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ABSTRACT: Activation of heterotrimeric G proteins, such as G(12/13) and G(q), by cell surface receptors is coupled to the regulation of numerous cellular functions controlled by activated Rho GTPases. Previous studies have implicated the Rho guanine nucleotide exchange factor (RhoGEF) leukemia-associated RhoGEF (LARG) as a regulatory protein receiving stimulatory inputs from activated Gα(12/13) and Gα(q). However, the molecular mechanisms of the Gα(q)-mediated LARG activation are not fully understood and the structural elements of LARG involved in this process have remained unclear. In the present work, the specific coupling of the histamine H1 receptor (HRH1) exogenously expressed in COS-7 cells to G(q), but not to G(12/13), was used to conduct a detailed analysis of receptor- and Gα(q)-mediated LARG activation and to define its structural requirements. The results show that HRH1-mediated activation of the strictly Rho-dependent transcriptional activity of serum response factor requires the PDZ domain of LARG and can be mimicked by activated Gα(q)(Q209L). The functional interaction between activated Gα(q) and LARG requires no more than the catalytic DH-PH tandem of LARG, and is independent of PLCβ activation and distinct from the mechanisms of Gα(q)-mediated p63RhoGEF and PLCβ(3) activation. Activated Gα(q) physically interacts with the relevant portions of LARG in COS-7 cells and histamine causes activation of LARG in native HeLa cells endogenously expressing HRH1, G(q), and LARG. This work is the first positive demonstration of a stimulatory effect of LARG on the ability of a strictly G(q)-coupled receptor to cause activation of a Rho-GTPase-dependent signaling pathway.
Cellular signalling 11/2011; 24(3):652-63. · 4.09 Impact Factor
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Katy L Everett,
Anja Buehler,
Tom D Bunney,
Anca Margineanu,
Rhona W Baxendale, Petra Vatter,
Michael Retlich,
Claudia Walliser,
Hugh B Manning,
Mark A A Neil,
Christopher Dunsby,
Paul M W French,
Peter Gierschik,
Matilda Katan
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ABSTRACT: We performed analyses of the molecular mechanisms involved in the regulation of phospholipase Cγ2 (PLCγ2). We identified several regions in the PLCγ-specific array, γSA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLCγ2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells. We further tested the possibility that the interaction of PLCγ2 with its activator protein Rac2 was sufficient for activation through the release of autoinhibition. However, we found that Rac2 binding in the absence of lipid surfaces was not able to activate PLCγ2. Together with other observations, these data suggest that an important consequence of Rac2 binding and translocation to the membrane is that membrane proximity, on its own or together with Rac2, has a role in the release of autoinhibition, resulting in interfacial activation.
Molecular and cellular biology 01/2011; 31(6):1240-51. · 6.06 Impact Factor
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Tom D Bunney,
Olaniyi Opaleye,
S Mark Roe, Petra Vatter,
Rhona W Baxendale,
Claudia Walliser,
Katy L Everett,
Michelle B Josephs,
Carolin Christow,
Fernando Rodrigues-Lima,
Peter Gierschik,
Laurence H Pearl,
Matilda Katan
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ABSTRACT: Rho family GTPases are important cellular switches and control a number of physiological functions. Understanding the molecular basis of interaction of these GTPases with their effectors is crucial in understanding their functions in the cell. Here we present the crystal structure of the complex of Rac2 bound to the split pleckstrin homology (spPH) domain of phospholipase C-gamma(2) (PLCgamma(2)). Based on this structure, we illustrate distinct requirements for PLCgamma(2) activation by Rac and EGF and generate Rac effector mutants that specifically block activation of PLCgamma(2), but not the related PLCbeta(2) isoform. Furthermore, in addition to the complex, we report the crystal structures of free spPH and Rac2 bound to GDP and GTPgammaS. These structures illustrate a mechanism of conformational switches that accompany formation of signaling active complexes and highlight the role of effector binding as a common feature of Rac and Cdc42 interactions with a variety of effectors.
Molecular cell 05/2009; 34(2):223-33. · 14.61 Impact Factor
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Claudia Walliser,
Michael Retlich,
Richard Harris,
Katy L Everett,
Michelle B Josephs, Petra Vatter,
Diego Esposito,
Paul C Driscoll,
Matilda Katan,
Peter Gierschik,
Tom D Bunney
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ABSTRACT: Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCgamma isoforms, only PLCgamma(2) has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLCgamma(2) required for activation. Based on reconstitution experiments with isolated PLCgamma variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLCgamma(2) by Rac2. We also demonstrate that Rac2 directly binds to PLCgamma(2) as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.
Journal of Biological Chemistry 10/2008; 283(44):30351-62. · 4.77 Impact Factor
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ABSTRACT: Expression of the human cytomegalovirus (HCMV)-encoded chemokine receptor homologue pUS28 in mammalian cells results in ligand-dependent and -independent changes in the activity of multiple cellular signal transduction pathways. The ligand-dependent signalling activity of pUS28 has been shown to be predominantly mediated by heterotrimeric G proteins of the G(i/o) and G(12/13) subfamilies. Ligand-independent constitutive activity of pUS28 causing stimulation of inositol phosphate formation has been correlated with the coupling of pUS28 to G proteins of the G(q) family. It is well known that activation of G(q) proteins by cell surface receptors is coupled to activation of the Rho GTPase RhoA. Activated RhoA regulates numerous cellular functions, including the activity of the transcription factor serum response factor (SRF). The marked activation of G(q) proteins by pUS28 in transfected and HCMV-infected cells prompted us to investigate its effect on SRF activity. The results presented herein demonstrate that expression of pUS28 in COS-7 cells caused a vigorous induction of SRF activity. This effect was observed in the absence of chemokines known to interact with pUS28, and was specifically mediated by endogenous G(q) and/or G(11) as well as RhoA and/or a closely related Rho GTPase. The stimulatory effect of pUS28 and Galpha(q/11) was independent of phospholipase C-beta (PLCbeta) activation and was markedly sensitive to inhibition by wild-type, but not by constitutively active Galpha(16), thus identifying Galpha(16) as a modulator of Galpha(q/11) function likely to act by competing with Galpha(q/11) for and thus uncoupling Galpha(q/11) from activation by pUS28.
Cellular Signalling 09/2008; 20(8):1528-37. · 4.06 Impact Factor
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ABSTRACT: G-protein-coupled receptor kinases (GRK) are known to phosphorylate agonist-occupied G-protein-coupled receptors. We expressed and functionally characterized mouse GRK6 proteins encoded by four distinct mRNAs generated by alternative RNA splicing from a single gene, mGRK6-A to mGRK6-D. Three isoforms, mGRK6-A to mGRK6-C differ in their C-terminal-most portion, which is known to mediate membrane and/or receptor interaction and regulate the activity of GRK4-like kinases. One isoform, mGRK6-D, is identical to the other mGRK6 variants in the N-terminal region, but carries an incomplete catalytical domain. Mouse GRK6-D was catalytically inactive and specifically present in the nucleus of transfected cells. Recombinant mouse GRK6-A to mGRK6-C were found to be membrane-associated in cell-free systems and in transfected COS-7 cells, suggesting that the very C-terminus of GRK6-A, lacking in GRK6-B and mGRK6-C and carrying consensus sites for palmitoylation, is not required for membrane interaction. Interestingly, the shortest catalytically active variant, mGRK6-C, was conspicuously more active in phosphorylating light-activated rhodopsin than mGRK6-A and mGRK6-B, implying that the C-terminus of the latter two variants may fulfil an autoinhibitory function. Mutation and removal of C-terminal-most region of mGRK6-A by site-directed mutagenesis revealed that this region contains three autoregulatory elements: two discontinuous inhibitory elements consisting of a single residue, D560, and the sequence between residues S566 and L576, and an intervening stimulatory element. The results suggest that mGRK6-C may be considered a basic, prototypic representative of the GRK4-like kinases, which is capable of interacting with both plasma membrane and its receptor substrate, but is resistant to further regulatory modification conferred to the prototype via C-terminal extension.
FEBS Journal 01/2006; 272(23):6039-51. · 3.79 Impact Factor
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ABSTRACT: The regulation of the two isoforms of phospholipase C-gamma, PLCgamma(1) and PLCgamma(2), by cell surface receptors involves protein tyrosine phosphorylation as well as interaction with adapter proteins and phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)) generated by inositol phospholipid 3-kinases (PI3Ks). All three processes may lead to recruitment of the PLCgamma isozymes to the plasma membrane and/or stimulation of their catalytic activity. Recent evidence suggests that PLCgamma may also be regulated by Rho GTPases. In this study, PLCgamma(1) and PLCgamma(2) were reconstituted in intact cells and in a cell-free system with Rho GTPases to examine their influence on PLCgamma activity. PLCgamma(2), but not PLCgamma(1), was markedly activated in intact cells by constitutively active Rac1(G12V), Rac2(G12V), and Rac3(G12V) but not by Cdc42(G12V) and RhoA(G14V). The mechanism of PLCgamma(2) activation was apparently independent of phosphorylation of tyrosine residues known to be modified by PLCgamma(2)-activating protein-tyrosine kinases. Activation of PLCgamma(2) by Rac2(G12V) in intact cells coincided with a translocation of PLCgamma(2) from the soluble to the particulate fraction. PLCgamma isozyme-specific activation of PLCgamma(2) by Rac GTPases (Rac1 approximately Rac2 > Rac3), but not by Cdc42 or RhoA, was also observed in a cell-free system. Herein, activation of wild-type Rac GTPases with guanosine 5'-(3-O-thio)triphosphate caused a marked stimulation of PLCgamma(2) but had no effect on the activity of PLCgamma(1). PLCgamma(1) and PLCgamma(2) have previously been shown to be indiscriminately activated by PtdInsP(3) in vitro. Thus, the results suggest a novel mechanism of PLCgamma(2) activation by Rac GTPases involving neither protein tyrosine phosphorylation nor PI3K-mediated generation of PtdInsP(3).
Journal of Biological Chemistry 11/2005; 280(47):38923-31. · 4.77 Impact Factor
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Claudia Walliser,
Michael Retlich,
Richard Harris,
Katy L Everett,
Michelle B Josephs, Petra Vatter,
Diego Esposito,
Paul C Driscoll,
Matilda Katan,
Peter Gierschik,
Tom D Bunney
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ABSTRACT: Expression of the human cytomegalovirus (HCMV)-encoded chemokine receptor homologue pUS28 in mammalian cells results in ligand-dependent and -independent changes in the activity of multiple cellular signal transduction pathways. The ligand-dependent signalling activity of pUS28 has been shown to be predominantly mediated by heterotrimeric G proteins of the Gi/o and G12/13 subfamilies. Ligand-independent constitutive activity of pUS28 causing stimulation of inositol phosphate formation has been correlated with the coupling of pUS28 to G proteins of the Gq family. It is well known that activation of Gq proteins by cell surface receptors is coupled to activation of the Rho GTPase RhoA. Activated RhoA regulates numerous cellular functions, including the activity of the transcription factor serum response factor (SRF). The marked activation of Gq proteins by pUS28 in transfected and HCMV-infected cells prompted us to investigate its effect on SRF activity. The results presented herein demonstrate that expression of pUS28 in COS-7 cells caused a vigorous induction of SRF activity. This effect was observed in the absence of chemokines known to interact with pUS28, and was specifically mediated by endogenous Gq and/or G11 as well as RhoA and/or a closely related Rho GTPase. The stimulatory effect of pUS28 and Gαq/11 was independent of phospholipase C-β (PLCβ) activation and was markedly sensitive to inhibition by wild-type, but not by constitutively active Gα16, thus identifying Gα16 as a modulator of Gαq/11 function likely to act by competing with Gαq/11 for and thus uncoupling Gαq/11 from activation by pUS28.
Cellular Signalling. 20(8):1528-1537.
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Petra Vatter
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ABSTRACT: Kinasen G-Protein-gekoppelter Rezeptoren (GRKs) phosphorylieren Agonist-besetzte G-Protein-gekoppelte Rezeptoren, ein Vorgang, der als homologe Desensibilisierung bezeichnet wird. In unserer Arbeitsgruppe konnten von der GRK6 der Maus (mGRK6) vier verschiedene cDNAs isoliert werden, welche für die Varianten mGRK6-A, -B, -C und -D kodieren. In der vorliegenden Arbeit wurde gezeigt, dass diese vier mGRK6-Varianten Produkte eines Gens darstellen und aus diesem durch alternatives Spleißen hervorgehen. Mit Hilfe von Northern blot-, RT-PCR (reverse Transkriptase-polymerase chain reaction)-, SNuPE (single nucleotide primer extension)-Analysen und der RNA in situ-Hybridisierung wurde in Geweben adulter Mäuse eine differentielle Expression der verschiedenen mGRK6-Transkripte nachgewiesen. Sowohl das mRNA-Expressionsmuster als auch die Verteilung der mGRK6-Isoformen A, B und C in lymphatischen Geweben der Maus im Western blot lässt vermuten, dass die GRK6-Varianten in vivo an der Regulation G-Protein-gekoppelter Rezeptoren in Leukozyten beteiligt sind. Für die in Insektenzellen exprimierten Varianten mGRK6-A, -B und -C wurde gezeigt, dass diese Isoformen im Gegensatz zu der innerhalb der katalytischen Domäne trunkierten mGRK6-D katalytisch aktiv sind. Die enzymatische Aktivität der GRKs setzt die Translokation an die Plasmamembran voraus, wobei für den Carboxyterminus der GRKs eine entscheidende Rolle bei der Membranverankerung und somit auch bei der Aktivierung diskutiert wird. Obwohl der carboxyterminal kürzesten Variante mGRK6-C im Bereich des Carboxyterminus Motive fehlen, die eine Membraninteraktion vermitteln könnten, war neben den Proteinen mGRK6-A und mGRK6-B auch diese mGRK6-Isoform membranassoziiert. Zudem wies die mGRK6-C eine deutlich höhere Phosphorylierungsaktivität für das Substrat Rhodopsin auf als die Varianten A und B. Diese Befunde stützen die Hypothese, dass für die GRKs nicht nur, wie bisher angenommen, die C-terminalen Motive bzw. Modifikationen für die Translokation entscheidend sind, sondern darüber hinaus andere Mechanismen existieren, welche die Assoziation mit der Membran und die Aktivierung der Rezeptorkinasen vermitteln.
