Compensatory increase of VE-cadherin expression through ETS1 regulates endothelial barrier function in response to TNFα.

VE-cadherin plays a central role in controlling endothelial barrier function, which is transiently disrupted by proinflammatory cytokines such as tumor necrosis factor (TNFα). Here we show that human endothelial cells compensate VE-cadherin degradation in response to TNFα by inducing VE-cadherin de novo synthesis. This compensation increases adherens junction turnover but maintains surface VE-cadherin levels constant. NF-κB inhibition strongly reduced VE-cadherin expression and provoked endothelial barrier collapse. Bacterial lipopolysaccharide and TNFα upregulated the transcription factor ETS1, in vivo and in vitro, in an NF-κB dependent manner. ETS1 gene silencing specifically reduced VE-cadherin protein expression in response to TNFα and exacerbated TNFα-induced barrier disruption. We propose that TNFα induces not only the expression of genes involved in increasing permeability to small molecules and immune cells, but also a homeostatic transcriptional program in which NF-κB- and ETS1-regulated VE-cadherin expression prevents the irreversible damage of endothelial barriers.

G-protein-coupled receptor kinase 2 safeguards epithelial phenotype in head and neck squamous cell carcinomas.

Head and neck squamous cell carcinoma (HNSCC) arises from the mucosal lining of the upper aerodigestive tract and display few treatment options in advanced stages. Despite increased knowledge of HNSCC molecular biology, the identification of new players involved in triggering HNSCC recurrence and metastatic disease is needed. We uncover that G-protein-coupled receptor kinase-2 (GRK2) expression is reduced in undifferentiated, high-grade human HNSCC tumors, whereas its silencing in model human HNSCC cells is sufficient to trigger epithelial-to-mesenchymal transition (EMT) phenotypic features, an EMT-like transcriptional program and enhanced lymph node colonization from orthotopic tongue tumors in mice. Conversely, enhancing GRK2 expression counteracts mesenchymal cells traits by mechanisms involving phosphorylation and decreased functionality of the key EMT inducer Snail1. Our results suggest that GRK2 safeguards the epithelial phenotype, whereas its downregulation contributes to the activation of EMT programs in HNSCC.

G protein-coupled receptor kinase 2 (GRK2) as a multifunctional signaling hub.

Accumulating evidence indicates that G protein-coupled receptor kinase 2 (GRK2) is a versatile protein that acts as a signaling hub by modulating G protein-coupled receptor (GPCR) signaling and also via phosphorylation or scaffolding interactions with an extensive number of non-GPCR cellular partners. GRK2 multifunctionality arises from its multidomain structure and from complex mechanisms of regulation of its expression levels, activity, and localization within the cell, what allows the precise spatio-temporal shaping of GRK2 targets. A better understanding of the GRK2 interactome and its modulation mechanisms is helping to identify the GRK2-interacting proteins and its substrates involved in the participation of this kinase in different cellular processes and pathophysiological contexts.

G protein-coupled receptor kinases (GRKs) in tumorigenesis and cancer progression: GPCR regulators and signaling hubs.

Increasing evidences point to G protein-coupled receptor kinases (GRKs), a subfamily of protein kinase A/G/C-like kinases, as relevant players in cancer progression, in a cell-type and tumor-specific way. Alterations in the expression and/or activity of particular GRKs have been identified in several types of tumors, and demonstrated to modulate the proliferation, survival or invasive properties of tumor cells by acting as integrating signaling nodes. GRKs are able to regulate the functionality of both G protein-coupled receptors (GPCR) and growth factor receptors and to directly control cytosolic, cytoskeletal or nuclear signaling components of pathways relevant for these processes. Furthermore, many chemokines as well as angiogenic and inflammatory factors present in the tumor microenvironment act through GPCR and other GRK-modulated signaling modules. Changes in the dosage of certain GRKs in the tumor stroma can alter tumor angiogenesis and the homing of immune cells, thus putting forward these kinases as potentially relevant modulators of the carcinoma-fibroblast-endothelial-immune cell network fostering tumor development and dissemination. A better understanding of the alterations in different GRK isoforms taking place during cancer development and metastasis in specific tumors and cell types and of its impact in signaling pathways would help to design novel therapeutic strategies.

Calpains mediate isoproterenol-induced hypertrophy through modulation of GRK2.

Inhibition of the Ca2+-dependent proteases calpains attenuates post-infarction remodeling and heart failure. Recent data suggest that calpain activity is elevated in non-ischemic cardiomyopathies and that upregulation of the key cardiac G-protein-coupled receptor kinase 2 (GRK2) signaling hub promotes cardiac hypertrophy. However, the functional interactions between calpains and GRK2 in this context have not been explored. We hypothesized that calpain modulates GRK2 levels in myocardial hypertrophy of non-ischemic cause, and analyzed the mechanisms involved and the potential therapeutic benefit of inhibiting calpain activity in this situation. The oral calpain inhibitor SNJ-1945 was administered daily to male Sprague-Dawley rats or wild-type and hemizygous GRK2 mice treated with 5 mg/Kg/day isoproterenol intraperitoneally for 1 week. In isoproterenol-treated animals, calpains 1 and 2 were overexpressed in myocardium and correlated with increased calpain activity and ventricular hypertrophy. Oral co-administration of SNJ-1945 attenuated calpain activation and reduced heart hypertrophy as assessed using morphological and biochemical markers. Calpain activation induced by isoproterenol increased GRK2 protein levels, while genetic downregulation of GRK2 expression prevented isoproterenol-mediated hypertrophy independently of calpain inhibition. GRK2 upregulation was associated to calpain-dependent degradation of the GRK2 ubiquitin ligase MDM2 and to enhanced NF-κB-dependent GRK2 gene expression in correlation with calpain-mediated IĸB proteolysis. These results demonstrate that calpain mediates isoproterenol-induced myocardial hypertrophy by modulating GRK2 protein content through mechanisms involving the control of GRK2 stability and expression. Sustained calpain inhibition attenuates isoproterenol-induced myocardial hypertrophy and could be an effective therapeutic strategy to limit ventricular remodeling of non-ischemic origin.

Protein Kinase C ζ Interacts with a Novel Binding Region of Gαq to Act as a Functional Effector.

Heterotrimeric G proteins play an essential role in the initiation of G protein-coupled receptor (GPCR) signaling through specific interactions with a variety of cellular effectors. We have recently reported that GPCR activation promotes a direct interaction between Gαq and protein kinase C ζ (PKCζ), leading to the stimulation of the ERK5 pathway independent of the canonical effector PLCß. We report herein that the activation-dependent Gαq/PKCζ complex involves the basic PB1-type II domain of PKCζ and a novel interaction module in Gαq different from the classical effector-binding site. Point mutations in this Gαq region completely abrogate ERK5 phosphorylation, indicating that Gαq/PKCζ association is required for the activation of the pathway. Indeed, PKCζ was demonstrated to directly bind ERK5 thus acting as a scaffold between Gαq and ERK5 upon GPCR activation. The inhibition of these protein complexes by G protein-coupled receptor kinase 2, a known Gαq modulator, led to a complete abrogation of ERK5 stimulation. Finally, we reveal that Gαq/PKCζ complexes link Gαq to apoptotic cell death pathways. Our data suggest that the interaction between this novel region in Gαq and the effector PKCζ is a key event in Gαq signaling.

Protein kinase C (PKC)ζ-mediated Gαq stimulation of ERK5 protein pathway in cardiomyocytes and cardiac fibroblasts.

Gq-coupled G protein-coupled receptors (GPCRs) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gα(q)-mediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gα(q) acts as an adaptor protein that facilitates PKCζ-mediated activation of ERK5 in epithelial cells. Because the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gq-dependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gq-coupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNA-mediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal cardiomyocytes isolated from PKCζ-deficient mice. Moreover, upon chronic challenge with angiotensin II, these mice fail to promote the changes in the ERK5 pathway, in gene expression patterns, and in hypertrophic markers observed in wild-type animals. Taken together, our results show that PKCζ is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts and indicate a key cardiac physiological role for the Gα(q)/PKCζ/ERK5 signaling axis.

G protein-coupled receptor kinase 2 positively regulates epithelial cell migration.

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration.

Gq Signaling in Autophagy Control: Between Chemical and Mechanical Cues.

All processes in human physiology relies on homeostatic mechanisms which require the activation of specific control circuits to adapt the changes imposed by external stimuli. One of the critical modulators of homeostatic balance is autophagy, a catabolic process that is responsible of the destruction of long-lived proteins and organelles through a lysosome degradative pathway. Identification of the mechanism underlying autophagic flux is considered of great importance as both protective and detrimental functions are linked with deregulated autophagy. At the mechanistic and regulatory levels, autophagy is activated in response to diverse stress conditions (food deprivation, hyperthermia and hypoxia), even a novel perspective highlight the potential role of physical forces in autophagy modulation. To understand the crosstalk between all these controlling mechanisms could give us new clues about the specific contribution of autophagy in a wide range of diseases including vascular disorders, inflammation and cancer. Of note, any homeostatic control critically depends in at least two additional and poorly studied interdependent components: a receptor and its downstream effectors. Addressing the selective receptors involved in autophagy regulation is an open question and represents a new area of research in this field. G-protein coupled receptors (GPCRs) represent one of the largest and druggable targets membrane receptor protein superfamily. By exerting their action through G proteins, GPCRs play fundamental roles in the control of cellular homeostasis. Novel studies have shown Gαq, a subunit of heterotrimeric G proteins, as a core modulator of mTORC1 and autophagy, suggesting a fundamental contribution of Gαq-coupled GPCRs mechanisms in the control of this homeostatic feedback loop. To address how GPCR-G proteins machinery integrates the response to different stresses including oxidative conditions and mechanical stimuli, could provide deeper insight into new signaling pathways and open potential and novel therapeutic strategies in the modulation of different pathological conditions.

G alpha(q) acts as an adaptor protein in protein kinase C zeta (PKCzeta)-mediated ERK5 activation by G protein-coupled receptors (GPCR).

G(q)-coupled G protein-coupled receptors (GPCR) mediate the actions of a variety of messengers that are key regulators of different cellular functions. These receptors can regulate a highly interconnected network of biochemical routes that control the activity of several members of the mitogen-activated protein kinase (MAPK) family. The ERK5 MAPK has been shown to be activated by G(q)-coupled GPCR via unknown mechanisms. We find that the atypical protein kinase C (PKCzeta), previously reported to interact with the ERK5 activator MEK5 and to be involved in epidermal growth factor-mediated ERK5 stimulation, plays a crucial role in the activation of the ERK5 pathway by G(q)-coupled GPCR. Stimulation of ERK5 by G(q)-coupled GPCR is abolished upon pharmacological inhibition of PKCzeta as well as in embryonic fibroblasts obtained from PKCzeta-deficient mice. Both PKCzeta and MEK5 associate to G alpha(q) upon activation of GPCR, thus forming a ternary complex that seems essential for the activation of ERK5. These data put forward a novel function of G alpha(q) as a scaffold protein involved in the modulation of the ERK5 cascade by GPCR that could be relevant in G(q)-mediated physiological functions.

Gαq activation modulates autophagy by promoting mTORC1 signaling.

The mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gαq subunit, a known transducer of plasma membrane G protein-coupled receptors signaling, as a core modulator of mTORC1 and autophagy. Cells lacking Gαq/11 display higher basal autophagy, enhanced autophagy induction upon different types of nutrient stress along with a decreased mTORC1 activation status. They are also unable to reactivate mTORC1 and thus inactivate ongoing autophagy upon nutrient recovery. Conversely, stimulation of Gαq/11 promotes sustained mTORC1 pathway activation and reversion of autophagy promoted by serum or amino acids removal. Gαq is present in autophagic compartments and lysosomes and is part of the mTORC1 multi-molecular complex, contributing to its assembly and activation via its nutrient status-sensitive interaction with p62, which displays features of a Gαq effector. Gαq emerges as a central regulator of the autophagy machinery required to maintain cellular homeostasis upon nutrient fluctuations.

Membrane interactions of G proteins and other related proteins.

Guanine nucleotide-binding proteins, G proteins, propagate incoming messages from receptors to effector proteins. They switch from an inactive to active state by exchanging a GDP molecule for GTP, and they return to the inactive form by hydrolyzing GTP to GDP. Small monomeric G proteins, such as Ras, are involved in controlling cell proliferation, differentiation and apoptosis, and they interact with membranes through isoprenyl moieties, fatty acyl moieties, and electrostatic interactions. This protein-lipid binding facilitates productive encounters of Ras and Raf proteins in defined membrane regions, so that signals can subsequently proceed through MEK and ERK kinases, which constitute the canonical MAP kinase signaling cassette. On the other hand, heterotrimeric G proteins undergo co/post-translational modifications in the alpha (myristic and/or palmitic acid) and the gamma (farnesol or geranylgeraniol) subunits. These modifications not only assist the G protein to localize to the membrane but they also help distribute the heterotrimer (Galphabetagamma) and the subunits generated upon activation (Galpha and Gbetagamma) to appropriate membrane microdomains. These proteins transduce messages from ubiquitous serpentine receptors, which control important functions such as taste, vision, blood pressure, body weight, cell proliferation, mood, etc. Moreover, the exchange of GDP by GTP is triggered by nucleotide exchange factors. Membrane receptors that activate G proteins can be considered as such, but other cytosolic, membranal or amphitropic proteins can accelerate the rate of G protein exchange or even activate this process in the absence of receptor-mediated activation. These and other protein-protein interactions of G proteins with other signaling proteins are regulated by their lipid preferences. Thus, G protein-lipid interactions control the features of messages and cell physiology.

An Overview on G Protein-coupled Receptor-induced Signal Transduction in Acute Myeloid Leukemia.

BACKGROUND: Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by uncontrolled proliferation of precursor myeloid-lineage cells in the bone marrow. AML is also characterized by patients with poor long-term survival outcomes due to relapse. Many efforts have been made to understand the biological heterogeneity of AML and the challenges to develop new therapies are therefore enormous. G Protein-coupled Receptors (GPCRs) are a large attractive drug-targeted family of transmembrane proteins, and aberrant GPCR expression and GPCR-mediated signaling have been implicated in leukemogenesis of AML. This review aims to identify the molecular players of GPCR signaling, focusing on the hematopoietic system, which are involved in AML to help developing novel drug targets and therapeutic strategies. METHODS: We undertook an exhaustive and structured search of bibliographic databases for research focusing on GPCR, GPCR signaling and expression in AML. RESULTS AND CONCLUSION: Many scientific reports were found with compelling evidence for the involvement of aberrant GPCR expression and perturbed GPCR-mediated signaling in the development of AML. The comprehensive analysis of GPCR in AML provides potential clinical biomarkers for prognostication, disease monitoring and therapeutic guidance. It will also help to provide marker panels for monitoring in AML. We conclude that GPCR-mediated signaling is contributing to leukemogenesis of AML, and postulate that mass spectrometrybased protein profiling of primary AML cells will accelerate the discovery of potential GPCR related biomarkers for AML.

The G protein-coupled receptor kinase (GRK) interactome: role of GRKs in GPCR regulation and signaling.

G protein-coupled receptor kinases (GRKs) and arrestins are key participants in the canonical pathways leading to phosphorylation-dependent GPCR desensitization, endocytosis, intracellular trafficking and resensitization as well as in the modulation of important intracellular signaling cascades by GPCR. Novel studies have revealed a phosphorylation-independent desensitization mechanism operating through their RGS-homology (RH) domain and the recent determination of the crystal structures of GRK2 and GRK6 has uncovered interesting details on the structure-function relationships of these kinases. Emerging evidence indicates that the activity of GRKs is tightly modulated by mechanisms including phosphorylation by different kinases and interaction with several cellular proteins such as calmodulin, caveolin or RKIP. In addition, GRKs are involved in multiple interactions with non-receptor proteins (PI3K, Akt, GIT or MEK) that point to novel GRK cellular roles. In this article, our purpose is to describe the ever increasing map of functional interactions for GRK proteins as a basis to better understand its contribution to cellular processes.

G protein-coupled receptor kinase 2 (GRK2) in migration and inflammation.

G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors and other plasma membrane receptors stimulated by chemotactic messengers. On top of that, GRK2 has been reported to interact with a variety of signal transduction proteins related to cell migration such as MEK, Akt, PI3Kgamma or GIT. Interestingly, the levels of expression and activity of this kinase are altered in a number of inflammatory disorders (as rheumatoid arthritis or multiple sclerosis), thus suggesting that it may play an important role in the onset or development of these pathologies. This review summarizes the mechanisms involved in the control of GRK2 expression and function and highlights novel functional interactions of this protein that might help to explain how altered GRK2 levels affects cell migration in different cell types and pathological settings.

Tyrosine phosphorylation of G-protein-coupled-receptor kinase 2 (GRK2) by c-Src modulates its interaction with Galphaq.

G-protein-coupled-receptor kinase 2 (GRK2) plays a key role in the modulation of G-protein-coupled-receptor (GPCR) signaling by both phosphorylating agonist-occupied GPCRs and by directly binding to activated Galphaq subunits, inhibiting downstream effectors activation. The GRK2/Galphaq interaction involves the N-terminal region of the kinase that displays homology to regulators of G-protein signaling (RGS) proteins. We have previously reported that upon GPCR stimulation, GRK2 can be phosphorylated by c-Src on tyrosine residues that are present in the RGS-homology (RH) region of this kinase. Here, we demonstrate that c-Src kinase activity increases the interaction between GRK2 and Galphaq. Tyrosine phosphorylation of GRK2 appears to be critically involved in the modulation of this interaction since the stimulatory effect of c-Src is not observed with a GRK2 mutant with impaired tyrosine phosphorylation (GRK2 Y13,86,92F), whereas a mutant that mimics GRK2 tyrosine phosphorylation in these residues displays an increased interaction with Galphaq. As evidence for a physiological role of this modulatory mechanism, activation of the muscarinic receptor M1, a Galphaq-coupled receptor, promotes an increase in GRK2/Galphaq co-immunoprecipitation that parallels the enhanced GRK2 phosphorylation on tyrosine residues. Moreover, c-Src activation enhances inhibition of the Galphaq/phospholipase Cbeta signaling pathway in intact cells, in a GRK2-tyrosine-phosphorylation-dependent manner. Our results suggest a feedback mechanism by which phosphorylation of GRK2 by c-Src increases both GRK2 kinase activity towards GPCRs and its specific interaction with Galphaq subunits, leading to a more rapid switch off of Galphaq-mediated signaling.

Mechanisms of regulation of G protein-coupled receptor kinases (GRKs) and cardiovascular disease.

The G protein-coupled receptor kinases (GRKs) participate with arrestins in the regulation and signal propagation of multiple G protein-coupled receptors (GPCR) of key physiological and pharmacological relevance in the cardiovascular system. The complex mechanisms of regulation of GRK expression, degradation and function are being unveiled gradually. The levels of these kinases are known to change in pathological situations such as heart failure, hypertrophy and hypertension, and in animal models of these diseases. A better understanding of the mechanisms underlying these changes and of how these alterations participate in the triggering or progression of cardiovascular disease may contribute to the design of novel diagnostic and therapeutic strategies.

BMP-7 attenuates left ventricular remodelling under pressure overload and facilitates reverse remodelling and functional recovery.

AIMS: TGF-ß regulates tissue fibrosis: TGF-ß promotes fibrosis, whereas bone morphogenetic protein (BMP)-7 is antifibrotic. To demonstrate that (i) left ventricular (LV) remodelling after pressure overload is associated with disequilibrium in the signalling mediated by these cytokines, and (ii) BMP-7 exerts beneficial effects on LV remodelling and reverse remodelling. METHODS AND RESULTS: We studied patients with aortic stenosis (AS) and mice subjected to transverse aortic constriction (TAC) and TAC release (de-TAC). LV morphology and function were assessed by echocardiography. LV biopsies were analysed by qPCR, immunoblotting, and histology. Pressure overload reduced BMP-7 and pSmad1/5/8 and increased TGF-ß and pSmad2/3 in AS patients and TAC mice. BMP-7 correlated inversely with collagen, fibronectin, and ß-MHC expressions, and with hypertrophy and diastolic dysfunction, and directly with the systolic function. Multiple linear regression disclosed BMP-7 and TGF-ß as hypertrophy predictors, negative and positive, respectively. BMP-7 prevented TGF-ß-elicited hypertrophic program in cardiomyocytes, and Col1A1 promoter activity in NIH-3T3 fibroblasts. The treatment of TAC mice with rBMP-7 attenuated the development of structural damage and dysfunction, and halted ongoing remodelling. The reverse remodelling after pressure overload release was facilitated by rBMP-7, and hampered by disrupting BMP-7 function using a neutralizing antibody or genetic deletion. CONCLUSION: The disequilibrium between BMP-7 and TGF-ß signals plays a relevant role in the LV remodelling response to haemodynamic stress in TAC mice and AS patients. Our observations may provide new important insights aimed at developing novel therapies designed to prevent, halt, or reverse LV pathological remodelling in pressure overload cardiomyopathy.

Mechanisms of regulation of the expression and function of G protein-coupled receptor kinases.

G protein-coupled receptor kinases (GRKs) are key modulators of G protein-coupled receptor signalling. Increasing evidence points to the occurrence of complex mechanisms able to modulate the subcellular localization, activity and expression levels of GRKs, revealing new functional interactions of these kinases with different cellular proteins and transduction cascades. GRK activity and subcellular targeting is tightly regulated by interaction with receptor domains, G protein subunits, lipids, anchoring proteins, caveolin and calcium-sensing proteins. In addition, GRK phosphorylation by several other kinases has recently been shown to modulate its functionality, thus putting forward new feedback mechanisms connecting different signalling pathways to G protein-coupled receptors (GPCR) regulation. On the other hand, the mechanisms governing GRK expression at both transcriptional and protein stability levels are just beginning to be unveiled. Namely, GRK2 has been shown to be rapidly degraded by the proteasome pathway in a process dependent on beta-arrestin and c-Src function, and also to be proteolyzed by m-calpain. A better knowledge of GRK regulatory mechanisms would contribute to greater understanding of GRK physiological function and also its reported alterations in different pathological situations, such as congestive heart failure, hypertension or inflammation.

Gαq signalling: the new and the old.

In the last few years the interactome of Gαq has expanded considerably, contributing to improve our understanding of the cellular and physiological events controlled by this G alpha subunit. The availability of high-resolution crystal structures has led the identification of an effector-binding region within the surface of Gαq that is able to recognise a variety of effector proteins. Consequently, it has been possible to ascribe different Gαq functions to specific cellular players and to identify important processes that are triggered independently of the canonical activation of phospholipase Cß (PLCß), the first identified Gαq effector. Novel effectors include p63RhoGEF, that provides a link between G protein-coupled receptors and RhoA activation, phosphatidylinositol 3-kinase (PI3K), implicated in the regulation of the Akt pathway, or the cold-activated TRPM8 channel, which is directly inhibited upon Gαq binding. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has also been described as a novel PLCß-independent signalling axis that relies upon the interaction between this G protein and two novel effectors (PKCζ and MEK5). Additionally, the association of Gαq with different regulatory proteins can modulate its effector coupling ability and, therefore, its signalling potential. Regulators include accessory proteins that facilitate effector activation or, alternatively, inhibitory proteins that downregulate effector binding or promote signal termination. Moreover, Gαq is known to interact with several components of the cytoskeleton as well as with important organisers of membrane microdomains, which suggests that efficient signalling complexes might be confined to specific subcellular environments. Overall, the complex interaction network of Gαq underlies an ever-expanding functional diversity that puts forward this G alpha subunit as a major player in the control of physiological functions and in the development of different pathological situations.