Petra Vatter

Isozyme-specific Stimulation of Phospholipase C-γ2 by Rac GTPases

The regulation of the two isoforms of phospholipase C-γ, PLCγ1 and PLCγ2, by cell surface receptors involves protein tyrosine phosphorylation as well as interaction with adapter proteins and phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) generated by inositol phospholipid 3-kinases (PI3Ks). All three processes may lead to recruitment of the PLCγ isozymes to the plasma membrane and/or stimulation of their catalytic activity. Recent evidence suggests that PLCγ may also be regulated by Rho GTPases. In this study, PLCγ1 and PLCγ2 were reconstituted in intact cells and in a cell-free system with Rho GTPases to examine their influence on PLCγ activity. PLCγ2, but not PLCγ1, was markedly activated in intact cells by constitutively active Rac1G12V, Rac2G12V, and Rac3G12V but not by Cdc42G12V and RhoAG14V. The mechanism of PLCγ2 activation was apparently independent of phosphorylation of tyrosine residues known to be modified by PLCγ2-activating protein-tyrosine kinases. Activation of PLCγ2 by Rac2G12V in intact cells coincided with a translocation of PLCγ2 from the soluble to the particulate fraction. PLCγ isozyme-specific activation of PLCγ2 by Rac GTPases (Rac1 ≈ 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 PLCγ2 but had no effect on the activity of PLCγ1. PLCγ1 and PLCγ2 have previously been shown to be indiscriminately activated by PtdInsP3 in vitro. Thus, the results suggest a novel mechanism of PLCγ2 activation by Rac GTPases involving neither protein tyrosine phosphorylation nor PI3K-mediated generation of PtdInsP3.

Biologically Inactive Leptin and Early-Onset Extreme Obesity

Mutations in the gene encoding leptin (LEP) typically lead to an absence of circulating leptin and to extreme obesity. We describe a 2-year-old boy with early-onset extreme obesity due to a novel homozygous transversion (c.298G→T) in LEP, leading to a change from aspartic acid to tyrosine at amino acid position 100 (p.D100Y) and high immunoreactive levels of leptin. Overexpression studies confirmed that the mutant protein is secreted but neither binds to nor activates the leptin receptor. The mutant protein failed to reduce food intake and body weight in leptin-deficient ob/ob mice. Treatment of the patient with recombinant human leptin (metreleptin) rapidly normalized eating behavior and resulted in weight loss.

Structural insights into formation of an active signaling complex between Rac and phospholipase C gamma 2.

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.

Rac Regulates Its Effector Phospholipase Cγ2 through Interaction with a Split Pleckstrin Homology Domain

Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCγ isoforms, only PLCγ2 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 PLCγ2 required for activation. Based on reconstitution experiments with isolated PLCγ 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 PLCγ2 by Rac2. We also demonstrate that Rac2 directly binds to PLCγ2 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 PLCγ1 and PLCγ2 and the implications of our findings for their respective signaling roles.

Constitutive serum response factor activation by the viral chemokine receptor homologue pUS28 is differentially regulated by Gαq/11 and Gα16

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.

Membrane Environment Exerts an Important Influence on Rac-Mediated Activation of Phospholipase Cγ2

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.

LARG links histamine-H1-receptor-activated Gq to Rho-GTPase-dependent signaling pathways.

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.

The variable C‐terminal extension of G‐protein‐coupled receptor kinase 6 constitutes an accessorial autoregulatory domain

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.

Ligand‐dependent serum response factor activation by the human CC chemokine receptors CCR2a and CCR2b is mediated by G proteins of the Gq family

Expression of the human CCR2 receptors, CCR2a and CCR2b, in mammalian cells results in ligand‐dependent changes in the activity of multiple cellular signal transduction pathways, mediated in most cases by pertussis toxin‐sensitive heterotrimeric G proteins of the Gi/o subfamily. In addition, CCR2a and CCR2b receptors have been shown to couple to Gq family members, triggering the canonical activation of phospholipase Cβ isoenzymes. Activation of pertussis toxin‐insensitive Gq proteins by cell‐surface receptors is not only coupled to activation of phospholipase isoenzymes but also to Rho guanine nucleotide exchange factors, which in turn mediate activation of the Rho GTPases. Activated Rho GTPases regulate numerous cellular functions, including the organization of the actin cytoskeleton and gene transcription, such as the transcription factor serum response factor. These findings prompted us to investigate whether CCR2a and/or CCR2b stimulate serum response factor activity. The results presented herein demonstrate that stimulation of human CCR2a‐ or CCR2b‐expressing COS‐7 cells caused a vigorous induction of serum response factor activity. This effect was specifically mediated by Gq and/or G14, as well as Rho A and/or a closely related Rho GTPase. Furthermore, the stimulatory effect of CCR2a and CCR2b and Gαq was sensitive to coexpression of the Gαq‐interacting leukemia‐associated Rho guanine nucleotide exchange factor. The findings of the work indicate a role for Gαq and/or Gα14 and in CCR2a/CCR2b‐stimulated Rho A GTPase‐mediated serum response factor activation and introduce a noncanonical pathway activated by CCR2 receptors by coupling to Gq proteins.

Cool-temperature-mediated activation of phospholipase C-γ2 in the human hereditary disease PLAID

Deletions in the gene encoding signal-transducing inositol phospholipid-specific phospholipase C-γ2 (PLCγ2) are associated with the novel human hereditary disease PLAID (PLCγ2-associated antibody deficiency and immune dysregulation). PLAID is characterized by a rather puzzling concurrence of augmented and diminished functions of the immune system, such as cold urticaria triggered by only minimal decreases in temperature, autoimmunity, and immunodeficiency. Understanding of the functional effects of the genomic alterations at the level of the affected enzyme, PLCγ2, is currently lacking. PLCγ2 is critically involved in coupling various cell surface receptors to regulation of important functions of immune cells such as mast cells, B cells, monocytes/macrophages, and neutrophils. PLCγ2 is unique by carrying three Src (SH) and one split pleckstrin homology domain (spPH) between the two catalytic subdomains (spPHn-SH2n-SH2c-SH3-spPHc). Prevailing evidence suggests that activation of PLCγ2 is primarily due to loss of SH-region-mediated autoinhibition and/or enhanced plasma membrane translocation. Here, we show that the two PLAID PLCγ2 mutants lacking portions of the SH region are strongly (>100-fold), rapidly, and reversibly activated by cooling by only a few degrees. We found that the mechanism(s) underlying PLCγ2 PLAID mutant activation by cool temperatures is distinct from a mere loss of SH-region-mediated autoinhibition and dependent on both the integrity and the pliability of the spPH domain. The results suggest a new mechanism of PLCγ activation with unique thermodynamic features and assign a novel regulatory role to its spPH domain. Involvement of this mechanism in other human disease states associated with cooling such as exertional asthma and certain acute coronary events appears an intriguing possibility.