Current Medicinal Chemistry, 2000, 7, 911-943 911 Signal Transduction Pathways of G Protein-coupled Receptors and Their Cross-Talk with Receptor Tyrosine Kinases: Lessons from Bradykinin Signaling

Claus Liebmann*a and Frank-D. Böhmerb

Institute of Biochemistry and Biophysics, Biological and Pharmaceutical Faculty (a) and Research Group `Molecular Cell Biology`, Medical Faculty (b), Friedrich-Schiller- University Jena, D-07743 Jena, Germany

Abstract: G protein-coupled receptors (GPCRs) represent a major class of drug targets. Recent investigation of GPCR signaling has revealed interesting novel features of their signal transduction pathways which may be of great relevance to drug application and the development of novel drugs. Firstly, a single class of GPCRs such as the bradykinin type 2 receptor (B2R) may couple to different classes of G proteins in a cell-specific and time-dependent manner, resulting in simultaneous or consecutive initiation of different signaling chains. Secondly, the different signaling pathways emanating from one or several GPCRs exhibit extensive cross-talk, resulting in positive or negative signal modulation. Thirdly, GPCRs including B2R have the capacity for generation of mitogenic signals. GPCR-induced mitogenic signaling involves activation of the p44/p42 "mitogen activated protein kinases" (MAPK) and frequently "transactivation" of receptor tyrosine kinases (RTKs), an unrelated class of receptors for mitogenic polypeptides, via currently only partly understood pathways. Cytoplasmic tyrosine kinases and protein-tyrosine phosphatases (PTPs) which regulate RTK signaling are likely mediators of RTK transactivation in response to GPCRs. Finally, GPCR signaling is the subject of regulation by RTKs and other tyrosine kinases, including tyrosine phosphorylation of GPCRs itself, of G proteins, and of downstream molecules such as members of the protein kinase C family. In conclusion, known agonists of GPCRs are likely to have unexpected effects on RTK pathways and activators of signal-mediating enzymes previously thought to be exclusively linked to RTK activity such as tyrosine kinases or PTPs may be of much interest for modulating GPCR-mediated biological responses.

1. Introduction signaling routes [1]. In other cases, multiple receptors can converge on a single G protein which has the G Protein-coupled receptors (GPCRs) constitute a capability of integrating different signals [1]. Stimulation superfamily of transmembrane proteins that transduce of a particular GPCR may result not only in activation of a extracellular signals to the intracellular level. More than single signaling pathway but also to subsequent 1000 of GPCRs are known up to now - tendency interactions with those activated by the other GPCRs. increasing. Their agonists differ in size and structure These interactions between different receptor-coupled ranging from large glycoproteins to simple molecules signal transduction pathways are termed cross-talk. In such as amines or nucleosides. Agonist binding to a that way, synergistic interactions may be produced GPCR leads to activation of a heterotrimeric G protein, which result in an amplification of a coincident signal which in turn is linked to either activating or inhibiting within the same cell playing a role in "fine-tuning" of second messenger pathways. The resulting change in multiple receptor-signaling pathways [2]. On the other second messenger concentration then leads to further hand, the signal transduction of one receptor may be downstream effector events, frequently activation of also negatively regulated by that of another receptor, protein kinases. by feedback effects or by initiation of a parallel inhibitory pathway. In many cases a single receptor can activate different G proteins and thereby induce dual or multiple Meanwhile it has become clear that GPCRs are not only involved in the regulation of metabolic or excitatory *Address correspondence to this author at the Institute of Biochemistry cellular responses but are also implicated in cell and Biophysics, Friedrich-Schiller-University Jena, Philosophenweg 12, proliferation [3] (Fig. 1 ). Many ligands of GPCRs which D-07743 Jena, Germany; Tel.: +49-3641-949357; Fax : +49-3641- 949352; E-mail: [email protected] are known as classical hormones or neurotransmitters,

0929-8673/00 $19.00+.00 © 2000 Bentham Science Publishers B.V. 912 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer

Fig. (1). Schematic representation of GPCR-induced transmembrane signaling. such as vasopressin, angiotensin II, endothelin, and subsequently activated [8]. GPCRs that mediate substance P, bradykinin, acetylcholin or serotonin, growth stimulatory effects activate key effector were found to elicit a mitogenic response in various molecules of the MAPK pathway, including the RTK, cells [3,4]. Thus, for example, the nonapeptide Ras, Raf, or MAPK. Although MAPK activation may not bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) be required for all GPCR-mediated growth responses, which regulates blood pressure and smoooth muscle MAPK could be a convergence point of growth- tone has been found to stimulate growth in small cell promoting signals arriving from both RTKs and different lung cancer (SCLC) cells [5]. There is increasing GPCRs. Bradykinin, for example again, was also evidence indicating that GPCRs can function as reported to stimulate MAPK activation, e.g. in agonist-dependent oncoproteins [6]. fibroblasts [9], SCLC cells [10], or ventricular myocytes [11]. What are the mechanisms by which a GPCR can modulate cellular growth? One answer is cross-talk: In this review, we discuss principle signaling routes Beside the interactions between different GPCR linking GPCRs to the MAPK pathway and, vice versa, signaling pathways, stimulation of GPCRs may namely leading to modulation of GPCR signalling pathways by also result in activation of a pathway which receptor RTK-induced tyrosine phosphorylation. As an example tyrosine kinases (RTK) employ for stimulation of cell we will discuss the recent progress in the evaluation of growth, the so-called "MAP kinase (MAPK) pathway" cross-talk mechanisms between the bradykinin B2 [2,4,6,7]. Growth factor RTKs frequently stimulate receptor (B2R) and the EGF receptor (EGFR). MAPK via recruitment of a complex of Shc, Grb2 and Sos proteins to the cell membrane, leading to The majority of the modern pharmaca is targeted to subsequent activation of the small GTP-binding protein GPCRs [12]. Uncovering of the complexity of GPCR Ras, the two protein kinases Raf and MEK and finally of signaling, of cross-talk mechanisms and of interaction MAPK [8]. Once activated MAPK translocates to the with RTKs has interesting implications for drug nucleus where transcription factors are phosphorylated development. Previously known GPCR effectors may Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 913 have unexpected effects via affecting complex 6. GPCRs with protease ligands that act through signaling networks. This may lead to unwanted side cleavage of the N-terminal segment which in turn effects but also to novel applications of known drugs. activates the receptor (e.g. thrombin) Compounds affecting recently discovered GPCR signal transduction pathways as RTK transactivation may Within these subfamilies, the receptors for the present paradigms for novel principles to modulate various endogenous ligands may occur as receptor GPCR signaling. subtypes which are distiguished by their different pharmacological agonist and antagonist profiles, differences in their (cDNA-deduced) primary 2. Basic Principles of GPCR Signal sequences, and in many cases in different signal Transduction transduction mechanisms [17]. For example, molecular exploration has revealed the existence of 9 subtypes 2.1. GPCR Subfamilies and Receptor of the adrenergic receptor (a 1A, a 1B, a 1D, a 2A, a 2B, Subtypes a 2C, b1, b2, b3), or 5 subtypes of the muscarinic receptor (M1-M5), or 7 subtypes of the opioid receptor (m1, m2, All GPCRs consist of an extracellular N-terminal d1, d2, k1, k2, k3). segment, seven transmembrane helices (TM1-TM7), which form the TM core, three extracellular loops, and For bradykinin, three receptor subtypes with distinct three loops and the C-terminal segment exposed to pharmacological profiles have been cloned [18-20] and the cytoplasm. A fourth cytoplasmic loop may be characterized. The bradykinin B2 receptor (B2R) is formed when the C-terminal segment is palmitoylated at constitutively expressed in various cells and mediates a Cys-residue. The seven TMs are arranged into a the physiological and pathophysiological effects of membrane-spanning boundle in the counterclockwise bradykinin such as vasodilation, bronchoconstriction, direction from TM1 to TM7 as viewed from the inflammatory reactions or pain sensations. Expression extracellular surface [13]. The N-terminal segment is of the B1 receptor is only induced under the site of glycosylation and ligand binding, the C- pathophysiological conditions such as injury (for review terminal segment allows palmitoylation and see [21]). In addition, there are presumably tissue- phosphorylation as prerequisites for desensitization specific splice variants of the B2R [22]. Bradykinin and and internalization. The intracellular loops transmit the kallidin ([Lys0]BK) are equipotent natural agonist of the signal from the receptor to G protein. The TM core does B2R but do not act at the B1R. The principal B1R not form a pocket or tunnel structure as conceivable agonist is [des-Arg9]BK. For both subtypes specific but is tightly packed by hydrogen bonds and salt antagonists have been developed such as Hoe 140 (D- bridges [13]. There are several excellent reviews Arg[Hyp3,Thi5, D-Tic7, Oic8]BK) for the B2R and focussing on GPCR structure, conformation, and [Leu8,des-Arg9]BK for the B1R. Recently, a third type activation [13,14-16]. On the basis of ligand binding of kinin receptor was described in chicken and termed and receptor activation several GPCR subfamilies have ornithokinin receptor [20] where bradykinin is inactive been classified [14,16]: but the B2R antagonist Hoe 140 acts as full agonist.

1. GPCRs with ligand binding to the core The existence of receptor subtypes represent the exclusively (photons, biogenic amines, first level of specificity in GPCR signal transduction: a nucleosides, lysophosphatidic acid, single agonist may activate distinct signaling pathways eicosanoids) via receptor subtypes which are encoded by different genes and display distinct coupling specificities. 2. GPCRs with ligand binding partially in both TM core and exoloops (short peptides) 2.2. Posttranslational Modifications of 3. GPCRs with ligand binding to the exoloops and GPCRs: Glycosylation, Palmitoylation and the N-terminal segment (large polypeptides, e.g. Phosphorylation glucagon) The N-terminal segments of most GPCRs are 4. GPCRs whose ligands bind to the N-terminus glycosylated on asparagyl residues. Glycosylation may exclusively (glycoproteins, e.g. LH, TSH) be important for the functional expression and cell surface localization of receptors [23] and may 5. GPCRs whose ligands bind to a long N-terminal contribute to the ligand binding [24], depending on the segment that subsequently interacts with the cell type. membrane-associated receptor domaine (small neurotransmitters) and Most GPCRs contain one or two conserved cysteine residues in their C-terminal cytoplasmic domain that appear to be generally palmitoylated. Palmitoylation is 914 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer unique among lipid modification of proteins: it is occupied receptor but also by other factors such as reversible and adjustable [25]. There are several lines PKC or calmodulin [30]. Therefore, homologous or of evidence indicating that palmitoylation is important heterologous receptor phosphorylation on Ser- or Thr- for intracellular trafficking of GPCRs and their cell residues might be an important mechanism in cross-talk surface expression but not for ligand binding or G regulation. protein activation [26,27]. Tyrosine phosphorylation of GPCRs will be Phosphorylation of GPCRs on seryl or threonyl discussed in a later chapter of this article. residues represents a mechanism for the rapid control of receptor function and regulates the association of a GPCR with other proteins as well as their subcellular 2.3. Heterotrimeric G Proteins as Signal localization. This type of covalent modification Mediators terminates or attenuates receptor signaling via desensitization [28]. Receptor desensitization by The heterotrimeric guanine-nucleotide-binding phosphorylation is mediated either by G protein- regulatory proteins (G proteins) are composed of a 36- coupled receptor kinases (GRKs) or by second 52 kDa a -subunit, a 35-36 kDa b-subunit, and an 8-10 messenger kinases [28,29]. Heterologous kDa g-subunit. The b-and g-subunits are assembled desensitization of a particular GPCR is a feed back into bg-complexes that act as functional units. G regulation by second messenger kinases such as proteins are usually classified into four major protein kinase A (PKA) or protein kinase C (PKC) which subfamilies that share some common features: Gs, Gi, may be activated via a signaling pathway of the target Gq/11 and G12 (Table 1). Multiple isoformes of each receptor or via a separate GPCR. Phosphorylation subunit have been identified up to date including 20 leads to changes in the receptor conformation and, different a -, 6 b- and 12 g-subunits. Interestingly, not all subsequently, results in an impaired interaction with G the possible combinations of b- and g- subunits can be proteins. formed. G proteins underlie a cycle of activation and deactivation that transmits the signal from the receptor Ligand-induced or homologous desensitization to the effectors (reviewed in [31, 34]). Receptor requires GRKs and their functional cofactors, the activation triggers the exchange of GDP (bound in the arrestins. Both the GRK family and the arrestin family inactive state) for GTP on the a -subunit of a G protein include at least six members [29]. In a first step the coupled to the receptor. This results in dissociation of agonist-occupied receptors are phosphorylated by a the complex into receptor, GTP-liganded Ga - and Gbg- GRK. Then, an arrestin binds to the phosphorylated units. The free receptor has a reduced affinity for its GPCR and disrupt the interaction between receptors agonist which is released. Ga -GTP and/or Gbg can and G proteins. Desensitization represents the first interact with their target effectors. The slow intrinsic step of internalization and down-regulation [28]. The GTPase activity of Ga terminates the a -induced cytosolically localized GRKs may be translocated to the effector association. Ga -GDP re-associates the free bg- membrane and thereby activated by the agonist- complexes thereby also terminating bg-mediated

Table 1. Classification of G Proteins. Data are Summarized from [1,31-34]

Class Subtyp Toxin Effectors Receptors

Gs a s, a olf CTX AC stimulation; Ca2+-channels b1/b2-AR; V2-R; D1-R; A2-R; odorant- R

Gi a i1-3, a o, PTX AC inhibition; a 2-AR; M2/M4-R; SSTR;

a t1-2; a gust PTX regulation of K+- and Ca2+ m-/d-OR; TR; D2-R;

a z - -channels; cGMP-PDE A1-R; LPA-R

Gq a q, a 11, - PLCb activation AT II-R; ET-R; B2R;

a 14-16 - M1/M3-R; V1-R; P2Y-R

G12 a 12,13 - Na+/K+-exchange; Bruton`s TxA2-R; LPA-R tyrosine Kinase/ras GAP

Abbreviations used are explained in the text. Additional abbreviations: a/b-AR= adrenergic receptors; V1/2-R= vasopressin receptors; D1/2-R= dopamine receptors; A1/2-R=adenosine receptors; M1-4-R= muscarinic receptors; SSTR= somatostatin receptors; m/d-OR= opioid receptors; TR= taste receptor; LPA-R= lysophosphatidic acid receptor; AT II-R= angiotensin II receptor; ET-R= endothelin receptors; B2R= bradykinin receptor; P2Y= purinergic receptor; TxA2-R= thromboxane A2 receptor Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 915 signaling. The reconstituted heterotrimeric G protein The functional properties of G proteins are interacts again with the receptor and can begin the influenced by the covalent attachment of three types of cycle anew. lipids (for review see [35,36]). All a -subunits (with exception of transducin) are reversibly palmitoylated by Several of the a -subunits are substrates for labile thioester bonds to N-terminal cysteine residues. covalent modifications by either cholera toxin (CTX) or In addition, a -subunits of the G family are + i/o pertussis toxin (PTX). CTX catalyzes the NAD - myristoylated at conserved N-terminal glycine residues. dependent ADP-ribosylation of a conserved arginine This lipid modification is formed by a stable amide residue of Ga s and Ga t proteins, resulting in an linkage and irreversible. The g-subunits of all G proteins inhibition of GTPase activity and, therefore, constitutive are prenylated by a stabile thioether bond between activation of Ga . PTX induces ADP-ribosylation of most prenyl groups and cysteine residues. The precise Ga -subunits of the Gi/o-family (except of Ga z), which function of these lipid modifications is not yet known results in inhibition of receptor-G protein-coupling. but they appear to facilitate the targeting of G proteins These toxins are important tools for identifying and to the membrane and the localization of G proteins to discriminating G protein-mediated responses. specific membrane subdomains, e.g. caveolae [36].

Fig. (2). Adenylate cyclase isoform II (A) and phospholipase Cg (B) as typical examples for convergence and integration of signals at the level of single key molecules within signaling networks. Abbreviations are explained in the text. 916 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer

The a -subunits of G proteins have different contact two forms, R-I and R-II. The PKA-RI complex is regions to receptors, bg-subunits, and effectors. There cytosolically localized and its immobilization needs the is accumulating evidence suggesting that a region of interaction with additional regulatory proteins, the A- the C-terminal segment of Ga is important for the kinase-anchoring proteins (AKAPs). The PKA-RII coupling selectivity in receptor-G protein-interaction complex is particulate-associated. Thus, multiple [37]. For example, in elegant molecular and isoforms of AC, PDE and PKA combined with spatial biochemical studies has been shown that in a - i/o and temporal control provide an immense cross-talk subunits the C-terminal residues -4 (cysteine), -3 potential of the cAMP system. (glycine), and -1 (phenylalanine/tyrosine) and in a q/11- subunits the C-terminal residues -3 (asparagine) and -5 2.4.2. Phospholipases, Lipidkinases, and (glutamate) play key roles in determining the receptor Phospholipid-derived Second Messenger selectivity [38,39]. The first 25 amino acids in the N- terminal part of a -subunits are essential for bg-binding Systems Constitute Their own Signaling whereas the effector binding region partially overlaps Network the putative bg-binding region. Therefore, the a - The membrane lipid phosphatidylinositol 4,5- subunit cannot simultaneously bind effector and bg [31]. In addition to the a -subunit, the C-terminal region bisphosphate (PIP2) represents the substrate for phospholipase C (PLC). The hydrolysis of PIP results of Gb and the C-terminal part of Gg may be involved in 2 receptor coupling and specificity [40]. in the simultaneous production of two second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which mediate intracellular Ca2+- 2.4. The Main Effector Systems of GPCR release and the activation of PKC, respectively [44]. To Signaling: Isoforms, Multiple Second date 3 subfamilies of PLC are known including 4 PLCb Messengers, and Second Messenger- isozymes, 2 PLCg isozymes, and 4 PLCd isoforms (for derived Mediators review see [45]). The PLCb isozymes are activated by a -subunits of the G family but may be also 2.4.1. Adenylate cyclase (AC) q/11 stimulated by bg-complexes of Gq/11, Gi or Gz proteins. Classically, adenylate cyclase responds to GPCR- The biological importance of PLCd isozymes is not yet induced stimulatory or inhibitory regulation, mediated clear. It is assumed that activation of PLCd might be an either by Gsa or Gia , respectively. Meanwhile at least 9 event secondary to receptor-mediated activation of mammalian AC isoforms have been cloned and other PLCs or Ca2+-channels [45]. The two PLCg functionally characterized (for review see [41-43]). All isozymes are structurally distinct from the other PLCs isozymes can be stimulated by Ga s and, because they contain two SH2 domains and one SH3 experimentally, with the plant diterpene forskolin domain (SH = Src homology; see next chapter). Thus, (except AC-IX) but they differ in their response to other PLCg is activated by RTKs of growth factors via binding regulatory molecules. Based on their sequence to their autophosphorylated tyrosine residues and the homology, the ACs have been divided into several subsequent phosphorylation of PLCg at the tyrosines subfamilies with similar patterns of regulation. 771, 783, and 1254. The PLCg isozymes may be also Adenylate cyclase type I (AC-I) can be stimulated by activated by nonreceptor phosphotyrosine kinases 2+ Ca /calmodulin and inhibited by Gia - and bg-subunits. (PTKs) such as Src or Pyk2 (see also chapter 3). In AC-II may be also activated by Ca2+/calmodulin but is various cells, these PTKs can be activated by GPCRs. not inhibited by bg-complexes. AC-VIII defines ist own In addition, PLCg isozymes may be activated directly by subfamily although its regulatory properties correspond several lipid-derived second messengers of GPCRs in to those of AC-III. Type V and VI AC constitute a the absence of tyrosine phosphorylation. Thus, subfamily that is only activated by Gsa and may be GPCRs coupled to PLD or PI3-kinases may stimulate inhibited by Gia as well as submicromolar PLCg through generation of their second messengers concentrations of Ca2+ but is insensitive to calmodulin phosphatidic acid (PA) or PIP3, respectively [45]. Like and is not affected by bg-complexes. The subfamily of AC-II, PLCg represents a typical point of signal AC-II, AC-IV, and AC-VII is insensitive towards Ca2+, can integration and may play a key role in cross-talk (Fig. be stimulated by activated PKC and is sensitive to bg- 2B). complexes which are capable of enhancing the In addition to PLCs, PIP2 serves also as a substrate stimulatory effect of Gsa (Fig. 2A). The regulation of for the receptor-regulated class I phosphoinositide 3- AC-IX is still unclear. In addition, cyclic nucleotide kinases (PI3Ks) which phosphorylate PIP2 to phosphodiesterases (PDEs) that play a crucial role in PtdIns(3,4,5)P (PIP ) [46,47]. One subtype of PI3K, cAMP signal termination, are protein products of a 3 3 the p110 PI3Kg, has been shown to be directly multigene family displaying arround 30 isoforms [43]. activated by bg-complexes from G proteins [48]. In The intracellular target of cAMP is protein kinase A i addition to PIP3 production, this enzyme has the (PKA) which consists of two cAMP-binding regulatory capacity to activate the MAPK pathway [49]. Two other subunits (R) and two catalytic subunits (C). R exists in Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 917 isoforms, the PI3Ks a and b are dimeric molecules PLA2 to the membrane phospholipid substrate and consisting of the p110 catalytic subunit and a p85 requires also phosphorylation that is obviously adaptor protein that contains two SH2 domains and catalyzed by activated MAPK (ERK2). PKC and PKA links the p110 subunit to RTK signaling pathways. can phosphorylate PLA2 in vitro but there is no Recently it was reported that also the PI3Kb can be evidence for a direct phosphorylation in vivo [60]. alternatively activated by bg-complexes from G proteins Nevertheless, PKC activation can lead indirectly to [50]. The PI3K-produced second messenger PIP3 is PLA2 activation by triggering the MAPK pathway (see rapidly degradated to PtdIns (3,4)P2, both molecules chapter 4). Activation of PLA2 represents the direct act as second messengers and can directly activate pathway of AA release. Alternatively, AA may be several PKC isoforms and/or the serine/threonine released by a DAG-lipase from DAG which may be protein kinases PDK and PKB/Akt. Activated PKB/Akt produced either via PLCb, PLCg, or the PLD pathway. provides a survival signal that protects cells from AA from the various sources may be converted via the apoptosis [51]. cyclooxygenase pathway , for example, to PGE2 that can bind to and activate 4 different prostanoid receptor PIP2 is not only substrate for PLCs and PI3Ks but subtypes (EP ). EP and EP couple to G and also cofactor for the phospholipase D (PLD) which 1-4 2 4 s stimulate adenylate cyclase activity. EP1 couples to represents another GPCR-regulated effector enzyme G and activates PLC EP is able to regulate three [52]. PLD hydrolyzes phosphatidylcholine (PC) to q/11 b. 3 different sets of G proteins including Gq/11, Gi/o, and Gs. phosphatidic acid (PA) and choline in a cell-specific This is another example for a signaling mechanism response to various stimuli including thrombin, where second messengers can produce mediator vasopressin, endothelin, angiotensin II or bradykinin. molecules acting via GPCRs anew and utilize the same At least 3 mammalian PLD isoforms have been set of effectors as their first messengers. identified up to now (for review see [52]). Downstream of GPCRs, PLD regulation occurs by activation via PKC We have to go back once more to the Gq/11/PLCb- or by activation via small G proteins such as ARF (ADP- pathway which is chiefly producing the multiple second ribosylation factor) or Rho (a monomeric G protein). messengers IP3 and DAG. IP3 binds to a tetrameric IP3 PLD could contribute to the intracellular signaling by receptor in the endoplasmatic reticulum (ER) and several mechanism. Firstly, PA has been implicated as a triggers the release of Ca2+ from the ER resulting in an lipid second messenger in the regulation of protein intracellular increase in cytosolic Ca2+ from kinases [54] activation of PLCg [45], and other approximately100 nM to approximately 1 mM. In terms signaling molecules, including the protein-tyrosine of signalling, Ca2+ has to bind to trigger proteins such phosphatase SHP-1 [55]. Secondly, in most cells PA is as calmodulin that realize the messenger function of rapidly converted to DAG through the action of Ca2+ (for review see [62,63]). phosphadidate phosphohydrolase. Thus, PLD-derived DAG is implicated in the late phase activation of PKC The intracellular target for DAG is protein kinase C [56]. Thirdly, PA is converted to lysophosphatidic acid (PKC) that belongs to the serine/threonine protein (LPA) by a specific PLA2. LPA is known as an kinases. PKC is thought to be essentially involved in intercellular signaling molecule that is rapidly released the regulation of cellular proliferation and and affects cells by acting on ist own GPCR. LPA may differentiation. The PKC superfamily consists of several be involved in the regulation of a variety of cellular isoforms. Based on their domain structure and their responses, e.g. cell proliferation, smooth muscle regulation they have to be divided into at least 3 contraction, platelet aggregation, or neurotransmitter subfamilies: "convential" cPKCs (a , bI, bII, and g), which release [57]. Interestingly, the LPA receptor is capable are sensitive to Ca2+, DAG, and phorbol esters; "novel" of coupling to both Gi and Gq/11 proteins thus activating nPKCs (d, e, h, and q), which are activated by DAG and dual pathways [57]. Therefore, LPA generated from PA phorbol esters but are independent of Ca2+; and is a typical example that a second messenger of a given "atypical" aPKCs (z, l , and m), which are insensitive to GPCR signalling pathway may produce a mediator Ca2+, DAG and phorbol esters and may be regulated by which acts through another GPCR in an autocrine or phospholipids, such as PA (for review see [56, 64-66]. paracrine manner. Recently, the PKC superfamily was supplemented by the newly discovered PKC kinases (PRKs) consisting Another phospholipase that is stimulated by of at least 3 members (PRK1-3) [66]. Like the aPKCs, numerous agonists of GPCRs but also by growth they are insensitive to Ca2+, DAG and phorbol esters. factors in a variety of cells is the cytosolic 85 kDa Phorbol esters are potent tumor promoters and can phospholipase A2 (PLA2) (for review see [58-60]). substitute for DAG in activating cPKCs and nPKCs. Stimulation of PLA2 leads to the release of arachidonic They are not metabolized like DAG, evoke a prolonged acid (AA) and, subsequently, to the production of activation of PKC and are useful tools in studying PKC- eicosanoids [61]. Activation of PLA2 by GPCRs regulated pathways in vivo. requires increase in cytosolic Ca2+ for the association of 918 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer Table 2. Typical Bradykinin Signalling Pathways : Multiple Coupling and Cell Specificity

Cell/Tissue B2R Effector Mechanism Ref.

A431 cells, guinea pig ileum, others PLCb (stimulation) via Gq/11 [71,72]

fibroblasts, rat myometrial cells PLA2 (stimulation) partially via Gi [73.74] tracheal cells PLD (activation) unknown [75]

A431 cells AC (stimulation) via Gs [76]

airway smooth muscle cells AC (stimulation) via Gq/11, PKC, MAPK, PLA2, PGE2 [77] 2+ PC-12 cells AC (stimulation) via Gq/11, Ca /calmodulin [78]

Rat uterus, GPI AC (inhibiton) via Gi [79, 80] 2+ + 2+ NG 108-15 cells Ca -activated K channels (activation) via Gq/11, IP3, Ca [81]

NG 108-15 cells N-type Ca2+ channels (inhibition) via G13, Rac, p38 MAPK [82]

Thus, all signaling routes that generate DAG may stimulation of AC whereas higher concentrations result in the activation of distinct PKC isoforms. In induce activation of PLCb [84]. The activation of addition, the PKC isoforms z, e, and d are known as multiple coupling GPCRs, consequently results in targets for PIP3, the second messenger of PI3Ks multifunctional signaling. Interactions between the [67,68]. particular pathways of a single GPCR with multiple signal transduction within the same cell could be Finally, it should be noted that not only membrane classified as homologous cross-talk. Such interactions + 2+ enzymes but also K and Ca channels may be may occur at different levels of signal transduction. effectors for GPCRs (for review see [69]). Recently, Recently, the group of Lefkowitz [85] has also cross-talk at the level of Ca2+ channels was demonstrated that the b-AR can "switch" from Gs to Gi. reported resulting from PKC-mediated phosphorylation The "switch on" of Gi requires the proceeding that antagonizes bg-induced inhibition of Ca2+ activation of Gs and the stimulation of the cAMP channels [70]. This cross-talk mechanism provides a pathway. Activated PKA phosphorylates the b-AR first example that not only effector enzymes but also leading to receptor coupling to Gi. Gbg-complexes ion channels have the potential for the integration of released from Gi then activate MAPK in a Src- and Ras- multiple signaling inputs. dependent manner [85]. Thus, this "switch on" mechanism induces cross-talk at the receptor level. 2.4.3. Bradykinin B2 Receptor-mediated Another example representing a "switch off" Signal Transduction mechanism comes from our own work about BK In dependency on the cell or tissue investigated the signaling in A431 cells [76]. In this cell line, BK induces pleiotropic hormone bradykinin has been shown to be a rapid Gq/11-mediated activation of PLCb resulting in capable of activating different G proteins and multiple stimulation of PKC translocation. In a dual pathway BK signaling pathways (Table 2). In the majority of cases, slowly activates Gs followed by an increase in cAMP however, and in most cells bradykinin stimulates PLCb production and PKA activation that leads to an inhibiton of PKC translocation (Fig. 3 ). via a Gq/11 protein. In addition, numerous interactions between 2.5.Complexity of GPCR Signaling: Multiple signaling pathways of different GPCRs have been Coupling, Switching, and Cross-talk described (for review see [2]). This type of interaction, therefore, could be classified as "heterologous cross- It is well known that many individual receptors are talk". Synergistic cross-talk between Gi- and Gq- capable of interacting with different G proteins [1,83]. coupled receptors, e.g. adenosine A1- or For example, the human thyrotropin (TSH) receptor or neuropeptide Y (NPY)-receptors and a 1-adrenergic also the bradykinin B2 receptor (Table 1) can interact receptors often result in augmentation of physiological with members from each of the four major subfamilies of responses such as smooth muscle contractions [2]. a -subunits. The specificity of interaction may be Such synergistic interactions seem to play an important determined by the agonist concentration or by different role in the "fine tuning" of multiple receptor signaling kinetics. Thus, low concentrations of TSH lead to pathways [2]. Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 919

Fig. (3). Dual signaling pathway of the bradykinin B2 receptor (B2R) in A431 cells: a rapid Gq/11-mediated activation of the PLCb/PKC pathway is negatively modulated by a slowly induced but independent and Gs-mediated activation of the cAMP/PKA pathway [76].

On the other hand, a single G protein can be the independent control of multiple signaling pathways activated by multiple receptors (Table 1) thus acting as of GPCRs. convergence point [1,83]. The specificity of receptor coupling to a mutual effector system may be determined by the composition of heterotrimeric G 3. Receptor Tyrosine Kinase (RTK) proteins. For example, using the antisense Signaling and MAP Kinase Pathways oligonucleotide technique Kleuss et al. [86, 87] 3.1. Ligand-dependent Activation of RTKs showed in excellent studies that in GH3 cells inhibition of Ca2+- channels by somatostain receptors is mediated by G whereas inhibition by M4 muscarinic Protein tyrosine kinases (PTKs) have central o2b1g3 functions for the regulation of cell proliferation and receptors is mediated by Go1b1g4. Another mechanism for selective regulation of GPCR signaling pathways other cell functions. Many polypeptidic mitogenic may be provided by the newly discovered RGS factors ("growth factors") elicit their cellular responses (regulators of G protein signaling) proteins [88, 89]. via receptors of the transmembrane receptor tyrosine These RGS proteins function obviously as GAPS kinase (RTK) family [90-92]. Well known examples are (GTPase-activating proteins) for heterotrimeric G the receptors for epidermal growth factor [93,94], the platelet derived growth factors (PDGFs) [95,96] or the proteins and and enhance the speed of GTP hydrolysis fibroblast growth factors (FGFs) [97-99]. The biological thereby controling the kinetics of G protein signaling. effects of RTK stimulation include effects, largely Members of the RGS family have been shown to positive, on cell proliferation ("growth"), cell display selectivity for different G -subunits such as a differentiation, locomotion and cell survival (Fig. 4 ). In RGS2 for a q or RGS1/3 for a i [89]. Thus, the cell- specific expression of different GRS proteins may alow contrast, receptors for cytokines, a class of growth- and 920 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer differentiation-modulating polypeptides for frequently been observed in high level overexpression hematopoietic and immune cells, are devoid of intrinsic experiments [109,110] and in cancer cells enzymatic activity and need to recruit cytoplasmic PTKs overexpressing an RTK. The receptors in the insulin for signaling [100]. While many aspects of signaling receptor family [111,112] present an exception since from RTKs and cytokine receptors have similarities, the they consist of disulfid-bridged dimers in the absence latter class of receptors shall not be discussed further in of the ligand. Ligand bindig initiates signaling by this review. RTKs consist of an extracellular ligand triggering dimer/oligomer formation or, in case of the binding domain, a single transmembrane domain and insulin receptor family, by triggering interaction of a an intracellular domain which harbors the conserved preformed receptor dimer. Various molecular means tyrosine kinase subdomain and variable regulatory are employed by RTK ligands to accomplish receptor sequences [101]. The hitherto known molecules in this dimer stabilization [105]. Monomeric ligands as EGF receptor family fall into 13 classes, assigned largely on drive dimerization by interacting with receptor subunits the basis of homology within their intracellular tyrosine in a 1:1 stoichiometry [113]. Dimerization may be kinase domain [102]. More than 100 genes for RTKs consequence of conformational changes in the ligand- may exist in the human genome [101]. The functional occupied receptors or may result from a bivalent unit of an RTK consists of at least two receptor interaction of both ligands with both receptor subunits molecules or higher oligomers [103-105]. Receptor in the dimer [114]. Other ligands are dimeric and dimers or oligomers can be homomers or heteromers of interact with two receptor subunits at the time, as in related [106,107], possibly also of unrelated RTKs . In case of PDGFs [104]. the absence of ligand, receptor dimer (oligomer) formation apparantly occurs spontaneously with low As a consequence of receptor dimerization the two efficiency [108]. Solely overexpression of a given RTK subunits trans-phosphorylate each other on tyrosine may trigger sufficient dimer formation leading to residues. Two types of phosphorylations can be signaling in the absence of a ligand [107]. This has distinguished: Phosphorylations in the "activation

Fig. (4). Multiple signaling pathways activated by RTKs and negatively controlled by PTPs in absence and presence of an RTK ligand. The mechanisms as well as abbreviations are explained in the text. Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 921 loop" in the kinase subdomain which lead to elevation 3.2. Specificity and Redundancy in RTK of kinase activity and phosphorylations in other parts of Downstream Signaling the intracellular domain which create binding sites for signaling molecules. Crystal structures of the insulin Downstream signaling molecules which bind to receptor catalytic domain [115] and the FGF-receptor 1 trans-phosphorylated RTKs fall in two classes: catalytic domain [116] have revealed the mechanism of enzymes and adaptor molecules. An example for the activation of these kinases [117]. The activation loop, a former is the phospholipase Cg [125], which binds via flexible structure identified in many protein kinases, in tandem SH2-domains to different activated RTKs, is in its unphosphorylated state can occupy partially (FGF turn phosphorylated and itself activated. An example receptor -1) or completely (insulin receptor) the active for the latter is the p85 subunit of PI3kinase a , which site of the kinase. Phosphorylation leads to docks to several growth factor receptors and permits in displacement of the activation loop and in turn provides turn binding and thus membrane recruitment of the PI3 access for substrates to the kinase active site. A kinase catalytic subunit p110a [126, 127]. Adaptor phosphorylation site corresponding to tyrosine 1162 in molecules can also recruit further adaptors, which the insulin receptor kinase is conserved in RTKs recruit other singaling molecules and so forth [122]. An suggesting that this mechanism of activation is a example is the family of Shc adaptor molecules, which common principle. However, for example in case of the are phosphorylated subsequent to RTK binding and in PDGFb-receptor substantial residual kinase activity can their phosphorylated form are able to bind the adaptor be detected in a receptor variant with the 857 tyrosine molecule Grb2 [128]. Thus, RTKs present scaffolding (corresponds to 1162 in insulin receptor) mutated molecules for the assembly of multiple signaling [118] or selectively blocked by a kinase inhibitor [119]. proteins which can interact in a chain of signal Thus, stringency of regulation by activation loop transduction events [129]. phosphorylation varies among the different RTKs. Multiple tyrosine residues can further be Many RTKs have been shown to interact with the phosphorylated in the RTK cytoplasmic domains, both same set of signaling proteins. For example, Shc and C- or N-terminally of the catalytic subdomain or, in RTKs Grb2 have been demonstrated to bind to multiple RTKs with a split kinase domain as PDGF receptors, also in including the EGFR [130], the EGFR-related receptor the "kinase insert" [95, 96]. These sites, in their HER2/erbB2 [131] and the hepatocyte growth factor phosphorylated form, present recognition and binding (HGF) receptor Met [132]. On the other hand, there are sites for signaling molecules posessing different types also pronounced specificities. Thus, PLCg cannot bind of phosphotyrosine binding domains as SH2-domains to the insulin receptor [111] or Grb2 binds only with low [120-122], or PTB domains [123, 124]. SH2 ("src- affinity directly to the PDGFb-receptor [133]. Certain homology 2"-domains) are protein modules of about RTKs have even quite specific interaction partners. 100 amino acids which are found in numerous proteins The insulin receptor family RTKs accomplish most of and recognize phosphotyrosine in the context of up to their signaling activity via recruitment of relatively 6 C-terminal amino acids. In contrast, PTB specific docking proteins, the insulin receptor ("phosphotyrosine binding") domains recognize substrates (IRS), which in their phosphorylated form phosphotyrosine and 1-6 N-terminally situated amino then interact with further signaling molecules [111]. acids. Another type of protein domains involved in Other examples are the FGF receptor 1 which elicits signal transduction are SH3 ("src-homology 3") much of its signaling via the FRS2 adaptor [134, 135] domains. They consist of about 60 amino acids and and the Met/HGF receptor RTK which exhibits a interact with other proteins in a phosphorylation- particularly strong interaction with the docking protein independent manner recognizing proline-rich Gab1 [136]. sequences. These domains and a still increasing number of recently discovered protein-protein 3.3. RTK Interaction with Further Protein interaction domains are apparently required to achieve Tyrosine Kinases efficiency and specificity in highly ordered signal transduction complexes. Multiple interactions of the Another level of complexity of RTK signaling has RTK cytoplasmic domains with signaling molecules emerged when it was found that RTKs utilize also form the basis for initiation of multiple intracellular cytoplasmic PTKs for signal transmission [137]. signaling events (Fig. 4 ). Several signaling chains may Cytoplasmic PTKs fall into 9 classes [102, 138]. be necessary to elicit together a particular biological Members of the Src-family of PTKs, comprising for response. On the other hand, they form the basis for example the intensely investigated PTK pp60c-src different types of biological responses which can be (designated as "Src" throughout this review), its mediated via the same receptor as stimulation of cell oncogenic counterpart v-Src and the PTKs Yes (pp62c- growth, cell locomotion, cell differentiation and yes), Fyn (p59fyn), Lyn (p56lyn) and Lck (p56lck) harbor prevention of apoptosis. one SH2-domain and one SH3 domain in addition to 922 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer the catalytic domain. Negative regulation of these exist which allow termination of signaling, and, evenly kinases occurs by phosphorylation on tyrosine important, silencing of the RTK basal signaling activity residues in the C-terminus, leading to interaction with in the absence of a ligand. Ligand-independent the intramolecular SH2-domain. Positive regulation is activation of RTKs [107] (discussed below) by various then possible by dephosphorylation of this and quite diverse cell treatments including stimulation phosphotyrosine by protein-tyrosine phosphatases of GPCRs clearly shows that RTKs may have a signaling (PTPs, see chapter 3.4). Src, Fyn and Yes may have activity in the absence of ligand which is normally apparently very similar functions. Several assays used silenced. There are paradigms for negative regulation for detecting involvement of Src in cellular functions of RTKs at all levels of RTK activation, including cannot differentiate between these kinases. Another interference with ligand binding [156], with receptor family of cytoplasmic PTKs is named after its prototype dimerization [157] and with the tyrosine focal adhesion kinase (Fak), a 125kDa enzyme which phosphorylations executed by the receptor kinase cooperates with Src in integrin signaling and regulation [158]. Also, receptor internalization [159-162] and of the assembly of cell-matrix adhesion complexes. A phosphorylation of the receptor cytoplasmic domains relative of Fak is Pyk2, a PTK which is activated by Ca2+ by heterologous serin-threonine kinases are means of and likely to be involved in downstream signaling of RTK inactivation, which are utilized by the cell. The some GPCRs. Finally, the family of JAK ("janus EGFR is subject to phosphorylation by PKC at Thr 654 kinases") should be mentioned here, which includes [163], leading to attenuation of receptor signaling. also Tyk2 as a member. These PTKs function Also, phosphorylations at Thr 669, Ser 1002, 1046 and downstream of cytokine receptors but may also interact 1047 [164-167] may modulate EGF RTK negatively. A with some RTKs. A paradigm for interaction of an RTK modulation of signaling by PKC-dependent with cytoplasmic PTKs present the PDGF-receptors phosphorylation has also been shown for other RTKs which have the capacity to recruit Src family kinases, [168] and may be an important general regulation including p60c-src and activate them in turn [139-141]. principle. Src family kinase activation has been suggested to be essential for mitogenic signaling of the PDGFb-receptor Another means or negative RTK control are the [142]. Similar observations have been made with the actions of protein-tyrosine phosphatases (PTPs) (Fig. EGFR, although RTK- Src kinase complexes are less 4 ). Since this type of interactions may provide an readily demonstrable in this case [142-145]. Src-family important link for cross-talk with the GPCR family of kinases do appear, however, to not only simply mediate receptors (see chapter 4.4), we will describe it in some a signal but, to also phosphorylate the RTKs and detail. Ligand-activated RTKs are rapidly thereby modulate signaling. Src phosphorylation sites dephosphorylated by cellular PTPs, shown and have been mapped on the EGFR, the PDGFb-receptor investigated in some detail for example for the EGFR, and the IGF-1 receptor [146-151]. Mutation of a main PDGFb-receptor or the insulin receptor [169-173]. Src-phosphorylation site on the PDGFb-receptor leads Dephosphorylation parameters for not ligand-activated to a reduced mitogenic signaling and a more receptors are difficult to evaluate, however, treatment pronounced stimulation of chemotaxis by PDGF via the of cells with PTP inhibitors as vanadate [174], mutant receptor [150]. For the EGFR and the IGF-1 phenylarsineoxide [175, 176], hydrogen peroxide receptor, increased RTK activity subsequent to [177] or diamide [178, 179] leads to phosphorylation of phosphorylation by Src has been shown [146, 151]. many RTKs. Thus, "basal" activity of RTKs is probably Other cytoplasmic tyrosine kinases which have been quite significant and under negative control of PTPs. shown to interact with RTKs are for example FER, a Over the last 10 years PTPs have emerged as a large member of the fes/fps family of nontransmembrane family of quite diverse molecules [180-182]. More than receptor tyrosine kinases [152, 153] and possibly 100 PTP species may exist [183] and hitherto almost members of the JAK/Tyk family [154]. 50 different individual PTPs have been shown to be expressed in a single cell type [184]. PTPs can broadly be classified in cytoplasmic and transmembrane, or 3.4. Inactivation Mechanisms for RTKs "receptor-like" enzymes [185]. The former posess one PTP catalytic domain and various types of protein- Tyrosine phosphorylation needs to be tightly protein interaction or membrane targeting domains. regulated. Loss of this tight regulation may lead to The tramsmembrane PTPs have frequently two aberrant cell behaviour as uncontrolled proliferation. catalytic domains and variable extracellular domains. For many tyrosine kinases including RTKs oncogenic Depending on the PTP subclass, these may recognize variants exist which have constitutive activity as a cell surface proteins, matrix components or soluble common property [155]. In case of RTKs, tight ligands. For example, the PTPs RPTPµ and k have regulation is on the one hand provided by the been shown to interact homophilically [186-188]. necessity of ligand stimulation for activation. On the RPTPb has a carboanhydrase-like motif in the other hand, several negative regulation mechanisms extracellular domain which can interact with contactin, a Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 923 neuronal cell surface protein [189]. Also, RPTPb has and soft agar colony forming capacity [210]. Thus, been shown to interact with the matrix protein tenascin, RPTPs appears to control aspects of EGFR signaling in cellular adhesion molecules (CAMs) and pleiotrophin, a A431 cells. Understanding regulation of PTP activity is heparin-binding neurite-promoting factor (reviewed in only in its beginning. This concerns effects of known or [190]). Hitherto, it is unclear whether these interactions putative ligands for RPTPs, mentioned above as well as can affect intracellular PTP activity. From crystal possible effects of intracellular effectors. Activity of a structure data for RPTPa and from experiments with a number of PTPs has been shown to be modulated by chimeric molecule consisting of the EGFR extracellular Ser/Thr phosphorylation. For example, phos- domain and the RPTP CD45 intracellular domain it has phorylation of SHP-1 by PKC leads to inhbition [211], been proposed that for certain RPTPs dimerization may PKC-phosphorylation of the transmembrane PTP lead to inactivation of the first (membrane-proximal) RPTPa to elevation [212] of PTP activity. Various PTPs catalytic domain, which harbors most of PTP activity are also phosphorylated on tyrosine residues, which [191-193]. Among the cytoplasmic PTPs much has likewise been suggested do modulate activity attention has been devoted to SHP-1 (HCP, SH-PTP1, [213]. Several cytosolic and transmembrane PTPs PTP1C) and SHP-2 (syp, SH-PTP2), the only known undergo partial proteolytic cleavages which modify mammalian PTPs which posess SH2-domains [194, cellular localization and may also affect activity towards 195]. Their tandem SH2-domains target these PTPs to cellular substrates [214]. Finally, lipid second cellular phosphotyrosine-containing proteins including messengers can modulate PTP activity as shown for RTKs. Both PTPs exist in a relatively inactive "closed" phosphatidic acid and the PTP SHP-1 [55]. In conformation, with the N-terminal SH2-domain blocking conclusion, PTPs and regulation of their activity the active site. Occupation of the SH2-domain leads to provide important but hitherto poorly understood activation of PTP activity [196, 197]. SHP-2 is pathways to modulate RTK signaling activity in a ubiquitously expressed and mediates positive signals positive and negative manner. for many RTKs and also cytokine receptors by a hitherto unknown mechanism involving the PTP catalytic activity. For some receptors, however, SHP-2 3.5. RTK-mediated MAP-Kinase Activation may also negatively control signaling steps (reviewed in [198]). SHP-1 negatively regulates various types of An important signaling pathway which links RTKs to receptors in hematopoietic cells, including cell proliferation and possibly other biological immunoreceptors [199, 200], cytokine receptors [201], endpoints is the so-called "MAP-kinase cascade". This and the RTKs Kit/SCF-receptor [202] and CSF-1 term refers to a signal transmission chain from the receptor [203]. Apart from hematopoietic cells, SHP-1 membrane to the nucleus, which is conserved from is also expressed in epithelial cells [204]. It can yeast to mammals and consists of a small G-protein associate with the EGFR and negatively regulate followed by three consecutively activated protein signaling of this receptor at least in cells with relatively kinases (for recent reviews see [215-217]). In high EGFR levels [55, 205, 206]. While SHP-1 has multicellular organisms RTKs have aquired the capacity been assigned to various RTKs as a likely or possible to activate one variant of this chain, whose last negative regulator, in most cases the important PTPs enzymes in this case are the closely related Ser/Thr- for a given RTK signaling regulation are unknown. It is specific; proline-directed "extracelluar signal regulated quite possible that several PTPs are involved in kinases" (ERKs) 1, and 2, also designated "mitogen- different aspects of signal regulation. The EGFR may activated protein kinases" (MAPK) p44 and p42, serve as an example of this. In transient coexpression respectively. In the literature frequently the term "MAP systems multiple PTPs have the capacity to kinase" is used as a synonym for ERK1/2, as we do in dephosphorylate the receptor [109]. Interaction of the the other chapters of this article. The main pathway for receptor has been described with the cytoplasmic mitogenic signaling from RTKs like the EGFR or the PTPs SHP-1 and SHP-2 (via SH2-domains) [205], PDGFb- receptor involves the following steps: PTP1B and T-cell PTP (with so-called "substrate Subsequent to RTK activation and trans- trapping mutants" of the PTPs, forming stable enzyme- phosphorylation occurs binding of a complex substrate complexes) [207, 208] and PTPs of the PTP- consisting of the adaptor molecule Grb2 and the PEST-family [209]. Also, transmembrane PTPs are guanine-nucleotide exchange factor (GEF) Sos for the likely to modulate EGFR signaling. Inducible small G-protein Ras. Ras, a 21kDa protein is the prototype for numerous small G-proteins in the cell (for overexpression of the transmembrane PTP RPTPs in A431 cells leads to reduced EGFR phosphorylation review see [218-222]). They all have in common that and a reduced capacity of the cells to form colonies in they are active, i.e. capable to interact with "effector proteins" in the GTP-loaded state. They have a small soft agar. Partial suppression of endogenous RPTPs intrinsic GTPase activity, which is greatly enhanced by by inducible expression of an RPTPs antisense- construct leads to elevated receptor phosphorylation specific GTPase-activating proteins (GAPs). GTP- loading of inactive, GDP-loaded small G-proteins is 924 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer accomplished with the help of GEFs as the MAP kinases ERK2/1 confers ERK activation. aforementioned Sos. Binding of the Grb2-Sos comples Activated ERKs can phosphorylate multiple cellular to the receptor is mediated by the Grb2 SH2-domain target proteins, including transcription factors and can be either directly to the tyrosine- (subsequent to nuclear translocation of Erks), the p90 phosphorylated RTK as in case of EGFR or indirectly via ribosomal S6 kinase (Rsk90), microtubule-associated the phosphorylated adaptor Shc as in case of the proteins (MAPs) and phospholipase A2. These PDGF receptor. Membrane-recruited Sos now enables phosphorylations link activation of the MAP kinase a GDP-GTP-exchange on Ras. All this occurs at the pathway to cell growth and transformation. It should be inner surface of the plasma membrane, where Ras is noted that several members in the pathway upstream bound via a farnesyl-anchor (Fig. 5 ). GTP-loaded Ras from Erks are transforming when overexpressed in a has a high affinity for the first kinase in the MAPK- constitutively active form, including Ras, Raf and MEK, cascade, the product of the cellular protooncogene c- emphasizing that this is a main pathway for mitgogenic raf, Raf-1 and recruits it to the membrane. Membrane signaling. recruitment of Raf-1 leads in an incompletely understood manner to activation of Raf-1. Probably Biological responses initiated via the MAPK pathway additional proteins [223] and membrane lipids [224] as seem to critically depend on the duration of MAPK well as phosphorylation [225] contribute to activation of activation. For example in PC12 cells, sustained MAPK Ras-bound Raf-1. Activated Raf-1 phosphorylates its stimulation via the nerve growth factor (NGF) receptor donwstream kinases MKK1or 2 (MAP-kinase kinase; TrkA leads to a differentiation response, while a short also termed MEK for MAP/ERK-kinase) on two Ser wave of MAPK activation via the EGFR mediates a residues, leading to activation of MKK. MKKs are mitogenic response. In other cells a sustained kinases with "dual specificity", i.e. they can activation of MAPK may be necessary for eliciting a phosphorylate Ser/Thr and Tyr residues. Dual mitogenic response [226]. An intersting feature of the phosphorylation on Thr and Tyr in the activation loop of MAPK pathway is negative feedback regulation. Erks

Fig. (5). MAP kinase pathways are routed by scaffold proteins and negatively controlled by multiple phosphatases. Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 925 can phosphorylate and thereby negatively regulate presents another level of regulation and possibilities for upstream molecules in the chain, notably Sos. Also, for cross-talk with signaling pathways emanating from example the EGFR can be phosphorylated on Thr 669 GPCR. by Erk2.

There are multiple variants of the MAPK signaling 3.6 Ligand-independent Activation of RTKs chain, starting with the existence of different isoforms of Ras, Raf, MKK, and ERK [217]. Parallel pathways on Various quite diverse agents and cell treatments the basis of distinct members of the MAPK-family with have been observed to induce activation of RTKs in corresponding upstream components and distinct the absence of ligand. These include cell treatments downstream targets and biological effects exist. In with "adverse agents", for example UV [235] or X-ray yeast probably 6 members of the MAPK family exist irradiation [236]. Also, activation of different GPCRs has [227]. They do not couple to RTKs (those do not exist been shown to lead to RTK activation. This pathway in yeast) but to nutrient sensors or the pheromone has been designated "transactivation" and is discussed receptor, a GPCR. In animal cells there are likewise in detail below (chapter 4.4). Finally, integrin and cell- multiple members of the MAPK-family. Other members, cell adhesion molecule activation have been shown to which are relatively well investigated apart from result in RTK activation [237]. ERK1/2, are the NH2-terminal Jun-kinase/stress activated protein kinase (JNK/SAPK) and p38. Many From the current knowledge of RTK activation and RTKs can activate ERKs, some growth factor receptors signaling control pathways different mechanisms could have also been shown to activate JNK. JNK and p38 be envisaged for ligand-independent RTK activation: are both activated by various stress factors. In addition ligand-independent RTK dimer/oligomer formation, to ERK1/2 further MAPK-family members may be heterologous phosphorylations and inactivation of important for RTK signaling. For example the mitogenic "silencing" mechanisms. signal of the EGFR has recently been shown to require Some evidence has been obtained for the last activation of ERK5 (also known as "big molecular possibility in case of UV-mediated RTK activation. PTPs weight kinase" BMK) [228]. which keep RTKs silent and inactivate them after ligand Specificity within a given MAP kinase cascade is on stimulation perform catalysis with particiation of a the one hand provided by specificities of the upstream reactive cysteine in their active center and are therefore and downstream elements for each other. On the other very susceptible to oxidation. UV treatment of cells hand, scaffolding of different components of the chain could recently been shown to inactivate membrane by specific scaffold proteins seems to be an additional bound PTPs for the EGFR and the PDGF receptor, mean of providing specificity and efficient coupling of most likely via an oxidative mechanism [110]. It seems the signal transducing components [229, 230]. For quite likely that various adverse agents can lead to example, MP1 and JIP1 are recently identified proteins generation of reactive oxygen species and which bind members of the ERK or JNK cascade, subsequently to PTP inactivation. These inactivation respectively, and may provide a scaffold for routing the mechanisms may in fact be partially reversible and play signal within the cascade (Fig. 5 ). even a role in ligand-activated signal transduction [238]. Hydrogen peroxide generation subsequent to MAPK signaling is subject to negative regulation by RTK activation has been shown for the PDGF receptor phosphatases (reviewed in [231]). One class of to be essential for eliciting a mitogenic signal [239]. phosphatases responsible for inactivation of MAP EGFR activation is accompanied by hydrogen peroxide kinases are the dual-specificity MAP kinase generation [240] and a reversible inactivation of the phosphatases (MKPs). Members of this family, for PTP PTP1B [241]. PTP activity, however, is likely to be example CL100/MKP1 are products of "immeadiate subject to regulation by further mechanisms, as early genes" which are rapidly activated upon growth phosphorylation or proteolysis, outlined above. This factor stimulation. Multiple MKPs have pronounced should in turn also modulate activity of PTP-regulated selectivities for certain MAP kinase members and thus RTKs. for feedback inhibition of the respective cascade [232, 233]. Also, PTPs can inactivate MAP kinases by An interesting question is to what extent dephosphorylating solely the phosphotyrosine in the heterologous tyrosine kinases may be capable of RTK MAPK activation loop [234]. Finally, the regulatory activation. RTK phosphorylation by Src-family kinases is phosphothreonine on MAP kinase familiy members can possible and may be sufficient to generate signal be subject to dephosphorylation by Ser/Thr specific molecule docking sites and subsequent signal phosphatases [231]. In conclusion, inactivation of the generation [137]. This would in principle be possible MAP kinase cascades by phosphatases, either dual- without participation of the RTK intrinsic tyrosine kinase specificity or tyrosine-specific or Ser/Thr-specific, activity. For the case of GPCR transactivation, however, 926 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer participation of RTK intrinsic activity has been cell type to cell type. However, stimulation of various demonstrated (see chapter 4.4). It is an interesting GPCRs was found to induce activation of key effector possibility that Src-family kinases or other heterologous molecules of RTK signaling, including Ras, Raf, and tyrosine kinases could also execute such RTK MAPK. These observations led to the assumption that phosphorylations that would lead to RTK activation. MAPK might be a point of convergence for proliferative Indeed, phosphorylation sites for Src on the EGFR are signals emanating from both different G proteins and partially within the kinase domain and may regulate RTKs (Fig. 6 ). kinase activation [146, 148]. Also, PDGFb-receptor was found to become phosphorylated on many sites It is thought that both the a -subunits and bg- including Tyr 857, the site presumably involved in complexes of heterotrimeric G proteins are capable of kinase activation, by Src kinase in vitro [150]. Thus, Src- mediating activation of MAPK. Obviously, in PTX- family kinases and possibly other tyrosine kinases may sensitive pathways MAPK is activated through bg- be capable of direct RTK activation although this complexes from Gi/o proteins whereas in PTX- possibility clearly requires further investigation. insensitive pathways the activation of MAPK is mediated via a q/11-subunits and PKC [4,7,8]. Gs- mediated regulatory effects on MAPK activity may be 4. Signaling Pathways from GPCRs to either stimulatory or inhibitory. The role of G12/13 MAP Kinase proteins in MAPK activation is not yet clear. Very recently, the binding of Ga 12 to Bruton`s tyrosine Like RTKs, many GPCRs can induce mitogenic kinase (Btk) and the stimulation of Btk and of Gap1m responses or contribute to neoplastic growth of human which is a RasGTPase-activating protein (rasGAP) was tumors (reviewed in [242]. Depending on the cell type, reported [33]. mitogenic responses can be mediated by Gi/o, Gq/11, Gs, These findings indicate, for the first time, the or G12 proteins. [4,6,79]. For example, constitutively active mutants of G protein a -subunits have been possibility of a direct link between heterotrimeric and identified in various tumor cells, and GTPase deficient monomeric G proteins. forms of G , G , and G have been demonstrated to s i/o 12 Intensive research led to the identification of various induce cellular growth after expression in several cell protein kinases that could make the link between G lines [243]. proteins and the MAPK cascade. Recently, a novel The pathways coupling GPCRs to nuclear principal pathway of MAPK activation has been responses are of high complexity and may differ from discovered termed transactivation [243, 244].

Fig. (6). MAP kinase activation via RTKs and by various GPCRs may involve Ras and different G protein subtypes. A schematic overview. Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 927

Fig. (7). Principle mechanisms of MAPK activation via Gi/o -coupled receptors. The different and possible biochemical routes are explained in the text.

Transactivation stands for ligand-independent addition, bg-stimulated MAPK activation is inhibited by activation of RTKs triggered by GPCRs [107]. Despite tyrosine kinase inhibitors such as genistein suggesting the efforts made in the last years the early mechanisms the involvement of a tyrosine kinase in this pathway of MAPK activation by agonists of GPCRs remain poorly [247]. Taken together, all experimental data led understood. However, in the following we will attempt assume that Ras or an effector molecule upstream of to describe the most likely biochemical routes Ras should be the target for the Gibg-mediated connecting GPCRs to MAPK. mitogenic signaling. The question remained how the bg-complexes might regulate Ras. First answer came 4.1. Principle Mechanisms of MAPK from the laboratory of Lefkowitz. This group Activation by G -coupled Receptors demonstrated that bg-subunits stimulated tyrosine i/o phosphorylation and thereby activation of the adaptor There are several lines of evidence suggesting that protein p52 Shc thus inducing the formation of Shc- Grb2 complexes [249, 250]. Interstingly, G - MAPK activation by Gi-coupled receptors involves the bg bg-complexes and is Ras-dependent. Thus, activation stimulated phosphorylation of Shc has been also found to be inhibited by Wortmannin, a specific inhibitor of of MAPK via Gi is attenuated by coexpression of the a - PI3K suggesting an additional involvement of PI3K subunits of transducin which acts to sequester Gbg [245]. This was supported by similar results obtained in upstream of Shc [249]. Then Luttrell et al. [251] Rat-1 cells with bARKct, a carboxy-terminal fragment of provided evidence that the non-receptor tyrosine the b-adrenergic receptor kinase that also acts as bg- kinase Src mediates the bg-induced tyrosine sequestrant and significantly inhibited activation of phosphorylation of Shc. Later, several studies described the involvement of more Src-like kinases both Ras and MAPK via the Gi-coupled LPA receptor such as Fyn, Lyn, and Yes [252] or also, recently, of an [246]. Further, overexpression of Gbg subunits results in activation of MAPK [245, 247] whereas a unknown non-Src cytosolic tyrosine kinase that induces the interaction of a p100 kDa protein with Grb2 constitutively active mutant of Gia did not increase MAPK activity in COS-7 cells [248]. It was [253]. demonstrated that MAPK activation by bg-subunits Another candidate that has been implicated in Shc- required neither PLC nor PKC (reviewed in [4]) but b Grb2-Sos complex formation is the FAK-related non- was blocked by dominant negative Ras [245]. In 928 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer receptor tyrosine kinase PYK2. This enzyme is independent or also Ras-dependent pathways [4,7]. predominantly expressed in neuronal cells and may link The Ras-independent pathway shows, e.g. in COS 2+ both Gi and Gq with Grb2-Sos in a Ca - and Src- cells or CHO cells expressing Gq-coupled receptors no dependent pathway as was shown for LPA- and BK- sensitivity to a dominant negative mutant of Ras and receptors in PC-12 cells [254]. may involve activation of PKC and Raf. Gq-mediated signalling in CHO cells has been demonstrated to be On the other hand, PI3Kg has been shown to play a inhibited by down-regulation of PKC by chronic major role in Gbg-mediated activation of MAPK. Under exposure to phorbol esters and by dominant negative endogenous conditions bg-sensitive PI3K activity has Raf [247]. Phorbol esters as direct activators of PKC been described in neutrophils and platelets [255, 256], have been reported to activate MAPK in various cells. and PI3Kg has been shown to activate MAPK when In addition, expression of constitutively active mutants expressend in COS-7 cells in response to Gi-derived of PKC showed that at least in vitro the PKC isoforms bg-complexes [49]. PI3Kg can be activated due to a ,b,d, e, and h have the potential to activate MAPK at direct interaction with bg-complexes [48] or due to a the level of Raf-1. The aPKC z cannot increase Raf-1 constitutive association with the p101 bg-sensitive activity and stimulates MEK by an independent protein [257]. PI3Kg was found to act upstream of Src- mechanism [263]. Recently, much interest has been like kinases suggesting another putative link to the focussed at a possible role of the PKC isoforms a and Shc-Grb2-Sos complex [49]. Furthermore, we [258] especially e as activators of Raf-1 in vivo. For example, a and others [50, 259] have recently shown that the dominant negative mutant of PKC e inhibited both p85/p110 PI3Kb may play a role downstream of Gi- or proliferation of NIH 3T3 cells and Raf activation in COS Gq-coupled receptor signalling, too. The putative cells whereas active PKC e overcame the inhibitory signaling pathways connecting Gi/o-coupled receptors effects of dominant negative Ras in NIH 3T3 cells [264]. to MAPK are summarized in Fig. (7 ). Furthermore, constitutively active mutants of PKCa as well as PKC e overcame the inhibitory effects of Very recently, once more the group of Lefkowitz dominant negative mutants of the other PKC isotype provided evidence which probably opened a new [264]. In addition, PKC e can be co-precipitated with dimension in our understanding of mitogenic signal Raf-1 from Sf-9 insect cells and PKC e transformed NIH transduction (reviewed in [260]). Firstly, stimulation of 3T3 cells [265]. PKC e was demonstrated to be b-adrenergic receptor expressed in HEK 293 cells activated by both phosphatidylinositol (PI)- and resulted in a G -coupled, bg-mediated and Ras- i phosphatidylcholin (PC)-derived DAG [266], and PC- dependent activation of MAPK. However, the hydrolysis was shown to induce Raf-1 activation [267]. interaction of b-AR with G required a prior receptor i Moreover, PKC e may be also activated via the PI3K coupling to G subsequently leading to cAMP s pathway thus representing a point of convergence for production and activation of PKA. PKA-induced several lipid-derived second messengers. Taken receptor phosphorylation was found to be a together, there is increasing evidence that Raf-1 prerequisite for switching the receptor from G to G s i activation by PKC e may play a critical role in mitogenic thereby activating another signaling pathway. [261]. signalling of G -coupled receptors. Furthermore, in HEK 293 cells additionally expressing q/11 dominant negative mutants of b-arrestin or dynamin On the other hand, agonists of Gq/11-coupled which are known to block receptor endocytosis the b- receptors such as bombesin, bradykinin, or AR-mediated activation of MAPK was prevented [262]. vasopressin as well as phorbol esters have been also The inhibitors of receptor internalization were found to described to stimulate Src family kinases transiently specifically block the interaction between Ras-bound thereby activating MAPK in a Ras-dependent manner Raf and the cytosolic MEK. Thus, GRKs and arrestin [268]. According to a hypothesis proposed by Della mediating the uncoupling and internalization of GPCRs Rocca et al. [269] activation of Src kinases by Gq/11- may play also an essential role in the GPCR-induced coupled receptors might be the final event of a cascade MAPK activation [260, 262]. including the increase in cytosolic Ca2+ in response to 2+ IP3 and the Ca -calmodulin mediated activation of Pyk2 or of a related tyrosine kinase which 4.2. Principle Mechanisms by which Gq/11- phosphorylates Src. Indeed, stimulation of Gq-coupled coupled Receptors May Activate MAPK BK receptors in PC-12 cells was demonstrated to Receptors coupled to PTX-insensitive G proteins of activate PyK2 and subsequently Src and MAPK [254]. the G family are thougth to mediate MAPK activation Src may phosphorylate either a RTK or Shc leading to q/11 the formation of a Shc-Grb2-Sos complex. The most via their a -subunits. Their signaling is mostly insensitive likely biochemical routes connecting Gq/11-coupled to bg-sequestrants (reviewed in [4,7]). Gq/11-coupled receptors may activate MAPK by either Ras- receptors to The MAPK pathway are summarized in Fig. (8 ). Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 929

Fig. (8). Principle mechanisms of MAPK activation in response to of Gq/11-coupled receptor stimulation. The putative links of Gq/11 to MAPK are explained in the text.

4.3. Gs-coupled Receptors and MAPK: released from Gi after switching of b-AR from Gs to Gi Differential and Controversial Effects of [261] should mediate the activation of MAPK. cAMP and bg-Subunits Very recently, a new family of cAMP-binding The role of Gs in the regulation of cell growth and proteins was discovered that exhibit properties of a MAPK activity appears to be extremely cell-type guanine necleotide exchange factor (GEF). These specific. In various cells such as smooth muscle cells cAMP-GEFs are capable of activating monomeric G [270], adipocytes [271], or fibroblasts [272], cAMP has proteins in a cAMP-dependent and PKA-independent been shown to inhibit the MAPK cascade. In these manner suggesting a direct coupling of cAMP- cells, cAMP activates PKA which in turn mediated signaling to Ras superfamily signaling [276]. phosphorylates Raf-1 thereby decreasing the affinity of Raf for Ras as well as Raf kinase catalytic activity [273]. In PC-12 cells, in contrast, cAMP stimulates the MAPK 4.4. A New Role for RTKs in GPCR Signaling: pathway and induces neuronal differentiation [274]. In Different Pathways Lead to Transactivation COS-7 cells, Faure et al. [248] reported that the RTKs are not only receptors for specific peptidic expression of a constitutively active mutant of G , s growth factors but also essentially involved in the treatment with forskolin or dibutyryl cAMP, or mitogenic signalling of GPCRs where they may act as stimulation of transiently expressed, G -coupled LH s scaffold proteins, signal mediaters, and signal receptor resulted in activation of MAPK. Contradictory integrators. First evidence for ligand-independent results have been presented by Crespo et al. [275] tyrosine phosphorylation of RTKs by a GPCR came in suggesting dual effects of b-adrenergic receptors on 1995 from reports demonstrating the tyrosine MAPK activity in COS-7 cells. In this study, stimulation phosphorylation of PDGFR by angiotensin II [277] in rat of b-AR simultaneously led to bg-dependent activation smooth muscle cells or of EGFR by bradykinin in human and cAMP-mediated inhibition of MAPK. The balance keratinocytes [278]. Then, in two excellent papers, between these two mechanisms was postulated do Daub et al. [243, 244] described the transactivation of determine the outcome of the signal to MAPK [275]. EGFR in diverse cell types such as Rat-1 cells, HaCat However, in view of the present knowledge it may be keratinocytes, mouse astrocytes as well as in COS-7 assumed that not bg-complexes from G but those s cells (Fig. 9A). In Rat-1 cells it was shown that mitogens 930 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer such as endothelin, LPA and thrombin mediate both was potently inhibited by a PKC inhibitor and the MAPK activation and DNA synthesis via activation of phorbol ester PMA could mimick the carbachol effect EGFR [243]. In COS-7 cells transiently expressing Gi- suggesting the PKC-dependency of EGFR tyrosine or Gq-coupled receptors was demonstrated that both phoshorylation [284] (Fig. 9B). A completely different types of GPCRs stimulated Shc phosphorylation as well mechanism of EGFR transactivation was found in GN4 as MAPK activity via EGFR transactivation [244]. Since rat liver epithelial cells where angiotensin II (A II) is inhibition of PI3K did not affect EGFR tyrosine capable of activating MAPK via two pathways. Under phosphorylation but abolished MAPK activation PI3K normal conditions, A II stimulates MAPK via a PKC- was supposed to act downstream of EGFR. Similar dependent, Ras-independent pathway. In PKC- results were obtained with the Src-inhibitor PP-1 depleted cells, the A II receptor can switch and leading to the assumption that also Src should be activates MAPK via an equipotent Ras-dependent involved in the signaling pathway downstream of pathway including EGFR tyrosine phosphorylation. EGFR. Independently, Luttrell et al. from the Thus, the latent transactivation route represents an Lefkowitz`group provided evidence that at least Gi- alternative pathway to MAPK that is masked by PKC coupled receptors may induce EGFR tyrosine and uncovered when the PKC pathway breaks down phosphorylation via Src-family tyrosine kinases [279]. [285] (Fig. 9C). They proposed, in contrast, that a PI3K-dependent step might lie upstream of Src kinase activation. Src Moreover, in COS-7 cells transiently co-transfected kinase, indeed, possesses a domain with high affinity with the human bradykinin B2 receptor and MAPK we found a pathway of MAPK activation that includes the to PIP3 the second messenger product of PI3K [280]. In contrast again, recently published data suggest that independent and equipotent stimulation of both PKC in HeLa cells the intrinsic EGFR tyrosine kinase and EGFR [329]. Activation of MAPK in response to BK contributes to the LPA-stimulated MAPK pathway and is prevented by inhibitors of PKC as well as EGFR. c-Src is probably not involved [281]. However, this Inhibitors of PI3K or Src failed to affect MAPK activation finding in HeLa cells does not exclude that members of by BK. PKC translocation studies and coexpression of the Src family may be involved in other cell types. inactive and constitutively active mutants of different Furthermore, the same GPCR may induce PKC isoforms provided evidence for a critical role of the transactivation of different RTKs depending on the cell PKC isozymes a and e in BK signalling towards MAPK. type. This has been recently demonstrated for LPA BK induced tyrosine phosphorylation of the EGFR that which transactivates EGFR in COS-7 cells but is also was independent of PKC. Since blockade of the EGFR capable of using the PDGFR in L cells that lack EGFR did also not influence BK-stimulated increase in [282]. phosphatidylinositol phosphate formation, PKC should act neither upstream nor downstream of EGFR but in a In neuronal cells EGFR transactivation appears to be permanent dual signalling pathway. MAPK activation by dependent on Ca2+. In PC-12 cells, for example, BK requires signals from both pathways which bradykinin-induced tyrosine phosphorylation of EGFR represent a two-key system for regulating an enzyme upstream of Shc and MAPK was reported [283]. This which is critically involved in the control of cell growth effect of BK was absent in PC-12 cells pretreated with (Fig. 9D). EGTA. In other cells, however, EGFR transactivation was found to be independent of Ca2+ [284]. In addition As described above, a common theme in the to the differences in Ca2+-sensitivity or the role of Src, literature on RTK transactivation by GPCR is the the high degree of cell specificity within the possible involvement of Src-family kinases. They may mechanisms involved in EGFR transactivation by induce signal transduction by either phosphorylating GPCRs is also reflected by different modes of action of RTKs on docking sites for signaling molecules or via PKC (Fig. 9 ). Although activation of PKC has been direct activation of intrinsic RTK activity. As outlined in widely shown to play a key role in MAPK activation by chapter 3, it is not entirely clear, how Src-family kinases can possibly accomplish RTK activation. One would Gq-coupled receptors, in COS-7 cells the Gq-mediated transactivation of EGFR was postulated without any have to assume that they execute phosphorylations of attempt to modify PKC activity in these cells [244]. This the RTK molecules which are functionally equivalent to is surprising all the more because in 1995 Coutant et al. trans-phosphorylations in RTK dimers. Such a model [278] already showed that in HaCaT human would be in agreement with some of the available data keratinocytes bradykinin induces tyrosine on Src-phosphorylation sites in different RTKs but this phosphorylation of EGFR via a PKC-dependent mechanism has not been fully established. Further, pathway. Similar results were reported for human 293 while tyrosine phosphorylation in the RTK "activation cells stably transfected with m1 muscarinic receptors. In loop" has been established as activation mechanism for these cells carbachol-induced EGFR transactivation some RTKs as FGF receptor, insulin receptor or Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 931

Fig. (9). Different modes of EGFR transactivation. (A) Principle mechanisms of RTK (EGFR, PDGFR) transactivation without consideration of PKC. Several authors implicate PI3Kg either upstream or downstream of RTK [244, 279]. (B) In human 293 cells PKC was found to be involved in M1AchR-mediated EGFR transactivation. It is not yet clear whether PKC induces activation of a cytosolic phosphotyrosine kinase (PTK) or inactivation of a phosphotyrosine phosphatase (PTP) subsequently leading to enhanced tyrosine phosphorylation of EGFR. In that model, activated EGFR was demonstrated to open a K+ channel in the membrane [284]. (C) Latent dual pathway of EGFR transactivation by angiotensin II. In GN4 cells AII activates MAPK dominantly via the PKC/Raf pathway . When the PKC pathway is cancelled MAPK is alternatively activated via EGFR transactivation [285]. (D) Activation of MAPK by BK via a permanent dual pathway. Both activation of PKC and EGFR transactivation are necessary for the stimulation of MAPK activity by BK.

Met/HGF receptor, respective evidence is still missing In contrast, it should be considered that a main for other RTKs. In fact, it is likely that for other RTKs, in pathway for RTK activation may be instead the particular EGFR regulation by activation loop interference with negative regulatory pathway phosphorylations is less important. Thus, the possible mechanisms (see chapter 3.4) in particular with the role of heterologous Src-kinase phosphorylations is activity of RTK-silencing PTPs. Their activity could well even more questionable. be affected by GPCR signaling events, as changed 932 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer

Ca2+-levels, lipid second messengers or various additional proteins of interest in COS-7 cells or other protein kinases including Src-family kinases. This kind transfectable cell lines. However, the procedure of of mechanism is not easy to demonstrate. EGF transfection might and the overexpression of certain receptor dephosphorylation is a very rapid process proteins will result in artificial and abnormal conditions [286-288] with t1/2 <<2min. It can be detected in intact within the cell compared with the native cell. Therefore, cells as a decay of phosphotyrosine on the EGFR after it should be considered that in general in quenching the kinase with specific cell-permeable overexpression models only signaling mechanisms can blockers, subsequent to ligand stimulation [288]. A be detected which represent putative pathways which moderate attenuation of the dephoshporylation rate, in may not necessarily reflect the real situation under particular if it would affect only the not ligand-stimulated endogenous conditions. In natural cells or tumor cell receptor, may be impossible to monitor with this lines expressing individual amounts of signal technique. Use of general PTP inhibitors, on the other transducing molecules and different isoforms of hand, will lead to rapid hyperphosphorylation of many effector molecules, for instance, completely different cellular proteins which will likewise make conclusions as links between GPCRs and the MAPK cascade may be to the possible involvement of PTP in transactivation observed compared with the signalling pathways after difficult. We believe, however, that the involvement of stimulation of the same GPCR in an expression system. PTPs in GPCR-mediated RTK activation requires much In order to demonstrate such conflicting findings in an attention and possibly the development of novel tools expression model and in tumor cell lines two examples to monitor RTK-directed PTP activity. from our own work may be mentioned. Thus, in COS-7 cells transiently transfected with the human B2R bradykinin activated MAPK by a dual pathway and 4.5. Expression Models versus Endogenous requires the independent signalling via both PKC and Conditions: Varying Mechanisms of MAPK EGFR transactivation as described above. In contrast, Activation by GPCRs in Tumor Cell Lines when we studied MAPK activation in response to BK in the human colon carcinoma cell line SW-480 we The majority of models describing mitogenic detected a hitherto unknown biochemical route to pathways from a GPCR to MAPK are based upon MAPK [258]. Both BK-induced stimulation of DNA experimental data obtained by coexpression of synthesis and activation of MAPK were abolished by epitope-tagged MAPK together with GPCRs and often two different inhibitors of PI3K, wortmannin and LY

Fig. (10). Bradykinin receptor signaling in the human colon carcinoma cell line SW-480. Here BK-induced activation of MAPK was found to be mediated via a pathway involving the consecutive stimulation of Gq/11 protein, PI3Kb, and PKCe [258]. Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 933 294002, as well as by two different inhibitors of PKC, resulting in EGFR desensitization [289]. It may be bisindolylmaleimide and Ro 31-8220. Furthermore, concluded that BK affects the EGFR via both a stimulation of SW-480 cells by BK led to both increased decrease of tyrosine phosphorylation by enhanced formation of PIP3 and translocation of PKC e that was PTPase activity and a decrease of EGFR sensitivity inhibited by wortmannin, too. Using subtype-specific towards EGF by PKC. Although the EGFR is antibodies, only the p110 and p85 subunits of PI3Kb transinactivated by BK in A 431 cells, a BK-induced and but not p110 a or p110 g were detectable in SW-480 PKC-mediated activation of MAPK was observed (Fig. cells. Finally, p110 b co-immunoprecipitated with PKC e 11). These results provide evidence that not in all indicating a physical association between the two cases EGFR transactivation is necessary for GPCR- proteins. These results suggest a novel pathway induced MAPK activation. involving the consecutive activation of Gq/11, PI3Kb, PKCe, and MAPK (Fig. 10). Additionally, a single agonist may also induce activation of MAPK by different signalling pathways Another cross-talk mechanism between B2R and which can vary between different cell types stimulated. the MAPK pathway we observed in A 431 cells [Graneß For example, in GN4 cells the angiotensin II (AII) A.; Hanke S.; Boehmer F.-D.; Liebmann C.; in press]. In receptor was reported to stimulate MAPK via a this cell line BK induced a decrease in both basal and dominant PKC pathway and a latent EGFR EGF-stimulated tyrosine phosphorylation of EGFR by transactivation pathway as already discussed [285]. In stimulating the activity of a phosphotyrosine rat cardiac myocytes, AII was found to activate MAPK via phosphatase. This effect can be demonstrated by PLCb, PKC, and Raf-1 independently of EGFR [290]. several experimental approaches including the In contrast, in rat vascular smooth muscle cells a respective Western blots of EGFR as well as direct pathway from AII receptor to MAPK was detected 32 measurement of PTPase activity using the [ P] involving the sequential activation of Gq/11, PI3K, PKC tyrosine-phosphorylated PTPase substrate raytide. z, and an association of PKCz with Ras suggesting a Moreover, BK was previously shown to also induce a PKC/Ras dependent pathway that can by-pass the PKC-mediated phosphorylation of EGFR at Thr-654 EGFR [291]. Further, in rabbit renal proximal tubular

Fig. (11). Bradykinin B2 receptor signaling in the human epidermoid carcinoma cell line A431. BK attenuates EGFR by both stimulation of PTP and of PKC. Activation of PTP results in a decreased tyrosine phosphorylation of EGFR and activated PKC induces threonine phosphorylation of EGFR that leads to its desensitization towards EGF. Nevertheless, BK is capable of stimulating MAPK activity via another PKC-dependent pathway. This is an example for a simultaneously occuring negative modulation of EGFR (transinactivation) and positive regulation of MAPK activity by a GPCR in a cell-specific manner. 934 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer epithelial cells, AII activates MAPK via the AT2 receptor Whereas the AT1 receptor preferentially couples to subtype and a signaling route involving stimulation of Gi Gq/11 proteins, in colonic epithelial cells also the Gi and, subsequently, PLA2, the release of arachidonic protein-coupled gastrin receptor was found to stimulate acid which in turn activates Shc and Ras [292]. In N1E- PLCg1 via Src [295]. It may be assumed, therefore, that 115 neuroblastoma cells AT2 receptors mediate both Gq/11- and Gi-coupled receptors have the ability to inhibition of serum- or EGF-induced MAPK activation, act without G proteins by forming signal transduction possibly via activation of a PTP (293). complexes which are thought to be typical for RTKs. On the other hand, with respect to ligand-induced These few examples may illustrate by which tyrosine phosphorylation of the B2 bradykinin receptor immense complexity and cell specificity the cross talk contradictory results have been reported. In human between GPCRs ant RTKs can occur. fibroblasts the B2R was shown to be phosphorylated at serine and threonine residues in response to BK and neither phosphoamino acid analysis nor Western 5. RTK-induced Tyrosine Phosphory- blotting with anti-phosphotyrosine antibodies revealed lation of GPCR Signaling Elements tyrosine phoyphorylation of the B2R [298]. In contrast, in endothelial cells the BK-induced IP formation was Although relatively much is known about the 3 reported to be dependent on a transient tyrosine interaction of GPCRs with RTKs and the MAPK phosphorylation of PLC 1 [299, 300]. In vascular cascade, the modulation of GPCR-coupled signal g endothelial cells, tyrosine phoyphorylation of PLC 1 transduction by RTK-induced tyrosine phosphorylation g could be correlated with its binding to the C-terminal is considerably less investigated. Meanwhile, however, intracellular domain of the B2R similar to the findings at there is mounting evidence that cross-talk includes not the AT receptor [300]. Unfortunately, there is no clear only the foreward regulation of the MAPK pathway by 1 evidence for a ligand-induced tyrosine phosphorylation GPCRs but also the regulation of GPCR signaling by of the B2R in this work. RTKs. Recently, at least three levels of GPCR signal transduction were implicated to be favoured targets of It is worth to speculate, however, that tyrosine tyrosine phosphorylation by RTKs. phosphorylation of GPCRs might occur not only ligand- induced (homologous) but also as a cross-talk event induced by activated RTKs (heterologous). In that way, 5.1. Tyrosine Phosphorylation of GPCRs a RTK could use tyrosine-phosphorylated GPCRs as G protein-coupled receptors lack intrinsic tyrosine scaffold proteins for ist own signal transduction kinase activity. Nevertheless, several agonists of GPCR machinery. such as angiotensin II [294] or gastrin [295] have been demonstrated to stimulate phosphatidylinositol 5.2. Tyrosine phosphorylation of G proteins metabolism by a signalling mechanism that involves PLC g and Src instead of PLCb. In vascular smooth Like other signalling proteins, also G proteins may muscle cells, for example, the AT1 receptor was be phosphorylated and thereby modulated by different demonstrated to induce a transient tyrosine protein kinases. Thus, phosphorylation of Gia by PKA phosphorylation of PLC 1 and a parallel IP formation g 3 appears to impair the dissociation of Gi into a - and bg- in a manner similar to that observed in response to subunits [301]. In contrast, the a -subunits of Gz as well growth factors [294]. In addition, electroporation of anti- as G12/13 are phosphorylated by various isoforms of Src antibodies into these cells eliminated both tyrosine PKC resulting in a prevention of their association with phosphorylation of PLCg and AII-induced stimulation of bg-subunits [302-304]. In addition, recombinant forms IP production suggesting that activation of PLC 3 g of Gsa were shown to be targets for PKC in vitro occurs downstream from activation of c-Src tyrosine suggesting a putative role of Gsa within the cross-talk kinase [296]. Very recently, the activation of PLCg by between receptors which activate PKCs and those AII was found to be accompanied by binding of PLC to g which stimulate adenylate cyclase via Gs [305]. the AT1 receptor in dependency on AII stimulation as well as tyrosine phosphorylation. A prerequisite of First evidence concerning tyrosine phosphorylation PLCg1 binding appears to be phosphorylation of of G proteins was provided in 1992 by Hausdorff et al. tyrosine 319 in a YIPP motif in the C-terminal [306]. They demonstrated in vitro the tyrosine intracellular domain of the AT1 receptor. This phosphorylation of Gsa as well as other Ga subtypes phosphorylated tyrosine residue can be recognized by (Gia , Goa ) by activated Src. As a functional a SH2-domain of PLCg1 [297]. It might be speculated consequence, phosphorylation of Gsa was found to that the receptor serves as a scaffold for PLCg1 increase receptor-induced GDP/GTP exchange and allowing its phosphorylation by Scr tyrosine kinase. GTPase activity. Three years later, Moyers et al. [307] identified the in vitro phosphorylation sites of Gsa Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 935 mediated by Src. In an excellent work two sites of the susceptibility of Gsa to GPCRs (Fig. 12). These phosphorylation were mapped, Tyr-37 located near the findings emphasized a differential recruitment of Gsa N-terminus and Tyr-377 located at the site of receptor by GPCRs and EGFR as a novel cross-talk mechanism. binding in the C-terminus. Tyrosine phosphorylation of Moreover, also G appears to be tyrosine Gsa , at these sites, therefore, could change its ability to q/11 interact with both bg-subunits and the receptor [307]. phosphorylated. Recently, stimulation of GPCRs Almost at the same time the tyrosine phosphorylation coupled to Gq/11 was shown to induce phosphorylation on Tyr356 and a PTK is required before G protein of recombinant Gsa by isolated EGF receptor tyrosine kinase was reported [308]. Tyrosine-phosphorylated activation. It was further demonstrated that this tyrosine phosphorylation is apparently essential for the Gsa showed enhanced GTP binding and GTPase activity and an increased degree of adenylate cyclase activation of Gq/11 by agonist stimulation [310]. These stimulation after reconstitution with S49 cyc- results demonstrate that tyrosine phosphorylation of membranes [308]. These findings suggested that also The Ga q/11 subunit by PTKs contributes to GPCR- a RTK has the ability to phosphorylate and thereby to mediated activation of Gq/11. activate G . In 1996, too, using several experimental sa Taken together, it may be assumed that depending approaches we provided the first evidence for tyrosine on the type of G protein and the type of cell both phosphorylation of G in A431 cells in vivo [309]. sa GPCR-induced ( probably mediated by Src, Pyk2 or Treatment of A431 cells with EGF abolished both others) and/or RTK-induced tyrosine phosphorylation bradykinin- and isoprenaline-induced binding of the of G -subunits might represent a mechanism to stabile GTP analogon [35S]GTPgS to G and a sa regulate the activation of G proteins. decreased the BK- and guanyl nucleotide-induced cAMP accumulation and adenylate cyclase stimulation. In contrast, the BK-induced and Gq/11-mediated 5.3. Tyrosine phosphorylation of PKCs formation of inositol phosphates was not affected by EGF. Thus, tyrosine phosphorylation of Gsa by EGF Tyrosine phosphorylation of PKC isoforms such as results in an activation of adenylate cyclase by EGF, on d, e, h, and z in response to Src family tyrosine kinases the one hand, and a simultaneously occuring loss of in vitro has been reported [311] but the biological

Fig. (12). EGFR-induced tyrosine phosphorylation of Gsa in A431 cells is accompanied by a loss of the susceptibility of a s to activation via GPCRs and its ability to stimulate adenylate cyclase in response to activated GPCRs [309]. 936 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer significance remains unclear. In transiently transfected on GPCR directed drug development. Cross-talk COS-7 cells overexpressing various PKC isoforms the beween different GPCRs and the discovery of multiple PKCs a , b1,g, d, e, and z were found to be tyrosine pathways initiated by a single class of GPCR predict that phosphorylated and catalytically activated in response GPCR agonists/antagonists will have side effects to H2O2 that is known to induce oxidative stress [312]. depending on the given coupling of the GPCR to However, the tyrosine kinases that phosphorylate PKC signaling pathways, even if they target a given receptor isoforms under these conditions are not yet known. subtype with absolute specificity. An example comes Among the PKC isoforms which are tyrosine from the bradykinin story. B2R antagonists have a phosphorylated in vitro some interest has been therapeutic potential as novel analgetic and anti- focussed to PKCd. This isoform has been shown to be inflammatory agents. Structural modifications of BK led phosphorylated on tyrosine in Ras-transformed cells to the discovery of the peptidic B2R antagonist Hoe [313] and in response to various stimuli such as 140 which is highly potent and specific in all cells and phorbol esters, carbachol, or substance P [314]. In tissues tested so far. When we screened various tumor addition, PKCd is tyrosine phosphorylated after cell lines for mitogenic effects of BK we used Hoe 140 stimulation of the EGFR signalling pathway involving as control. Very surprisingly, we found that in certain the activation of a member of Src kinase family and tumor cells the antagonist Hoe 140 induced stronger leading to inhibition of PKCd activity [311]. In v-Src- mitogenic effects than BK (C. Liebmann, unpublished transformed fibroblasts the formation of a Src-PKCd results). On the other hand, understanding of GPCR complex in which PKCd becomes tyrosine post-receptor signaling mechanisms opens up the phosphorylated and down-regulated was proposed as possibility to interfere with downstream signaling steps. a possible molecular mechanism [315]. As PKCd This concept has been put forward as "signal phosphorylation sites Tyr52 and Tyr187 in the N-terminal transduction therapy" initially mainly focussed on RTK part have been identified [316, 317]. However, the signaling [318], where development of efficient ligand functional consequences of PKCd tyrosine antagonists hitherto failed. Inhibitors of enzymes phosphorylation remain controversial and involve both mediating important steps of GPCR signaling would activation and inhibition of catalytic activity. block receptor effects. Alternatively, inhibition of negatively regulating steps could be used to augment Nevertheless, these findings implicate the signaling of a given GPCR. Interstingly, the recent possibility that PKC may be regulated by different findings on GPCR cross-talk with RTKs has brought signalling pathway in response to stimulation of GPCRs into play specific tyrosine kinase inhibitors as a novel as well as RTKs. One is the classical DAG-dependent class of effectors for GPCR signaling [319-321]. These pathway through activation of PLCb/g or PLD. Another drugs have actually been instrumental for clarifying the signalling route may occur DAG-independent but involvement of RTKs in aspects of GPCR signaling, involving activation of PI3K. A third pathway might be notably mitogenic signaling of GPCR. Such the tyrosine phosphorylation of PKCs in response to compounds are for example specific inhibitors for the RTKs stimulated directly by growth factors or EGFR of the anilino quinazoline family as AG1478 or transactivated via GPCRs. PD153035 (Table 3), specific inhibitors of the PDGF receptors as the quinoxaline AG1296 and the Nevertheless, these findings implicate the phenylaminopyrimidine CGP53716 [322] or the Src- possibility that PKC may be regulated by different family inhibitor PP1 [323]. The latter has, however, signaling pathways in response to stimulation of recently been found to block the PDGFb-receptor as GPCRs as well as RTKs. One is the classical DAG- well [287]. Another class of kinase inhibitors with dependent pathway through activation of PLC or b/g increasing relevance for GPCR signaling are blockers of PLD. Another signaling route may occur DAG- enzymes in the MAPK signaling cascades, including independent but involving activation of PI3K. A thrid the inhibitor of MEK1/2 PD98059 [288] or the specific pathway might be the tyrosine phosphorylation of p38-blocker SB203580 [324, 325]. One can anticipate PKCs in response to RTKs either stimulated directly by that the current rapid development in the field of kinase growth factors or transactivated via GPCRs. inhibitors will certainly be of great value for the pharmacological modulation of GPCR signaling.

6. Implications for Drug Development Several kinase blockers have recently been put forward to clinical trials, maily aiming at tumor therapy by Activity of GPCRs can be selectively modulated by targeting RTK pathways [326]. Since, however, as receptor-subtype specific agonists or antagonists. outlined above, pathways from GPCRs and tumor- Numerous drugs have been developed on the basis of relevant RTKs converge, overlap and cross-talk, one this principle. Progress in understanding of GPCR could anticipate that GPCR-related side effects may signal transduction pathways creates new perspectives become observed. Signal Transduction Pathways of G Protein-coupled Receptors Current Medicinal Chemistry, 2000, Vol. 7, No. 9 937

Table 3. Protein Kinase Inhibitors: Agents Which Block also GPCR-Mediated MAPK Activation and Mitogenesis Selected references: [143, 286-288, 319, 322, 323]

Compound Inhibited enzyme Concentration for near complete inhibition in Remarks intact cells by maintaining selectivity

AG1478 (AG1517) EGFR 10-300 nM may inhibit other HER-family kinases PD153053 as well

AG1295/6 PDGFa R, PDGFbR 5-10 µM

PP1/AGL1872 Src-family kinases, PDGFbR 1-3 µM

PD98059 MEK 10 µM also antagonist of aryl hydrocarbon receptor, inhibits cyclooxygenases

As we have tried to illustrate, GPCR signaling is a signaling mechanisms have been performed in pleiotropic response which, as a rule, involves multiple permanent cell lines or overexpression systems and signaling chains. To what extent these pathways are some if not many proposed mechanisms may reveal to activated strongly depends on the cell type concerned. be not physiologically relevant. In conclusion, Thus, interference with GPCR signaling is likely to predictions on selective in vivo effects of certain require a "cocktail" of signaling effectors which is effectors are currently hardly possible. Generation of tailored for a given target tissue. transgenic mice with inactivated genes for certain signal-mediating molecules has revealed, however, Frequently, pharmacological modulation of GPCR that the effects of even complete abolishment of signaling aims at stimulation or augmentation of certain signaling steps in all tissues may be much more receptor function. Compounds affecting enzymes selective than previously anticipated. An example is the which regulate signal transduction negatively would be recent inactivation of the gene for p85a , an adaptor excellent tools in this respects. However, effector molecule required for activation of the a and b- isoforms discovery for such enzymes is only in its beginnings. of PI3 kinase [328]. This knockout led not to lethality, This concerns inhibitors of PTPs, of MAPK inactivating as might have been anticipated but to a specific dual-specificity phosphatases, of inositolpolyphos- impairment of B-cell immunity. One has further to phate phosphatases, of phosphatidylinositolphos- consider, that application of a signal transduction phate phosphatases and others. An exception present enzyme inhibitor in vivo is unlikely to lead to complete inhibitors of phosphodiesterases, where a number of ablation of enzyme activity, rather more attenuation in compounds are in clinical trials and one drug has some tissues and less attenuation in others, recently been introduced to the market [327]. This depending on pharmacokinetic parameters and example illustrates the immense possibilities for drug expression levels. This leaves further possibilities for development aiming at interference with negatively specific effects. regulating signaling events. In conclusion, development of further signal One could assume that specificity for signal transduction effectors for GPCRs seems highly transduction therapy will be the more difficult to attain warranted although the only partially understood the further distal to the GPCR the targeted signaling complexity of signaling makes predictions of in vivo step is positioned. As outlined above, it becomes, effects and disease indications difficult. It can, however, increasingly clear that the pathways used by a however, be anticipated that many reasonably target given GPCR, the extent of cross-talk and feedback selective compounds will eventually find applications. reactions, are to a very high degree cell-specific. Thus, interference with a given enzyme is likely to have very different effects in different cell types and beneficial Note Added in Proof effects may be obtained in a particular tissue whithout negatively affecting others. It is difficult to predict to A novel mechanism for EGFR transactivation by what extent inhibition of a certain signaling step will GPCRs has been shown recently: GPCR-dependent abolish signaling or rather shift signaling to another activation of a metalloproteinase leads to processing of pathway. Also, partial inhibition of a pathway which pro-heparin-binding (HB)-EGF and EGFR activation by contains negative feeback elements may affect released HB-EGF. This process is suppressed by the feeback inhibition to a greater extent than positive metalloproteinase inhibitor batimastat. signal generation and thus, lead to even augmented [Prenzel, N.; Zwick, E.; Daub, H.; Laserer, M.; Abraham, signaling as net result. Further, most studies on R.; Wallasch, C., Ullrich, A. Nature, 1999, 402, 884]. 938 Current Medicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Böhmer References [32] Fields, T.A.; Casey, P.J. Biochem. J., 1997, 321, 561. [33] Jiang, Y.; Ma, W.; Wan, Y.; Kozasa, T., Hattori, S.; Huang, X.-Y. [1] Offermanns, S.; Schultz, G. Naunyn-Schmiedeberg`s Arch. Nature, 1998, 395, 808. Pharmacol., 1994, 350, 329. [34] Treisman, R. Curr. Opin. Cell Biol., 1996, 8, 205. [2] Selbie, L.A.; Hill, S.J. TiPS, 1998, 19, 87. [35] Casey, P.J. Curr. Opin. Cell Biol., 1994, 6, 219. [3] Rozengurt, E. Science, 1986, 234, 161. [36] Mumby, S.M. Curr. Opin. Cell Biol., 1997, 9, 148. [4] Van Biesen, T.; Luttrell, L.M.; Hawes, B.E.; Lefkowitz, R.J. Endocrin. Rev., 1996, 17, 698. [37] Sprang, S.R. Annu. Rev. Biochem., 1997, 66, 639.

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