The elevated intracellular concentration of one or more Calcium ion (Ca2 ) and several membrane-bound second messengers following binding of an external signaling phosphoinositides also act as second messengers. molecule triggers a rapid alteration in the activity of one or 542 CHAPTER 13 • Signaling at the Cell Surface mode of action of cAMP and other second messengers is dis- of GTP, favored by its high intracellular concentration, in- cussed in later sections. duces a conformational change in two segments of the pro- tein, termed switch I and switch II, allowing the protein to Many Conserved Intracellular Proteins bind to and activate other downstream signaling proteins (Figure 13-8). The intrinsic GTPase activity of the switch Function in Signal Transduction proteins then hydrolyzes the bound GTP to GDP and Pi, thus In addition to cell-surface receptors and second messengers, changing the conformation of switch I and switch II from the two groups of evolutionary conserved proteins function in active form back to the inactive form. The rate of GTP hy- signal-transduction pathways stimulated by extracellular sig- drolysis frequently is enhanced by a GTPase-accelerating nals. Here we briefly consider these intracellular signaling protein (GAP), whose activity also may be controlled by ex- proteins; their role in specific pathways is described elsewhere. tracellular signals. The rate of GTP hydrolysis regulates the length of time the switch protein remains in the active con- GTPase Switch Proteins We introduced the large group of formation and able to signal downstream. intracellular switch proteins that form the GTPase super- There are two classes of GTPase switch proteins: trimeric family in Chapter 3. These guanine nucleotide–binding pro- (large) G proteins, which as noted already directly bind to teins are turned “on” when bound to GTP and turned “off” and are activated by certain receptors, and monomeric when bound to GDP (see Figure 3-29). Signal-induced con- (small) G proteins such as Ras and various Ras-like proteins. version of the inactive to active state is mediated by a Ras is linked indirectly to receptors via adapter proteins and guanine nucleotide–exchange factor (GEF), which causes re- GEF proteins discussed in the next chapter. All G proteins lease of GDP from the switch protein. Subsequent binding contain regions like switch I and switch II that modulate the
(a) GTP-bound "on" state (b) GDP-bound "off" state
Gly-60 Thr-35
Switch II Switch I
P
GTP GDP GDP
Gly-60
Thr-35 Switch II
γ Switch I
GTP GDP