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    CurrentMedicinal Chemistry, 2000, 7, 911-943 911

    Claus Liebmann*aand Frank-D. Bhmerb

    Institute of Biochemistry and Biophysics, Biological and Pharmaceutical Faculty (a) andResearch 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 ofdrug targets. Recent investigation of GPCR signaling has revealedinteresting novel features of their signal transduction pathways which may beof great relevance to drug application and the development of novel drugs. Firstly, a single classof GPCRs such as the bradykinin type 2 receptor (B2R) may couple to different classes of Gproteins in a cell-specific and time-dependent manner, resulting in simultaneous or consecutiveinitiation of different signaling chains. Secondly, the different signaling pathways emanatingfrom 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 mitogenicsignals. GPCR-induced mitogenic signaling involves activation of the p44/p42 "mitogenactivated protein kinases" (MAPK) and frequently "transactivation" of receptor tyrosine kinases(RTKs), an unrelated class of receptors for mitogenic polypeptides, via currently only partlyunderstood 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 moleculessuch as members of the protein kinase C family. In conclusion, known agonists of GPCRs arelikely to have unexpected effects on RTK pathways and activators of signal-mediating enzymespreviously thought to be exclusively linked to RTK activity such as tyrosine kinases or PTPs maybe of much interest for modulating GPCR-mediated biological responses.

    *Address correspondence to this author at the Institute of Biochemistryand Biophysics, Friedrich-Schiller-University Jena, Philosophenweg 12,D-07743 Jena, Germany; Tel.: +49-3641-949357; Fax : +49-3641-949352; E-mail: [email protected]

    0929-8673/00 $19.00+.00 2000 Bentham Science Publishers B.V.

    Signal Transduction Pathways of G Protein-coupled Receptorsand Their Cross-Talk with Receptor Tyrosine Kinases: Lessonsfrom Bradykinin Signaling

    G Protein-coupled receptors (GPCRs) constitute asuperfamily of transmembrane proteins that transduceextracellular signals to the intracellular level. More than1000 of GPCRs are known up to now - tendencyincreasing. Their agonists differ in size and structureranging from large glycoproteins to simple moleculessuch as amines or nucleosides. Agonist binding to aGPCR leads to activation of a heterotrimeric G protein,which in turn is linked to either activating or inhibitingsecond messenger pathways. The resulting change in

    second messenger concentration then leads to furtherdownstream effector events, frequently activation ofprotein kinases.

    In many cases a single receptor can activatedifferent G proteins and thereby induce dual or multiple

    1. Introduction signaling routes [1]. In other cases, multiple receptorscan converge on a single G protein which has thecapability of integrating different signals [1]. Stimulationof a particular GPCR may result not only in activation of asingle signaling pathway but also to subsequeninteractions with those activated by the other GPCRsThese interactions between different receptor-coupledsignal transduction pathways are termed cross-talk. Inthat way, synergistic interactions may be producedwhich result in an amplification of a coincident signawithin the same cell playing a role in "fine-tuning" omultiple receptor-signaling pathways [2]. On the othe

    hand, the signal transduction of one receptor may bealso negatively regulated by that of another receptor,by feedback effects or by initiation of a parallel inhibitorypathway.

    Meanwhile it has become clear that GPCRs are noonly involved in the regulation of metabolic or excitatorycellular responses but are also implicated in celproliferation [3] (Fig. 1 ). Many ligands of GPCRs whichare known as classical hormones or neurotransmitters

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    912 CurrentMedicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Bhmer

    such as vasopressin, angiotensin II, endothelin,substance P, bradykinin, acetylcholin or serotonin,were found to elicit a mitogenic response in variouscells [3,4]. Thus, for example, the nonapeptidebradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg)which regulates blood pressure and smoooth muscletone has been found to stimulate growth in small celllung cancer (SCLC) cells [5]. There is increasingevidence indicating that GPCRs can function asagonist-dependent oncoproteins [6].

    and subsequently activated [8]. GPCRs that mediategrowth stimulatory effects activate key effectormolecules of the MAPK pathway, including the RTKRas, Raf, or MAPK. Although MAPK activation may nobe required for all GPCR-mediated growth responsesMAPK could be a convergence point of growth-promoting signals arriving from both RTKs and differenGPCRs. Bradykinin, for example again, was alsoreported to stimulate MAPK activation, e.g. infibroblasts [9], SCLC cells [10], or ventricular myocytes[11].

    What are the mechanisms by which a GPCR canmodulate cellular growth? One answer is cross-talk:

    Beside the interactions between different GPCRsignaling pathways, stimulation of GPCRs may namelyalso result in activation of a pathway which receptortyrosine kinases (RTK) employ for stimulation of cellgrowth, the so-called "MAP kinase (MAPK) pathway"[2,4,6,7]. Growth factor RTKs frequently stimulateMAPK via recruitment of a complex of Shc, Grb2 andSos proteins to the cell membrane, leading tosubsequent activation of the small GTP-binding proteinRas, the two protein kinases Raf and MEK and finally ofMAPK [8]. Once activated MAPK translocates to thenucleus where transcription factors are phosphorylated

    In this review, we discuss principle signaling routes

    linking GPCRs to the MAPK pathway and, vice versa,leading to modulation of GPCR signalling pathways byRTK-induced tyrosine phosphorylation. As an examplewe will discuss the recent progress in the evaluation ofcross-talk mechanisms between the bradykinin B2receptor (B2R) and the EGF receptor (EGFR).

    The majority of the modern pharmaca is targeted toGPCRs [12]. Uncovering of the complexity of GPCRsignaling, of cross-talk mechanisms and of interactionwith RTKs has interesting implications for drugdevelopment. Previously known GPCR effectors may

    Fig. (1) . Schematic representation of GPCR-induced transmembrane signaling.

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    have unexpected effects via affecting complexsignaling networks. This may lead to unwanted sideeffects but also to novel applications of known drugs.Compounds affecting recently discovered GPCR signaltransduction pathways as RTK transactivation maypresent paradigms for novel principles to modulateGPCR signaling.

    6. GPCRs with protease ligands that act throughcleavage of the N-terminal segment which in turnactivates the receptor (e.g. thrombin)

    Within these subfamilies, the receptors for thevarious endogenous ligands may occur as receptosubtypes which are distiguished by their differenpharmacological agonist and antagonist profiles,differences in their (cDNA-deduced) primarysequences, and in many cases in different signatransduction mechanisms [17]. For example, molecularexploration has revealed the existence of 9 subtypesof the adrenergic receptor (1A, 1B, 1D, 2A, 2B2C, 1, 2, 3), or 5 subtypes of the muscarinic receptor(M1-M5), or 7 subtypes of the opioid receptor (1, 21, 2, 1, 2, 3).

    2. Basic Principles of GPCR SignalTransduction

    2.1. GPCR Subfamilies and ReceptorSubtypes

    All GPCRs consist of an extracellular N-terminalsegment, seven transmembrane helices (TM1-TM7),which form the TM core, three extracellular loops, andthree loops and the C-terminal segment exposed tothe cytoplasm. A fourth cytoplasmic loop may beformed when the C-terminal segment is palmitoylated ata Cys-residue. The seven TMs are arranged into amembrane-spanning boundle in the counterclockwisedirection from TM1 to TM7 as viewed from theextracellular surface [13]. The N-terminal segment isthe site of glycosylation and ligand binding, the C-terminal segment allows palmitoylation andphosphorylation as prerequisites for desensitizationand internalization. The intracellular loops transmit thesignal from the receptor to G protein. The TM core doesnot form a pocket or tunnel structure as conceivablebut is tightly packed by hydrogen bonds and saltbridges [13]. There are several excellent reviews

    focussing on GPCR structure, conformation, andactivation [13,14-16]. On the basis of ligand bindingand receptor activation several GPCR subfamilies havebeen classified [14,16]:

    For bradykinin, three receptor subtypes with distinctpharmacological profiles have been cloned [18-20] andcharacterized. The bradykinin B2 receptor (B2R) isconstitutively expressed in various cells and mediatesthe physiological and pathophysiological effects obradykinin such as vasodilation, bronchoconstrictioninflammatory reactions or pain sensations. Expressionof the B1 receptor is only induced undepathophysiological conditions such as injury (for reviewsee [21]). In addition, there are presumably tissue-specific splice variants of the B2R [22]. Bradykinin andkallidin ([Lys0]BK) are equipotent natural agonist of theB2R but do not act at the B1R. The principal B1Ragonist is [des-Arg9]BK. For both subtypes specificantagonists have been developed such as Hoe 140 (D-Arg[Hyp3,Thi5, D-Tic7, Oic8]BK) for the B2R and

    [Leu8

    ,des-Arg9

    ]BK for the B1R. Recently, a third typeof kinin receptor was described in chicken and termedornithokinin receptor [20] where bradykinin is inactivebut the B2R antagonist Hoe 140 acts as full agonist.

    The existence of receptor subtypes represent thefirst level of specificity in GPCR signal transduction: asingle agonist may activate distinct signaling pathwaysvia receptor subtypes which are encoded by differengenes and display distinct coupling specificities.

    1. GPCRs with ligand binding to the coreexclusively (photons, biogenic amines,nucleosides, lysophosphatidic acid,eicosanoids)

    2. GPCRs with ligand binding partially in both TMcore and exoloops (short peptides)

    2.2. Posttranslational Modifications ofGPCRs: Glycosylation, Palmitoylation andPhosphorylation

    3. GPCRs with ligand binding to the exoloops and

    the N-terminal segment (large polypeptides, e.g.glucagon)

    The N-terminal segments of most GPCRs areglycosylated on asparagyl residues. Glycosylation maybe important for the functional expression and celsurface localization of receptors [23] and maycontribute to the ligand binding [24], depending on thecell type.

    4. GPCRs whose ligands bind to the N-terminusexclusively (glycoproteins, e.g. LH, TSH)

    5. GPCRs whose ligands bind to a long N-terminalsegment that subsequently interacts with themembrane-associated receptor domaine (smallneurotransmitters) and Most GPCRs contain one or two conserved cysteine

    residues in their C-terminal cytoplasmic domain thatappear to be generally palmitoylated. Palmitoylation is

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    914 CurrentMedicinal Chemistry, 2000, Vol. 7, No. 9 Liebmann and Bhmer

    unique among lipid modification of proteins: it isreversible and adjustable [25]. There are several linesof evidence indicating that palmitoylation is importantfor intracellular trafficking of GPCRs and their cellsurface expression but not for ligand binding or Gprotein activation [26,27].

    occupied receptor but also by other factors such asPKC or calmodulin [30]. Therefore, homologous oheterologous receptor phosphorylation on Ser- or Thrresidues might be an important mechanism in cross-talkregulation.

    Tyrosine phosphorylation of GPCRs will bediscussed in a later chapter of this article.Phosphorylation of GPCRs on seryl or threonyl

    residues represents a mechanism for the rapid controlof receptor function and regulates the association of aGPCR with other proteins as well as their subcellularlocalization. This type of covalent modificationterminates or attenuates receptor signaling viadesensitization [28]. Receptor desensitization byphosphorylation is mediated either by G protein-coupled receptor kinases (GRKs) or by secondmessenger kinases [28,29]. Heterologousdesensitization of a particular GPCR is a feed backregulation by second messenger kinases such asprotein kinase A (PKA) or protein kinase C (PKC) which

    may be activated via a signaling pathway of the targetreceptor or via a separate GPCR. Phosphorylationleads to changes in the receptor conformation and,subsequently, results in an impaired interaction with Gproteins.

    2.3. Heterotrimeric G Proteins as SignaMediators

    The heterotrimeric guanine-nucleotide-bindingregulatory proteins (G proteins) are composed of a 36-52 kDa -subunit, a 35-36 kDa -subunit, and an 8-10kDa -subunit. The -and -subunits are assembledinto -complexes that act as functional units. Gproteins are usually classified into four majorsubfamilies that share some common features: Gs, GiGq/11 and G12 (Table 1). Multiple isoformes of each

    subunit have been identified up to date including 20different -, 6 - and 12 -subunits. Interestingly, not althe possible combinations of - and - subunits can beformed. G proteins underlie a cycle of activation anddeactivation that transmits the signal from the receptoto the effectors (reviewed in [31, 34]). Receptoactivation triggers the exchange of GDP (bound in theinactive state) for GTP on the -subunit of a G proteincoupled to the receptor. This results in dissociation ofthe complex into receptor, GTP-liganded G-and Gunits. The free receptor has a reduced affinity for itsagonist which is released. G-GTP and/or G caninteract with their target effectors. The slow intrinsicGTPase activity of G terminates the -inducedeffector association. G-GDP re-associates the free complexes thereby also terminating -mediated

    Ligand-induced or homologous desensitizationrequires GRKs and their functional cofactors, thearrestins. Both the GRK family and the arrestin familyinclude at least six members [29]. In a first step theagonist-occupied receptors are phosphorylated by aGRK. Then, an arrestin binds to the phosphorylatedGPCR and disrupt the interaction between receptors

    and G proteins. Desensitization represents the firststep of internalization and down-regulation [28]. Thecytosolically localized GRKs may be translocated to themembrane and thereby activated by the agonist-

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

    Class Subtyp Toxin Effectors Receptors

    Gs s, olf CTX AC stimulation; Ca2+-channels 1/2-AR; V2-R; D1-R; A2-R; odorant-R

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

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

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

    Gq q, 11, - PLCactivation AT II-R; ET-R; B2R;

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

    G12 12,13 - Na+/K+-exchange; Bruton`styrosine Kinase/ras GAP

    TxA2-R; LPA-R

    Abbreviations used are explained in the text. Additional abbreviations: /-AR= adrenergic receptors; V1/2-R= vasopressin receptors; D1/2-R= dopaminereceptors; A1/2-R=adenosine receptors; M1-4-R= muscarinic receptors; SSTR= somatostatin receptors; /-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 A2receptor

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    Signal Transduction Pathw ays of G Protein-coupled Receptors Curren t Med ic inal Chem istry , 2000, Vol. 7, No. 9 915

    signaling. The reconstituted heterotrimeric G proteininteracts again with the receptor and can begin thecycle anew.

    The functional properties of G proteins areinfluenced by the covalent attachment of three types oflipids (for review see [35,36]). All -subunits (withexception of transducin) are reversibly palmitoylated bylabile thioester bonds to N-terminal cysteine residuesIn addition, -subunits of the Gi/o family aremyristoylated at conserved N-terminal glycine residues

    This lipid modification is formed by a stable amidelinkage and irreversible. The -subunits of all G proteinsare prenylated by a stabile thioether bond betweenprenyl groups and cysteine residues. The precisefunction of these lipid modifications is not yet knownbut they appear to facilitate the targeting of G proteinsto the membrane and the localization of G proteins tospecific membrane subdomains, e.g. caveolae [36].

    Several of the -subunits are substrates forcovalent modifications by either cholera toxin (CTX) orpertussis toxin (PTX). CTX catalyzes the NAD+-dependent ADP-ribosylation of a conserved arginineresidue of Gs and G t proteins, resulting in aninhibition of GTPase activity and, therefore, constitutiveactivation of G. PTX induces ADP-ribosylation of mostG-subunits of the Gi/o-family (except of Gz), whichresults in inhibition of receptor-G protein-coupling.These toxins are important tools for identifying anddiscriminating G protein-mediated responses.

    Fig. (2) . Adenylate cyclase isoform II (A) and phospholipase C (B) as typical examples for convergence and integration ofsignals at the level of single key molecules within signaling networks. Abbreviations are explained in the text.

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    The -subunits of G proteins have different contactregions to receptors, -subunits, and effectors. Thereis accumulating evidence suggesting that a region ofthe C-terminal segment of G is important for thecoupling selectivity in receptor-G protein-interaction[37]. For example, in elegant molecular andbiochemical studies has been shown that in i/o-subunits the C-terminal residues -4 (cysteine), -3

    (glycine), and -1 (phenylalanine/tyrosine) and in q/11-subunits the C-terminal residues -3 (asparagine) and -5(glutamate) play key roles in determining the receptorselectivity [38,39]. The first 25 amino acids in the N-terminal part of -subunits are essential for -bindingwhereas the effector binding region partially overlapsthe putative -binding region. Therefore, the -subunit cannot simultaneously bind effector and [31]. In addition to the -subunit, the C-terminal regionof Gand the C-terminal part of Gmay be involved inreceptor coupling and specificity [40].

    two forms, R-I and R-II. The PKA-RI complex iscytosolically localized and its immobilization needs theinteraction with additional regulatory proteins, the A-kinase-anchoring proteins (AKAPs). The PKA-RIcomplex is particulate-associated. Thus, multipleisoforms of AC, PDE and PKA combined with spatiaand temporal control provide an immense cross-talk

    potential of the cAMP system.2.4.2. Phosp hol ipases, Lipid kinases, and

    Phospho l ip id -der i ved Second Messenger

    Systems Const i tu te Their own Signaling

    N e t w o r k

    The membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) represents the substrate forphospholipase C (PLC). The hydrolysis of PIP2 resultsin the simultaneous production of two secondmessengers, inositol 1,4,5-trisphosphate (IP3) anddiacylglycerol (DAG), which mediate intracellular Ca2+

    release and the activation of PKC, respectively [44]. Todate 3 subfamilies of PLC are known including 4 PLCisozymes, 2 PLCisozymes, and 4 PLCisoforms (forreview see [45]). The PLCisozymes are activated by-subunits of the Gq/11 family but may be alsostimulated by -complexes of Gq/11, Gior Gz proteinsThe biological importance of PLCisozymes is not yetclear. It is assumed that activation of PLCmight be anevent secondary to receptor-mediated activation ofother PLCs or Ca2+-channels [45]. The two PLCisozymes are structurally distinct from the other PLCsbecause they contain two SH2 domains and one SH3domain (SH = Src homology; see next chapter). Thus

    PLCis activated by RTKs of growth factors via bindingto their autophosphorylated tyrosine residues and thesubsequent phosphorylation of PLCat the tyrosines771, 783, and 1254. The PLCisozymes may be alsoactivated by nonreceptor phosphotyrosine kinases(PTKs) such as Src or Pyk2 (see also chapter 3). Invarious cells, these PTKs can be activated by GPCRsIn addition, PLCisozymes may be activated directly byseveral lipid-derived second messengers of GPCRs inthe absence of tyrosine phosphorylation. ThusGPCRs coupled to PLD or PI3-kinases may stimulatePLC through generation of their second messengersphosphatidic acid (PA) or PIP3, respectively [45]. Like

    AC-II, PLC represents a typical point of signaintegration and may play a key role in cross-talk (Fig2B ).

    2.4. The Main Effector Systems of GPCR

    Signaling: Isoforms, Multiple SecondMessengers, and Second Messenger-derived Mediators

    2 . 4 .1 . Ad e n y l a t e c y c l a s e ( AC )

    Classically, adenylate cyclase responds to GPCR-induced stimulatory or inhibitory regulation, mediatedeither by Gsor Gi, respectively. Meanwhile at least 9mammalian AC isoforms have been cloned andfunctionally characterized (for review see [41-43]). Allisozymes can be stimulated by Gs and,experimentally, with the plant diterpene forskolin

    (except AC-IX) but they differ in their response to otherregulatory molecules. Based on their sequencehomology, the ACs have been divided into severalsubfamilies with similar patterns of regulation.Adenylate cyclase type I (AC-I) can be stimulated byCa2+/calmodulin and inhibited by Gi- and -subunits.AC-II may be also activated by Ca2+/calmodulin but isnot inhibited by -complexes. AC-VIII defines ist ownsubfamily although its regulatory properties correspondto those of AC-III. Type V and VI AC constitute asubfamily that is only activated by Gs and may beinhibited by Gi as well as submicromolarconcentrations of Ca2+but is insensitive to calmodulin

    and is not affected by -complexes. The subfamily ofAC-II, AC-IV, and AC-VII is insensitive towards Ca2+, canbe stimulated by activated PKC and is sensitive to -complexes which are capable of enhancing thestimulatory effect of Gs (Fig. 2A ). The regulation ofAC-IX is still unclear. In addition, cyclic nucleotidephosphodiesterases (PDEs) that play a crucial role incAMP signal termination, are protein products of amultigene family displaying arround 30 isoforms [43].The intracellular target of cAMP is protein kinase A(PKA) which consists of two cAMP-binding regulatorysubunits (R) and two catalytic subunits (C). R exists in

    In addition to PLCs, PIP2serves also as a substratefor the receptor-regulated class I phosphoinositide 3-kinases (PI3Ks) which phosphorylate PIP2 toPtdIns(3,4,5)P3(PIP3) [46,47]. One subtype of PI3Kthe p110 PI3K, has been shown to be directlyactivated by -complexes from Gi proteins [48]. Inaddition to PIP3 production, this enzyme has thecapacity to activate the MAPK pathway [49]. Two other

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    isoforms, the PI3Ks and are dimeric moleculesconsisting of the p110 catalytic subunit and a p85adaptor protein that contains two SH2 domains andlinks the p110 subunit to RTK signaling pathways.Recently it was reported that also the PI3K can bealternatively activated by -complexes from G proteins[50]. The PI3K-produced second messenger PIP3 is

    rapidly degradated to PtdIns (3,4)P2, both moleculesact as second messengers and can directly activateseveral PKC isoforms and/or the serine/threonineprotein kinases PDK and PKB/Akt. Activated PKB/Aktprovides a survival signal that protects cells fromapoptosis [51].

    PLA2 to the membrane phospholipid substrate andrequires also phosphorylation that is obviouslycatalyzed by activated MAPK (ERK2). PKC and PKAcan phosphorylate PLA2 in vitro but there is noevidence for a direct phosphorylation in vivo [60]Nevertheless, PKC activation can lead indirectly toPLA2activation by triggering the MAPK pathway (see

    chapter 4). Activation of PLA2 represents the directpathway of AA release. Alternatively, AA may bereleased by a DAG-lipase from DAG which may beproduced either via PLC, PLC, or the PLD pathwayAA from the various sources may be converted via thecyclooxygenase pathway , for example, to PGE2 thatcan bind to and activate 4 different prostanoid receptosubtypes (EP1-4). EP2 and EP4 couple to Gs andstimulate adenylate cyclase activity. EP1 couples toGq/11and activates PLC. EP3is able to regulate threedifferent sets of G proteins including Gq/11, Gi/o, and GsThis is another example for a signaling mechanismwhere second messengers can produce mediato

    molecules acting via GPCRs anew and utilize the sameset of effectors as their first messengers.

    PIP2 is not only substrate for PLCs and PI3Ks butalso cofactor for the phospholipase D (PLD) whichrepresents another GPCR-regulated effector enzyme[52]. PLD hydrolyzes phosphatidylcholine (PC) tophosphatidic acid (PA) and choline in a cell-specificresponse to various stimuli including thrombin,vasopressin, endothelin, angiotensin II or bradykinin.At least 3 mammalian PLD isoforms have beenidentified up to now (for review see [52]). Downstreamof GPCRs, PLD regulation occurs by activation via PKCor by activation via small G proteins such as ARF (ADP-ribosylation factor) or Rho (a monomeric G protein).PLD could contribute to the intracellular signaling byseveral mechanism. Firstly, PA has been implicated as alipid second messenger in the regulation of proteinkinases [54] activation of PLC [45], and othersignaling molecules, including the protein-tyrosinephosphatase SHP-1 [55]. Secondly, in most cells PA israpidly converted to DAG through the action of

    phosphadidate phosphohydrolase. Thus, PLD-derivedDAG is implicated in the late phase activation of PKC[56]. Thirdly, PA is converted to lysophosphatidic acid(LPA) by a specific PLA2. LPA is known as anintercellular signaling molecule that is rapidly releasedand affects cells by acting on ist own GPCR. LPA maybe involved in the regulation of a variety of cellularresponses, e.g. cell proliferation, smooth musclecontraction, platelet aggregation, or neurotransmitterrelease [57]. Interestingly, the LPA receptor is capableof coupling to both G iand Gq/11proteins thus activatingdual pathways [57]. Therefore, LPA generated from PAis a typical example that a second messenger of a given

    GPCR signalling pathway may produce a mediatorwhich acts through another GPCR in an autocrine orparacrine manner.

    We have to go back once more to the Gq/11/PLCpathway which is chiefly producing the multiple secondmessengers IP3and DAG. IP3binds to a tetrameric IP3receptor in the endoplasmatic reticulum (ER) andtriggers the release of Ca2+from the ER resulting in anintracellular increase in cytosolic Ca2+ fromapproximately100 nM to approximately 1 M. In termsof signalling, Ca2+has to bind to trigger proteins suchas calmodulin that realize the messenger function oCa2+(for review see [62,63]).

    The intracellular target for DAG is protein kinase C(PKC) that belongs to the serine/threonine proteinkinases. PKC is thought to be essentially involved inthe regulation of cellular proliferation anddifferentiation. The PKC superfamily consists of severaisoforms. Based on their domain structure and theirregulation they have to be divided into at least 3subfamilies: "convential" cPKCs (, I, II, and ), whichare sensitive to Ca2+, DAG, and phorbol esters; "novel"nPKCs (, , , and ), which are activated by DAG andphorbol esters but are independent of Ca2+; and"atypical" aPKCs (, , and ), which are insensitive to

    Ca2+, DAG and phorbol esters and may be regulated byphospholipids, such as PA (for review see [56, 64-66]Recently, the PKC superfamily was supplemented bythe newly discovered PKC kinases (PRKs) consistingof at least 3 members (PRK1-3) [66]. Like the aPKCsthey are insensitive to Ca2+, DAG and phorbol estersPhorbol esters are potent tumor promoters and cansubstitute for DAG in activating cPKCs and nPKCsThey are not metabolized like DAG, evoke a prolongedactivation of PKC and are useful tools in studying PKCregulated pathways in vivo.

    Another phospholipase that is stimulated bynumerous agonists of GPCRs but also by growthfactors in a variety of cells is the cytosolic 85 kDaphospholipase A2 (PLA2) (for review see [58-60]).Stimulation of PLA2leads to the release of arachidonicacid (AA) and, subsequently, to the production ofeicosanoids [61]. Activation of PLA2 by GPCRsrequires increase in cytosolic Ca2+for the association of

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    Table 2. Typical Bradykinin Signalling Pathways : Multiple Coupling and Cell Specificity

    Cell/Tissue B2R Effector Mechanism Ref.

    A431 cells, guinea pig ileum, others PLC(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]

    PC-12 cells AC (stimulation) via Gq/11, Ca2+/calmodulin [78]

    Rat uterus, GPI AC (inhibiton) via Gi [79, 80]

    NG 108-15 cells Ca2+-activated K+channels (activation) via Gq/11, IP3, Ca2+ [81]

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

    Thus, all signaling routes that generate DAG may

    result in the activation of distinct PKC isoforms. Inaddition, the PKC isoforms , , and are known astargets for PIP3, the second messenger of PI3Ks[67,68].

    stimulation of AC whereas higher concentrations

    induce activation of PLC [84]. The activation omultiple coupling GPCRs, consequently results inmultifunctional signaling. Interactions between theparticular pathways of a single GPCR with multiplesignal transduction within the same cell could beclassified as homologous cross-talk. Such interactionsmay occur at different levels of signal transductionRecently, the group of Lefkowitz [85] hasdemonstrated that the -AR can "switch" from Gsto GiThe "switch on" of Gi requires the proceedingactivation of Gs and the stimulation of the cAMPpathway. Activated PKA phosphorylates the -ARleading to receptor coupling to G i. G-complexesreleased from Gi then activate MAPK in a Src- and Ras-dependent manner [85]. Thus, this "switch on"mechanism induces cross-talk at the receptor levelAnother example representing a "switch off"mechanism comes from our own work about BKsignaling in A431 cells [76]. In this cell line, BK inducesa rapid Gq/11-mediated activation of PLC resulting instimulation of PKC translocation. In a dual pathway BKslowly activates Gs followed by an increase in cAMPproduction and PKA activation that leads to an inhibitonof PKC translocation (Fig. 3 ).

    Finally, it should be noted that not only membraneenzymes but also K+ and Ca2+ channels may beeffectors for GPCRs (for review see [69]). Recently,also cross-talk at the level of Ca2+ channels wasreported resulting from PKC-mediated phosphorylationthat antagonizes -induced inhibition of Ca2+

    channels [70]. This cross-talk mechanism provides afirst example that not only effector enzymes but also

    ion channels have the potential for the integration ofmultiple signaling inputs.

    2 .4 .3 . B radyk i n i n B2 Recep to r -med ia ted

    S i g n a l T r a n s d u c t i o n

    In dependency on the cell or tissue investigated thepleiotropic hormone bradykinin has been shown to becapable of activating different G proteins and multiplesignaling pathways (Table 2). In the majority of cases,however, and in most cells bradykinin stimulates PLCvia a Gq/11protein.

    In addition, numerous interactions betweensignaling pathways of different GPCRs have beendescribed (for review see [2]). This type of interactiontherefore, could be classified as "heterologous cross-talk". Synergistic cross-talk between Gi- and Gqcoupled receptors, e.g. adenosine A1- oneuropeptide Y (NPY)-receptors and 1-adrenergicreceptors often result in augmentation of physiologicaresponses such as smooth muscle contractions [2]Such synergistic interactions seem to play an importantrole in the "fine tuning" of multiple receptor signalingpathways [2].

    2.5.Complexity of GPCR Signaling: MultipleCoupling, Switching, and Cross-talk

    It is well known that many individual receptors arecapable of interacting with different G proteins [1,83].For example, the human thyrotropin (TSH) receptor oralso the bradykinin B2 receptor (Table 1) can interactwith members from each of the four major subfamilies of-subunits. The specificity of interaction may bedetermined by the agonist concentration or by differentkinetics. Thus, low concentrations of TSH lead to

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    Fig. (3) . Dual signaling pathway of the bradykinin B2 receptor (B2R) in A431 cells: a rapid Gq/11-mediated activation of thePLC/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 beactivated by multiple receptors (Table 1) thus acting asconvergence point [1,83]. The specificity of receptorcoupling to a mutual effector system may bedetermined by the composition of heterotrimeric Gproteins. For example, using the antisenseoligonucleotide technique Kleuss et al. [86, 87]showed in excellent studies that in GH3cells inhibitionof Ca2+- channels by somatostain receptors is mediatedby Go213 whereas inhibition by M4 muscarinic

    receptors is mediated by Go114. Another mechanismfor selective regulation of GPCR signaling pathwaysmay be provided by the newly discovered RGS(regulators of G protein signaling) proteins [88, 89].These RGS proteins function obviously as GAPS(GTPase-activating proteins) for heterotrimeric Gproteins and and enhance the speed of GTP hydrolysisthereby controling the kinetics of G protein signaling.Members of the RGS family have been shown todisplay selectivity for different G-subunits such asRGS2 for q or RGS1/3 for i [89]. Thus, the cell-specific expression of different GRS proteins may alow

    the independent control of multiple signaling pathwaysof GPCRs.

    3. Receptor Tyrosine Kinase (RTK)Signaling and MAP Kinase Pathways

    3.1. Ligand-dependent Activation of RTKs

    Protein tyrosine kinases (PTKs) have centrafunctions for the regulation of cell proliferation andother cell functions. Many polypeptidic mitogenicfactors ("growth factors") elicit their cellular responsesvia receptors of the transmembrane receptor tyrosinekinase (RTK) family [90-92]. Well known examples arethe receptors for epidermal growth factor [93,94], theplatelet derived growth factors (PDGFs) [95,96] or thefibroblast growth factors (FGFs) [97-99]. The biologicaeffects of RTK stimulation include effects, largelypositive, on cell proliferation ("growth"), celdifferentiation, locomotion and cell survival (Fig. 4 ). Incontrast, receptors for cytokines, a class of growth- and

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    differentiation-modulating polypeptides forhematopoietic and immune cells, are devoid of intrinsicenzymatic activity and need to recruit cytoplasmic PTKsfor signaling [100]. While many aspects of signalingfrom RTKs and cytokine receptors have similarities, thelatter class of receptors shall not be discussed further inthis review. RTKs consist of an extracellular ligand

    binding domain, a single transmembrane domain andan intracellular domain which harbors the conservedtyrosine kinase subdomain and variable regulatorysequences [101]. The hitherto known molecules in thisreceptor family fall into 13 classes, assigned largely onthe basis of homology within their intracellular tyrosinekinase domain [102]. More than 100 genes for RTKsmay exist in the human genome [101]. The functionalunit of an RTK consists of at least two receptormolecules or higher oligomers [103-105]. Receptordimers or oligomers can be homomers or heteromers ofrelated [106,107], possibly also of unrelated RTKs . Inthe absence of ligand, receptor dimer (oligomer)

    formation apparantly occurs spontaneously with lowefficiency [108]. Solely overexpression of a given RTKmay trigger sufficient dimer formation leading tosignaling in the absence of a ligand [107]. This has

    frequently been observed in high level overexpressionexperiments [109,110] and in cancer cellsoverexpressing an RTK. The receptors in the insulinreceptor family [111,112] present an exception sincethey consist of disulfid-bridged dimers in the absenceof the ligand. Ligand bindig initiates signaling bytriggering dimer/oligomer formation or, in case of the

    insulin receptor family, by triggering interaction of apreformed receptor dimer. Various molecular meansare employed by RTK ligands to accomplish receptordimer stabilization [105]. Monomeric ligands as EGFdrive dimerization by interacting with receptor subunitsin a 1:1 stoichiometry [113]. Dimerization may beconsequence of conformational changes in the ligandoccupied receptors or may result from a bivaleninteraction of both ligands with both receptor subunitsin the dimer [114]. Other ligands are dimeric andinteract with two receptor subunits at the time, as incase of PDGFs [104].

    As a consequence of receptor dimerization the twosubunits trans-phosphorylate each other on tyrosineresidues. Two types of phosphorylations can bedistinguished: Phosphorylations in the "activation

    Fig. (4) . Multiple signaling pathways activated by RTKs and negatively controlled by PTPs in absence and presence of an RTKligand. The mechanisms as well as abbreviations are explained in the text.

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    loop" in the kinase subdomain which lead to elevationof kinase activity and phosphorylations in other parts ofthe intracellular domain which create binding sites forsignaling molecules. Crystal structures of the insulinreceptor catalytic domain [115] and the FGF-receptor 1catalytic domain [116] have revealed the mechanism ofactivation of these kinases [117]. The activation loop, a

    flexible structure identified in many protein kinases, inits unphosphorylated state can occupy partially (FGFreceptor -1) or completely (insulin receptor) the activesite of the kinase. Phosphorylation leads todisplacement of the activation loop and in turn providesaccess for substrates to the kinase active site. Aphosphorylation site corresponding to tyrosine 1162 inthe insulin receptor kinase is conserved in RTKssuggesting that this mechanism of activation is acommon principle. However, for example in case of thePDGF-receptor substantial residual kinase activity canbe detected in a receptor variant with the 857 tyrosine(corresponds to 1162 in insulin receptor) mutated

    [118] or selectively blocked by a kinase inhibitor [119].Thus, stringency of regulation by activation loopphosphorylation varies among the different RTKs.Multiple tyrosine residues can further bephosphorylated in the RTK cytoplasmic domains, bothC- or N-terminally of the catalytic subdomain or, in RTKswith a split kinase domain as PDGF receptors, also inthe "kinase insert" [95, 96]. These sites, in theirphosphorylated form, present recognition and bindingsites for signaling molecules posessing different typesof phosphotyrosine binding domains as SH2-domains[120-122], or PTB domains [123, 124]. SH2 ("src-homology 2"-domains) are protein modules of about

    100 amino acids which are found in numerous proteinsand recognize phosphotyrosine in the context of up to6 C-terminal amino acids. In contrast, PTB("phosphotyrosine binding") domains recognizephosphotyrosine and 1-6 N-terminally situated aminoacids. Another type of protein domains involved insignal transduction are SH3 ("src-homology 3")domains. They consist of about 60 amino acids andinteract with other proteins in a phosphorylation-independent manner recognizing proline-richsequences. These domains and a still increasingnumber of recently discovered protein-proteininteraction domains are apparently required to achieveefficiency and specificity in highly ordered signaltransduction complexes. Multiple interactions of theRTK cytoplasmic domains with signaling moleculesform the basis for initiation of multiple intracellularsignaling events (Fig. 4 ). Several signaling chains maybe necessary to elicit together a particular biologicalresponse. On the other hand, they form the basis fordifferent types of biological responses which can bemediated via the same receptor as stimulation of cellgrowth, cell locomotion, cell differentiation andprevention of apoptosis.

    3.2. Specificity and Redundancy in RTKDownstream Signaling

    Downstream signaling molecules which bind totrans-phosphorylated RTKs fall in two classesenzymes and adaptor molecules. An example for theformer is the phospholipase C [125], which binds viatandem SH2-domains to different activated RTKs, is inturn phosphorylated and itself activated. An examplefor the latter is the p85 subunit of PI3kinase , whichdocks to several growth factor receptors and permits inturn binding and thus membrane recruitment of the PI3kinase catalytic subunit p110 [126, 127]. Adaptomolecules can also recruit further adaptors, whichrecruit other singaling molecules and so forth [122]. Anexample is the family of Shc adaptor molecules, whichare phosphorylated subsequent to RTK binding and intheir phosphorylated form are able to bind the adaptormolecule Grb2 [128]. Thus, RTKs present scaffoldingmolecules for the assembly of multiple signaling

    proteins which can interact in a chain of signatransduction events [129].

    Many RTKs have been shown to interact with thesame set of signaling proteins. For example, Shc andGrb2 have been demonstrated to bind to multiple RTKsincluding the EGFR [130], the EGFR-related receptorHER2/erbB2 [131] and the hepatocyte growth factor(HGF) receptor Met [132]. On the other hand, there arealso pronounced specificities. Thus, PLCcannot bindto the insulin receptor [111] or Grb2 binds only with lowaffinity directly to the PDGF-receptor [133]. CertainRTKs have even quite specific interaction partners

    The insulin receptor family RTKs accomplish most otheir signaling activity via recruitment of relativelyspecific docking proteins, the insulin receptorsubstrates (IRS), which in their phosphorylated formthen interact with further signaling molecules [111].Other examples are the FGF receptor 1 which elicitsmuch of its signaling via the FRS2 adaptor [134, 135and the Met/HGF receptor RTK which exhibits aparticularly strong interaction with the docking proteinGab1 [136].

    3.3. RTK Interaction with Further Protein

    Tyrosine Kinases

    Another level of complexity of RTK signaling hasemerged when it was found that RTKs utilize alsocytoplasmic PTKs for signal transmission [137]Cytoplasmic PTKs fall into 9 classes [102, 138]Members of the Src-family of PTKs, comprising forexample the intensely investigated PTK pp60c-src

    (designated as "Src" throughout this review), itsoncogenic counterpart v-Src and the PTKs Yes (pp62cyes), Fyn (p59fyn), Lyn (p56lyn) and Lck (p56lck) harborone SH2-domain and one SH3 domain in addition to

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    the catalytic domain. Negative regulation of thesekinases occurs by phosphorylation on tyrosineresidues in the C-terminus, leading to interaction withthe intramolecular SH2-domain. Positive regulation isthen possible by dephosphorylation of thisphosphotyrosine by protein-tyrosine phosphatases(PTPs, see chapter 3.4). Src, Fyn and Yes may have

    apparently very similar functions. Several assays usedfor detecting involvement of Src in cellular functionscannot differentiate between these kinases. Anotherfamily of cytoplasmic PTKs is named after its prototypefocal adhesion kinase (Fak), a 125kDa enzyme whichcooperates with Src in integrin signaling and regulationof the assembly of cell-matrix adhesion complexes. Arelative of Fak is Pyk2, a PTK which is activated by Ca2+and likely to be involved in downstream signaling ofsome GPCRs. Finally, the family of JAK ("januskinases") should be mentioned here, which includesalso Tyk2 as a member. These PTKs functiondownstream of cytokine receptors but may also interact

    with some RTKs. A paradigm for interaction of an RTKwith cytoplasmic PTKs present the PDGF-receptorswhich have the capacity to recruit Src family kinases,including p60c-srcand activate them in turn [139-141].Src family kinase activation has been suggested to beessential for mitogenic signaling of the PDGF-receptor[142]. Similar observations have been made with theEGFR, although RTK- Src kinase complexes are lessreadily demonstrable in this case [142-145]. Src-familykinases do appear, however, to not only simply mediatea signal but, to also phosphorylate the RTKs andthereby modulate signaling. Src phosphorylation siteshave been mapped on the EGFR, the PDGF-receptor

    and the IGF-1 receptor [146-151]. Mutation of a mainSrc-phosphorylation site on the PDGF-receptor leadsto a reduced mitogenic signaling and a morepronounced stimulation of chemotaxis by PDGF via themutant receptor [150]. For the EGFR and the IGF-1receptor, increased RTK activity subsequent tophosphorylation by Src has been shown [146, 151].Other cytoplasmic tyrosine kinases which have beenshown to interact with RTKs are for example FER, amember of the fes/fps family of nontransmembranereceptor tyrosine kinases [152, 153] and possiblymembers of the JAK/Tyk family [154].

    exist which allow termination of signaling, and, evenlyimportant, silencing of the RTK basal signaling activityin the absence of a ligand. Ligand-independentactivation of RTKs [107] (discussed below) by variousand quite diverse cell treatments including stimulationof GPCRs clearly shows that RTKs may have a signalingactivity in the absence of ligand which is normally

    silenced. There are paradigms for negative regulationof RTKs at all levels of RTK activation, includinginterference with ligand binding [156], with receptordimerization [157] and with the tyrosinephosphorylations executed by the receptor kinase[158]. Also, receptor internalization [159-162] andphosphorylation of the receptor cytoplasmic domainsby heterologous serin-threonine kinases are means ofRTK inactivation, which are utilized by the cell. TheEGFR is subject to phosphorylation by PKC at Thr 654[163], leading to attenuation of receptor signalingAlso, phosphorylations at Thr 669, Ser 1002, 1046 and1047 [164-167] may modulate EGF RTK negatively. A

    modulation of signaling by PKC-dependentphosphorylation has also been shown for other RTKs[168] and may be an important general regulationprinciple.

    Another means or negative RTK control are theactions of protein-tyrosine phosphatases (PTPs) (Fig4 ). Since this type of interactions may provide animportant link for cross-talk with the GPCR family ofreceptors (see chapter 4.4), we will describe it in somedetail. Ligand-activated RTKs are rapidlydephosphorylated by cellular PTPs, shown andinvestigated in some detail for example for the EGFR

    PDGF-receptor or the insulin receptor [169-173]Dephosphorylation parameters for not ligand-activatedreceptors are difficult to evaluate, however, treatmentof cells with PTP inhibitors as vanadate [174]phenylarsineoxide [175, 176], hydrogen peroxide[177] or diamide [178, 179] leads to phosphorylation omany RTKs. Thus, "basal" activity of RTKs is probablyquite significant and under negative control of PTPsOver the last 10 years PTPs have emerged as a largefamily of quite diverse molecules [180-182]. More than100 PTP species may exist [183] and hitherto almost50 different individual PTPs have been shown to beexpressed in a single cell type [184]. PTPs can broadly

    be classified in cytoplasmic and transmembrane, or"receptor-like" enzymes [185]. The former posess onePTP catalytic domain and various types of protein-protein interaction or membrane targeting domainsThe tramsmembrane PTPs have frequently twocatalytic domains and variable extracellular domainsDepending on the PTP subclass, these may recognizecell surface proteins, matrix components or solubleligands. For example, the PTPs RPTP and k havebeen shown to interact homophilically [186-188]RPTP has a carboanhydrase-like motif in theextracellular domain which can interact with contactin, a

    3.4. Inactivation Mechanisms for RTKs

    Tyrosine phosphorylation needs to be tightlyregulated. Loss of this tight regulation may lead toaberrant cell behaviour as uncontrolled proliferation.For many tyrosine kinases including RTKs oncogenicvariants exist which have constitutive activity as acommon property [155]. In case of RTKs, tightregulation is on the one hand provided by thenecessity of ligand stimulation for activation. On theother hand, several negative regulation mechanisms

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    neuronal cell surface protein [189]. Also, RPTPhasbeen shown to interact with the matrix protein tenascin,cellular adhesion molecules (CAMs) and pleiotrophin, aheparin-binding neurite-promoting factor (reviewed in[190]). Hitherto, it is unclear whether these interactionscan affect intracellular PTP activity. From crystalstructure data for RPTPand from experiments with a

    chimeric molecule consisting of the EGFR extracellulardomain and the RPTP CD45 intracellular domain it hasbeen proposed that for certain RPTPs dimerization maylead to inactivation of the first (membrane-proximal)catalytic domain, which harbors most of PTP activity[191-193]. Among the cytoplasmic PTPs muchattention has been devoted to SHP-1 (HCP, SH-PTP1,PTP1C) and SHP-2 (syp, SH-PTP2), the only knownmammalian PTPs which posess SH2-domains [194,195]. Their tandem SH2-domains target these PTPs tocellular phosphotyrosine-containing proteins includingRTKs. Both PTPs exist in a relatively inactive "closed"conformation, with the N-terminal SH2-domain blocking

    the active site. Occupation of the SH2-domain leads toactivation of PTP activity [196, 197]. SHP-2 isubiquitously expressed and mediates positive signalsfor many RTKs and also cytokine receptors by ahitherto unknown mechanism involving the PTPcatalytic activity. For some receptors, however, SHP-2may also negatively control signaling steps (reviewed in[198]). SHP-1 negatively regulates various types ofreceptors in hematopoietic cells, includingimmunoreceptors [199, 200], cytokine receptors [201],and the RTKs Kit/SCF-receptor [202] and CSF-1receptor [203]. Apart from hematopoietic cells, SHP-1is also expressed in epithelial cells [204]. It can

    associate with the EGFR and negatively regulatesignaling of this receptor at least in cells with relativelyhigh EGFR levels [55, 205, 206]. While SHP-1 hasbeen assigned to various RTKs as a likely or possiblenegative regulator, in most cases the important PTPsfor a given RTK signaling regulation are unknown. It isquite possible that several PTPs are involved indifferent aspects of signal regulation. The EGFR mayserve as an example of this. In transient coexpressionsystems multiple PTPs have the capacity todephosphorylate the receptor [109]. Interaction of thereceptor has been described with the cytoplasmicPTPs SHP-1 and SHP-2 (via SH2-domains) [205],PTP1B and T-cell PTP (with so-called "substratetrapping mutants" of the PTPs, forming stable enzyme-substrate complexes) [207, 208] and PTPs of the PTP-PEST-family [209]. Also, transmembrane PTPs arelikely to modulate EGFR signaling. Inducibleoverexpression of the transmembrane PTP RPTP inA431 cells leads to reduced EGFR phosphorylationand a reduced capacity of the cells to form colonies insoft agar. Partial suppression of endogenous RPTPby inducible expression of an RPTP antisense-construct leads to elevated receptor phosphorylation

    and soft agar colony forming capacity [210]. ThusRPTPappears to control aspects of EGFR signaling inA431 cells. Understanding regulation of PTP activity isonly in its beginning. This concerns effects of known oputative ligands for RPTPs, mentioned above as well aspossible effects of intracellular effectors. Activity of anumber of PTPs has been shown to be modulated by

    Ser/Thr phosphorylation. For example, phos-phorylation of SHP-1 by PKC leads to inhbition [211]PKC-phosphorylation of the transmembrane PTPRPTPto elevation [212] of PTP activity. Various PTPsare also phosphorylated on tyrosine residues, whichhas likewise been suggested do modulate activity[213]. Several cytosolic and transmembrane PTPsundergo partial proteolytic cleavages which modifycellular localization and may also affect activity towardscellular substrates [214]. Finally, lipid secondmessengers can modulate PTP activity as shown fophosphatidic acid and the PTP SHP-1 [55]. Inconclusion, PTPs and regulation of their activity

    provide important but hitherto poorly understoodpathways to modulate RTK signaling activity in apositive and negative manner.

    3.5. RTK-mediated MAP-Kinase Activation

    An important signaling pathway which links RTKs tocell proliferation and possibly other biologicaendpoints is the so-called "MAP-kinase cascade". Thisterm refers to a signal transmission chain from themembrane to the nucleus, which is conserved fromyeast to mammals and consists of a small G-protein

    followed by three consecutively activated proteinkinases (for recent reviews see [215-217]). Inmulticellular organisms RTKs have aquired the capacityto activate one variant of this chain, whose lastenzymes in this case are the closely related Ser/Thr-specific; proline-directed "extracelluar signal regulatedkinases" (ERKs) 1, and 2, also designated "mitogen-activated protein kinases" (MAPK) p44 and p42respectively. In the literature frequently the term "MAPkinase" is used as a synonym for ERK1/2, as we do inthe other chapters of this article. The main pathway formitogenic signaling from RTKs like the EGFR or thePDGFreceptor involves the following steps

    Subsequent to RTK activation and transphosphorylation occurs binding of a complexconsisting of the adaptor molecule Grb2 and theguanine-nucleotide exchange factor (GEF) Sos for thesmall G-protein Ras. Ras, a 21kDa protein is theprototype for numerous small G-proteins in the cell (forreview see [218-222]). They all have in common thatthey are active, i.e. capable to interact with "effectoproteins" in the GTP-loaded state. They have a smalintrinsic GTPase activity, which is greatly enhanced byspecific GTPase-activating proteins (GAPs). GTP-loading of inactive, GDP-loaded small G-proteins is

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    accomplished with the help of GEFs as theaforementioned Sos. Binding of the Grb2-Sos complesto the receptor is mediated by the Grb2 SH2-domainand can be either directly to the tyrosine-phosphorylated RTK as in case of EGFR or indirectly viathe phosphorylated adaptor Shc as in case of thePDGF receptor. Membrane-recruited Sos now enables

    a GDP-GTP-exchange on Ras. All this occurs at theinner surface of the plasma membrane, where Ras isbound via a farnesyl-anchor (Fig. 5 ). GTP-loaded Rashas a high affinity for the first kinase in the MAPK-cascade, the product of the cellular protooncogene c-raf, Raf-1 and recruits it to the membrane. Membranerecruitment of Raf-1 leads in an incompletelyunderstood manner to activation of Raf-1. Probablyadditional proteins [223] and membrane lipids [224] aswell as phosphorylation [225] contribute to activation ofRas-bound Raf-1. Activated Raf-1 phosphorylates itsdonwstream kinases MKK1or 2 (MAP-kinase kinase;also termed MEK for MAP/ERK-kinase) on two Ser

    residues, leading to activation of MKK. MKKs arekinases with "dual specificity", i.e. they canphosphorylate Ser/Thr and Tyr residues. Dualphosphorylation on Thr and Tyr in the activation loop of

    MAP kinases ERK2/1 confers ERK activationActivated ERKs can phosphorylate multiple cellulatarget proteins, including transcription factors(subsequent to nuclear translocation of Erks), the p90ribosomal S6 kinase (Rsk90), microtubule-associatedproteins (MAPs) and phospholipase A2. Thesephosphorylations link activation of the MAP kinase

    pathway to cell growth and transformation. It should benoted that several members in the pathway upstreamfrom Erks are transforming when overexpressed in aconstitutively active form, including Ras, Raf and MEKemphasizing that this is a main pathway for mitgogenicsignaling.

    Biological responses initiated via the MAPK pathwayseem to critically depend on the duration of MAPKactivation. For example in PC12 cells, sustained MAPKstimulation via the nerve growth factor (NGF) receptorTrkA leads to a differentiation response, while a shortwave of MAPK activation via the EGFR mediates a

    mitogenic response. In other cells a sustainedactivation of MAPK may be necessary for eliciting amitogenic response [226]. An intersting feature of theMAPK pathway is negative feedback regulation. Erks

    Fig. (5) . MAP kinase pathways are routed by scaffold proteins and negatively controlled by multiple phosphatases.

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    can phosphorylate and thereby negatively regulateupstream molecules in the chain, notably Sos. Also, forexample the EGFR can be phosphorylated on Thr 669by Erk2.

    presents another level of regulation and possibilities forcross-talk with signaling pathways emanating fromGPCR.

    3.6 Ligand-independent Activation of RTKsThere are multiple variants of the MAPK signalingchain, starting with the existence of different isoformsof Ras, Raf, MKK, and ERK [217]. Parallel pathways on

    the basis of distinct members of the MAPK-family withcorresponding upstream components and distinctdownstream targets and biological effects exist. Inyeast probably 6 members of the MAPK family exist[227]. They do not couple to RTKs (those do not existin yeast) but to nutrient sensors or the pheromonereceptor, a GPCR. In animal cells there are likewisemultiple members of the MAPK-family. Other members,which are relatively well investigated apart fromERK1/2, are the NH2-terminal Jun-kinase/stressactivated protein kinase (JNK/SAPK) and p38. ManyRTKs can activate ERKs, some growth factor receptorshave also been shown to activate JNK. JNK and p38are both activated by various stress factors. In additionto ERK1/2 further MAPK-family members may beimportant for RTK signaling. For example the mitogenicsignal of the EGFR has recently been shown to requireactivation of ERK5 (also known as "big molecularweight kinase" BMK) [228].

    Various quite diverse agents and cell treatments

    have been observed to induce activation of RTKs inthe absence of ligand. These include cell treatmentswith "adverse agents", for example UV [235] or X-rayirradiation [236]. Also, activation of different GPCRs hasbeen shown to lead to RTK activation. This pathwayhas been designated "transactivation" and is discussedin detail below (chapter 4.4). Finally, integrin and cell-cell adhesion molecule activation have been shown toresult in RTK activation [237].

    From the current knowledge of RTK activation andsignaling control pathways different mechanisms couldbe envisaged for ligand-independent RTK activation

    ligand-independent RTK dimer/oligomer formation,heterologous phosphorylations and inactivation of"silencing" mechanisms.

    Some evidence has been obtained for the lastpossibility in case of UV-mediated RTK activation. PTPswhich keep RTKs silent and inactivate them after ligandstimulation perform catalysis with particiation of areactive cysteine in their active center and are thereforevery susceptible to oxidation. UV treatment of cellscould recently been shown to inactivate membranebound PTPs for the EGFR and the PDGF receptor,most likely via an oxidative mechanism [110]. It seems

    quite likely that various adverse agents can lead togeneration of reactive oxygen species andsubsequently to PTP inactivation. These inactivationmechanisms may in fact be partially reversible and playeven a role in ligand-activated signal transduction[238]. Hydrogen peroxide generation subsequent toRTK activation has been shown for the PDGF receptoto be essential for eliciting a mitogenic signal [239]EGFR activation is accompanied by hydrogen peroxidegeneration [240] and a reversible inactivation of thePTP PTP1B [241]. PTP activity, however, is likely to besubject to regulation by further mechanisms, asphosphorylation or proteolysis, outlined above. Thisshould in turn also modulate activity of PTP-regulatedRTKs.

    Specificity within a given MAP kinase cascade is onthe one hand provided by specificities of the upstreamand downstream elements for each other. On the otherhand, scaffolding of different components of the chainby specific scaffold proteins seems to be an additionalmean of providing specificity and efficient coupling ofthe signal transducing components [229, 230]. Forexample, MP1 and JIP1 are recently identified proteinswhich bind members of the ERK or JNK cascade,respectively, and may provide a scaffold for routing thesignal within the cascade (Fig. 5 ).

    MAPK signaling is subject to negative regulation byphosphatases (reviewed in [231]). One class ofphosphatases responsible for inactivation of MAPkinases are the dual-specificity MAP kinasephosphatases (MKPs). Members of this family, forexample CL100/MKP1 are products of "immeadiateearly genes" which are rapidly activated upon growthfactor stimulation. Multiple MKPs have pronouncedselectivities for certain MAP kinase members and thusfor feedback inhibition of the respective cascade [232,233]. Also, PTPs can inactivate MAP kinases bydephosphorylating solely the phosphotyrosine in theMAPK activation loop [234]. Finally, the regulatoryphosphothreonine on MAP kinase familiy members canbe subject to dephosphorylation by Ser/Thr specificphosphatases [231]. In conclusion, inactivation of theMAP kinase cascades by phosphatases, either dual-specificity or tyrosine-specific or Ser/Thr-specific,

    An interesting question is to what extentheterologous tyrosine kinases may be capable of RTKactivation. RTK phosphorylation by Src-family kinases ispossible and may be sufficient to generate signamolecule docking sites and subsequent signageneration [137]. This would in principle be possiblewithout participation of the RTK intrinsic tyrosine kinaseactivity. For the case of GPCR transactivation, however

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    participation of RTK intrinsic activity has beendemonstrated (see chapter 4.4). It is an interestingpossibility that Src-family kinases or other heterologoustyrosine kinases could also execute such RTKphosphorylations that would lead to RTK activation.Indeed, phosphorylation sites for Src on the EGFR arepartially within the kinase domain and may regulate

    kinase activation [146, 148]. Also, PDGF-receptor wasfound to become phosphorylated on many sitesincluding Tyr 857, the site presumably involved inkinase activation, by Src kinase in vitro [150]. Thus, Src-family kinases and possibly other tyrosine kinases maybe capable of direct RTK activation although thispossibility clearly requires further investigation.

    cell type to cell type. However, stimulation of variousGPCRs was found to induce activation of key effectomolecules of RTK signaling, including Ras, Raf, andMAPK. These observations led to the assumption thatMAPK might be a point of convergence for proliferativesignals emanating from both different G proteins andRTKs (Fig. 6 ).

    It is thought that both the -subunits and complexes of heterotrimeric G proteins are capable ofmediating activation of MAPK. Obviously, in PTX-sensitive pathways MAPK is activated through complexes from Gi/o proteins whereas in PTX-insensitive pathways the activation of MAPK ismediated via q/11-subunits and PKC [4,7,8]. Gsmediated regulatory effects on MAPK activity may beeither stimulatory or inhibitory. The role of G12/13proteins in MAPK activation is not yet clear. Veryrecently, the binding of G12 to Bruton`s tyrosinekinase (Btk) and the stimulation of Btk and of Gap1m

    which is a RasGTPase-activating protein (rasGAP) wasreported [33].

    4. Signaling Pathways from GPCRs toMAP Kinase

    Like RTKs, many GPCRs can induce mitogenicresponses or contribute to neoplastic growth of human

    tumors (reviewed in [242]. Depending on the cell type,mitogenic responses can be mediated by Gi/o, Gq/11, Gs,or G12 proteins. [4,6,79]. For example, constitutivelyactive mutants of G protein -subunits have beenidentified in various tumor cells, and GTPase deficientforms of Gs, Gi/o, and G12have been demonstrated toinduce cellular growth after expression in several celllines [243].

    These findings indicate, for the first time, thepossibility of a direct link between heterotrimeric andmonomeric G proteins.

    Intensive research led to the identification of variousprotein kinases that could make the link between Gproteins and the MAPK cascade. Recently, a noveprincipal pathway of MAPK activation has beendiscovered termed transactivation [243, 244]

    The pathways coupling GPCRs to nuclearresponses are of high complexity and may differ from

    Fig. (6) . MAP kinase activation via RTKs and by various GPCRs may involve Ras and different G protein subtypes. Aschematic overview.

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

    Transactivation stands for ligand-independentactivation of RTKs triggered by GPCRs [107]. Despitethe efforts made in the last years the early mechanismsof MAPK activation by agonists of GPCRs remain poorlyunderstood. However, in the following we will attemptto describe the most likely biochemical routesconnecting GPCRs to MAPK.

    addition, -stimulated MAPK activation is inhibited bytyrosine kinase inhibitors such as genistein suggestingthe involvement of a tyrosine kinase in this pathway[247]. Taken together, all experimental data ledassume that Ras or an effector molecule upstream ofRas should be the target for the Gi-mediatedmitogenic signaling. The question remained how the-complexes might regulate Ras. First answer camefrom the laboratory of Lefkowitz. This groupdemonstrated that -subunits stimulated tyrosinephosphorylation and thereby activation of the adaptorprotein p52 Shc thus inducing the formation of Shc-Grb2 complexes [249, 250]. Interstingly, Gstimulated phosphorylation of Shc has been also foundto be inhibited by Wortmannin, a specific inhibitor oPI3K suggesting an additional involvement of PI3K

    upstream of Shc [249]. Then Luttrell et al. [251]provided evidence that the non-receptor tyrosinekinase Src mediates the -induced tyrosinephosphorylation of Shc. Later, several studiesdescribed the involvement of more Src-like kinasessuch as Fyn, Lyn, and Yes [252] or also, recently, of anunknown non-Src cytosolic tyrosine kinase thatinduces the interaction of a p100 kDa protein with Grb2[253].

    4.1. Principle Mechanisms of MAPKActivation by Gi/o-coupled Receptors

    There are several lines of evidence suggesting thatMAPK activation by G i-coupled receptors involves the-complexes and is Ras-dependent. Thus, activationof MAPK via Giis attenuated by coexpression of the -subunits of transducin which acts to sequester G

    [245]. This was supported by similar results obtained inRat-1 cells with ARKct, a carboxy-terminal fragment ofthe -adrenergic receptor kinase that also acts as -sequestrant and significantly inhibited activation ofboth Ras and MAPK via the G i-coupled LPA receptor[246]. Further, overexpression of G subunits resultsin activation of MAPK [245, 247] whereas aconstitutively active mutant of Gi did not increaseMAPK activity in COS-7 cells [248]. It wasdemonstrated that MAPK activation by -subunitsrequired neither PLC nor PKC (reviewed in [4]) butwas blocked by dominant negative Ras [245]. In

    Another candidate that has been implicated in Shc-Grb2-Sos complex formation is the FAK-related non

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    receptor tyrosine kinase PYK2. This enzyme ispredominantly expressed in neuronal cells and may linkboth Gi and Gq with Grb2-Sos in a Ca2+- and Src-dependent pathway as was shown for LPA- and BK-receptors in PC-12 cells [254].

    independent or also Ras-dependent pathways [4,7]The Ras-independent pathway shows, e.g. in COScells or CHO cells expressing Gq-coupled receptors nosensitivity to a dominant negative mutant of Ras andmay involve activation of PKC and Raf. Gq-mediatedsignalling in CHO cells has been demonstrated to beinhibited by down-regulation of PKC by chronic

    exposure to phorbol esters and by dominant negativeRaf [247]. Phorbol esters as direct activators of PKChave been reported to activate MAPK in various cellsIn addition, expression of constitutively active mutantsof PKC showed that at least in vitro the PKC isoforms,,, , and have the potential to activate MAPK atthe level of Raf-1. The aPKC cannot increase Raf-1activity and stimulates MEK by an independenmechanism [263]. Recently, much interest has beenfocussed at a possible role of the PKC isoforms andespecially as activators of Raf-1 in vivo. For example, adominant negative mutant of PKC inhibited bothproliferation of NIH 3T3 cells and Raf activation in COS

    cells whereas active PKC overcame the inhibitoryeffects of dominant negative Ras in NIH 3T3 cells [264]Furthermore, constitutively active mutants of PKC

    as well as PKC overcame the inhibitory effects ofdominant negative mutants of the other PKC isotype[264]. In addition, PKC can be co-precipitated withRaf-1 from Sf-9 insect cells and PKC transformed NIH3T3 cells [265]. PKC was demonstrated to beactivated by both phosphatidylinositol (PI)- andphosphatidylcholin (PC)-derived DAG [266], and PC-hydrolysis was shown to induce Raf-1 activation [267]Moreover, PKC may be also activated via the PI3Kpathway thus representing a point of convergence for

    several lipid-derived second messengers. Takentogether, there is increasing evidence that Raf-1activation by PKC may play a critical role in mitogenicsignalling of Gq/11-coupled receptors.

    On the other hand, PI3Khas been shown to play amajor role in G-mediated activation of MAPK. Underendogenous conditions -sensitive PI3K activity hasbeen described in neutrophils and platelets [255, 256],and PI3K has been shown to activate MAPK whenexpressend in COS-7 cells in response to Gi-derived-complexes [49]. PI3K can be activated due todirect interaction with -complexes [48] or due to aconstitutive association with the p101 -sensitiveprotein [257]. PI3Kwas found to act upstream of Src-like kinases suggesting another putative link to theShc-Grb2-Sos complex [49]. Furthermore, we [258]and others [50, 259] have recently shown that thep85/p110 PI3Kmay play a role downstream of Gi- or

    Gq-coupled receptor signalling, too. The putativesignaling pathways connecting Gi/o-coupled receptorsto MAPK are summarized in Fig. (7 ).

    Very recently, once more the group of Lefkowitzprovided evidence which probably opened a newdimension in our understanding of mitogenic signaltransduction (reviewed in [260]). Firstly, stimulation of-adrenergic receptor expressed in HEK 293 cellsresulted in a Gi-coupled, -mediated and Ras-dependent activation of MAPK. However, theinteraction of -AR with Gi required a prior receptorcoupling to Gs subsequently leading to cAMP

    production and activation of PKA. PKA-inducedreceptor phosphorylation was found to be aprerequisite for switching the receptor from Gs to Githereby activating another signaling pathway. [261].Furthermore, in HEK 293 cells additionally expressingdominant negative mutants of -arrestin or dynaminwhich are known to block receptor endocytosis the -AR-mediated activation of MAPK was prevented [262].The inhibitors of receptor internalization were found tospecifically block the interaction between Ras-boundRaf and the cytosolic MEK. Thus, GRKs and arrestinmediating the uncoupling and internalization of GPCRsmay play also an essential role in the GPCR-induced

    MAPK activation [260, 262].

    On the other hand, agonists of Gq/11-coupledreceptors such as bombesin, bradykinin, orvasopressin as well as phorbol esters have been alsodescribed to stimulate Src family kinases transientlythereby activating MAPK in a Ras-dependent manner[268]. According to a hypothesis proposed by DellaRocca et al. [269] activation of Src kinases by Gq/11coupled receptors might be the final event of a cascade

    including the increase in cytosolic Ca2+ in response toIP3 and the Ca2+-calmodulin mediated activation ofPyk2 or of a related tyrosine kinase whichphosphorylates Src. Indeed, stimulation of Gq-coupledBK receptors in PC-12 cells was demonstrated toactivate PyK2 and subsequently Src and MAPK [254].Src may phosphorylate either a RTK or Shc leading tothe formation of a Shc-Grb2-Sos complex. The moslikely biochemical routes connecting Gq/11-coupledreceptors to The MAPK pathway are summarized in Fig.(8 ).

    4.2. Principle Mechanisms by which Gq/11-

    coupled Receptors May Activate MAPK

    Receptors coupled to PTX-insensitive G proteins ofthe Gq/11family are thougth to mediate MAPK activationvia their -subunits. Their signaling is mostly insensitiveto -sequestrants (reviewed in [4,7]). Gq/11-coupledreceptors may activate MAPK by either Ras-

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

    4.3. Gs-coupled Receptors and MAPK:Differential and Controversial Effects of

    cAMP and -Subunits

    released from Giafter switching of -AR from Gs to G[261] should mediate the activation of MAPK.

    Very recently, a new family of cAMP-bindingproteins was discovered that exhibit properties of aguanine necleotide exchange factor (GEF). ThesecAMP-GEFs are capable of activating monomeric Gproteins in a cAMP-dependent and PKA-independentmanner suggesting a direct coupling of cAMPmediated signaling to Ras superfamily signaling [276].

    The role of Gs in the regulation of cell growth and

    MAPK activity appears to be extremely cell-typespecific. In various cells such as smooth muscle cells[270], adipocytes [271], or fibroblasts [272], cAMP hasbeen shown to inhibit the MAPK cascade. In thesecells, cAMP activates PKA which in turnphosphorylates Raf-1 thereby decreasing the affinity ofRaf for Ras as well as Raf kinase catalytic activity [273].In PC-12 cells, in contrast, cAMP stimulates the MAPKpathway and induces neuronal differentiation [274]. InCOS-7 cells, Faure et al. [248] reported that theexpression of a constitutively active mutant of Gs,treatment with forskolin or dibutyryl cAMP, or

    stimulation of transiently expressed, Gs-coupled LHreceptor resulted in activation of MAPK. Contradictoryresults have been presented by Crespo et al. [275]suggesting dual effects of -adrenergic receptors onMAPK activity in COS-7 cells. In this study, stimulationof -AR simultaneously led to -dependent activationand cAMP-mediated inhibition of MAPK. The balancebetween these two mechanisms was postulated dodetermine the outcome of the signal to MAPK [275].However, in view of the present knowledge it may beassumed that not -complexes from Gs but those

    4.4 . A New Role for RTKs in GPCR SignalingDifferent Pathways Lead to Transactivation

    RTKs are not only receptors for specific peptidicgrowth factors but also essentially involved in themitogenic signalling of GPCRs where they may act as

    scaffold proteins, signal mediaters, and signaintegrators. First evidence for ligand-independenttyrosine phosphorylation of RTKs by a GPCR came in1995 from reports demonstrating the tyrosinephosphorylation of PDGFR by angiotensin II [277] in rasmooth muscle cells or of EGFR by bradykinin in humankeratinocytes [278]. Then, in two excellent papersDaub et al. [243, 244] described the transactivation ofEGFR in diverse cell types such as Rat-1 cells, HaCatkeratinocytes, mouse astrocytes as well as in COS-7cells (Fig. 9A ). In Rat-1 cells it was shown that mitogens

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    such as endothelin, LPA and thrombin mediate bothMAPK activation and DNA synthesis via activation ofEGFR [243]. In COS-7 cells transiently expressing Gi-or Gq-coupled receptors was demonstrated that bothtypes of GPCRs stimulated Shc phosphorylation as wellas MAPK activity via EGFR transactivation [244]. Sinceinhibition of PI3K did not affect EGFR tyrosine

    phosphorylation but abolished MAPK activation PI3Kwas supposed to act downstream of EGFR. Similarresults were obtained with the Src-inhibitor PP-1leading to the assumption that also Src should beinvolved in the signaling pathway downstream ofEGFR. Independently, Luttrell et al. from theLefkowitz`group provided evidence that at least Gi-coupled receptors may induce EGFR tyrosinephosphorylation via Src-family tyrosine kinases [279].They proposed, in contrast, that a PI3K-dependentstep might lie upstream of Src kinase activation. Srckinase, indeed, possesses a domain with high affinityto PIP3 the second messenger product of PI3K [280].

    In contrast again, recently published data suggest thatin HeLa cells the intrinsic EGFR tyrosine kinasecontributes to the LPA-stimulated MAPK pathway andc-Src is probably not involved [281]. However, thisfinding in HeLa cells does not exclude that members ofthe Src family may be involved in other cell types.Furthermore, the same GPCR may inducetransactivation of different RTKs depending on the celltype. This has been recently demonstrated for LPAwhich transactivates EGFR in COS-7 cells but is alsocapable of using the PDGFR in L cells that lack EGFR[282].

    was potently inhibited by a PKC inhibitor and thephorbol ester PMA could mimick the carbachol effecsuggesting the PKC-dependency of EGFR tyrosinephoshorylation [284] (Fig. 9B ). A completely differenmechanism of EGFR transactivation was found in GN4rat liver epithelial cells where angiotensin II (A II) iscapable of activating MAPK via two pathways. Unde

    normal conditions, A II stimulates MAPK via a PKC-dependent, Ras-independent pathway. In PKC-depleted cells, the A II receptor can switch andactivates MAPK via an equipotent Ras-dependentpathway including EGFR tyrosine phosphorylationThus, the latent transactivation route represents analternative pathway to MAPK that is masked by PKCand uncovered when the PKC pathway breaks down[285] (Fig. 9C ).

    Moreover, in COS-7 cells transiently co-transfectedwith the human bradykinin B2 receptor and MAPK wefound a pathway of MAPK activation that includes the

    independent and equipotent stimulation of both PKCand EGFR [329]. Activation of MAPK in response to BKis prevented by inhibitors of PKC as well as EGFR.Inhibitors of PI3K or Src failed to affect MAPK activationby BK. PKC translocation studies and coexpression ofinactive and constitutively active mutants of differenPKC isoforms provided evidence for a critical role of thePKC isozymes and in BK signalling towards MAPKBK induced tyrosine phosphorylation of the EGFR thatwas independent of PKC. Since blockade of the EGFRdid also not influence BK-stimulated increase inphosphatidylinositol phosphate formation, PKC shouldact neither upstream nor downstream of EGFR but in a

    permanent dual signalling pathway. MAPK activation byBK requires signals from both pathways whichrepresent a two-key system for regulating an enzymewhich is critically involved in the control of cell growth(Fig. 9D ).

    In neuronal cells EGFR transactivation appears to bedependent on Ca2+. In PC-12 cells, for example,bradykinin-induced tyrosine phosphorylation of EGFRupstream of Shc and MAPK was reported [283]. Thiseffect of BK was absent in PC-12 cells pretreated withEGTA. In other cells, however, EGFR transactivationwas found to be independent of Ca2+ [284]. In additionto the differences in Ca2+-sensitivity or the role of Src,the high degree of cell specificity within themechanisms involved in EGFR transactivation byGPCRs is also reflected by different modes of action ofPKC (Fig. 9 ). Although activation of PKC has beenwidely shown to play a key role in MAPK activation by

    Gq-coupled receptors, in COS-7 cells the Gq-mediatedtransactivation of EGFR was postulated without anyattempt to modify PKC activity in these cells [244]. Thisis surprising all the more because in 1995 Coutant et al.[278] already showed that in HaCaT humankeratinocytes bradykinin induces tyrosinephosphorylation of EGFR via a PKC-dependentpathway. Similar results were reported for human 293cells stably transfected with m1 muscarinic receptors. Inthese cells carbachol-induced EGFR transactivation

    As described above, a common theme in theliterature on RTK transactivation by GPCR is thepossible involvement of Src-family kinases. They mayinduce signal transduction by either phosphorylatingRTKs on docking sites for signaling molecules or viadirect activation of intrinsic RTK activity. As outlined inchapter 3, it is not entirely clear, how Src-family kinases

    can possibly accomplish RTK activation. One wouldhave to assume that they execute phosphorylations othe RTK molecules which are functionally equivalent totrans-phosphorylations in RTK dimers. Such a modewould be in agreement with some of the available dataon Src-phosphorylation sites in different RTKs but thismechanism has not been fully established. Further,while tyrosine phosphorylation in the RTK "activationloop" has been established as activation mechanism forsome RTKs as FGF receptor, insulin receptor or

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    Fig. (9) . Different modes of EGFR transactivation. (A) Principle mechanisms of RTK (EGFR, PDGFR) transactivation withoutconsideration of PKC. Several authors implicate PI3Keither upstream or downstream of RTK [244, 279]. (B) In human 293 cellsPKC was found to be involved in M1AchR-mediated EGFR transactivation. It is not yet clear whether PKC induces activation ofa 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 themembrane [284]. (C) Latent dual pathway of EGFR transactivation by angiotensin II. In GN4 cells AII activates MAPKdominantly via the PKC/Raf pathway . When the PKC pathway is cancelled MAPK is alternatively activated via EGFRtransactivation [285]. (D) Activation of MAPK by BK via a permanent dual pathway. Both activation of PKC and EGFRtransactivation are necessary for the stimulation of MAPK activity by BK.

    Met/HGF receptor, respective evidence is still missingfor other RTKs. In fact, it is likely that for other RTKs, inparticular EGFR regulation by activation loopphosphorylations is less important. Thus, the possible

    In contrast, it should be considered that a mainpathway for RTK activation may be instead theinterference with negative regulatory pathwaymechanisms (see chapter 3.4) in particular with theactivity of RTK-silencing PTPs. Their activity could welbe affected by GPCR signaling events, as changed

    role of heterologous Src-kinase phosphorylations iseven more questionable.

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    Ca2+-levels, lipid second messengers or variousprotein kinases including Src-family kinases. This kindof mechanism is not easy to demonstrate. EGFreceptor dephosphorylation is a very rapid process[286-288] with t1/2

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    epithelial cells, AII activates MAPK via the AT2 receptorsubtype and a signaling route involving stimulation of Giand, subsequently, PLA2, the release of arachidonicacid which in turn activates Shc and Ras [292]. In N1E-115 neuroblastoma cells AT2 receptors mediateinhibition of serum- or EGF-induced MAPK activation,possibly via activation of a PTP (293).

    Whereas the AT1 receptor preferentially couples toGq/11 proteins, in colonic epithelial cells also the Gprotein-coupled gastrin receptor was found to stimulatePLC1 via Src [295]. It may be assumed, therefore, thatboth Gq/11- and Gi-coupled receptors have the ability toact without G proteins by forming signal transductioncomplexes which are thought to be typical for RTKs

    On the other hand, with respect to ligand-inducedtyrosine phosphorylation of the B2 bradykinin receptocontradictory results have been reported. In humanfibroblasts the B2R was shown to be phosphorylated aserine and threonine residues in response to BK andneither phosphoamino acid analysis nor Westernblotting with anti-phosphotyrosine antibodies revealedtyrosine phoyphorylation of the B2R [298]. In contrastin endothelial cells the BK-induced IP3 formation wasreported to be dependent on a transient tyrosinephosphorylation of PLC1 [299, 300]. In vascularendothelial cells, tyrosine phoyphorylation of PLC1could be correlated with its binding to the C-termina

    intracellular domain of the B2R similar to the findings athe AT1receptor [300]. Unfortunately, there is no clearevidence for a ligand-induced tyrosine phosphorylationof the B2R in this work.

    These few examples may illustrate by whichimmense complexity and cell specificity the cross talkbetween GPCRs ant RTKs can occur.

    5. RTK-induced Tyrosine Phosphory-lation of GPCR Signaling Elements

    Although relatively much is known about theinteraction of GPCRs with RTKs and the MAPKcascade, the modulation of GPCR-coupled signaltransduction by RTK-induced tyrosine phosphorylationis considerably less investigated. Meanwhile, however,there is mounting evidence that cross-talk includes notonly the foreward regulation of the MAPK pathway byGPCRs but also the regulation of GPCR signaling byRTKs. Recently, at least three levels of GPCR signaltransduction were implicated to be favoured targets oftyrosine phosphorylation by RTKs.

    It is worth to speculate, however, that tyrosinephosphorylation of GPCRs might occur not only ligandinduced (homologous) but also as a cross-talk eventinduced by activated RTKs (heterologous). In that waya RTK could use tyrosine-phosphorylated GPCRs asscaffold proteins for ist own signal transductionmachinery.

    5.1. Tyrosine Phosphorylation of GPCRs

    G protein-coupled receptors lack intrinsic tyrosinekinase activity. Nevertheless, several agonists of GPCRsuch as angiotensin II [294] or gastrin [295] have been

    demonstrated to stimulate ph