G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β₂-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling.     

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of histamine H2 receptor by G protein-coupled receptor kinase 2

It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by β-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a Gα(s)-coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrin-homology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domain-mediated regulation of GPCRs beyond Gα(q)-coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.     

Differential regulation of β2-adrenoceptor and adenosine A2B receptor signalling by GRK and arrestin proteins in arterial smooth muscle

Generation of cAMP through G_s-coupled G protein-coupled receptor (GPCR) [e.g. β₂-adrenoceptor (β₂AR), adenosine A_2B receptor (A_2BR)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors. G_s-coupled GPCR signalling is regulated by G protein-coupled receptor kinases (GRK) and arrestin proteins, and dysregulation of Gs/GPCR signalling is thought play a role in the development of hypertension, which may be a consequence of enhanced GRK2 and/or arrestin expression. However, despite numerous studies indicating that β₂AR and A_2BR can be substrates for GRK/arrestin proteins, currently little is known regarding GRK/arrestin regulation of these endogenous receptors in arterial smooth muscle. Here, endogenous GRK isoenzymes and arrestin proteins were selectively depleted using RNA-interference in rat arterial smooth muscle cells (RASM) and the consequences of this for β₂AR- and A_2BR-mediated adenylyl cyclase (AC) signalling were determined by assessing cAMP accumulation. GRK2 or GRK5 depletion enhanced and prolonged β₂AR/AC signalling, while combined deletion of GRK2/5 has an additive effect. Conversely, activation of AC by A_2BR was regulated by GRK5, but not GRK2. β₂AR desensitization was attenuated following combined GRK2/GRK5 knockdown, but not by depletion of individual GRKs, arrestins, or by inhibiting PKA. Arrestin3 (but not arrestin2) depletion enhanced A_2BR-AC signalling and attenuated A_2BR desensitization, while β₂AR-AC signalling was regulated by both arrestin isoforms. This study provides a first demonstration of how different complements of GRK and arrestin proteins contribute to the regulation of signalling and desensitization of these important receptors mediating vasodilator responses in arterial smooth muscle.     

Structural domains required for Caenorhabditis elegans G protein-coupled receptor kinase 2 (GRK-2) function in vivo

G protein-coupled receptor kinases (GRKs) are key regulators of signal transduction that specifically phosphorylate activated G protein-coupled receptors (GPCRs) to terminate signaling. Biochemical and crystallographic studies have provided great insight into mammalian GRK2/3 interactions and structure. However, despite extensive in vitro characterization, little is known about the in vivo contribution of these described GRK structural domains and interactions to proper GRK function in signal regulation. We took advantage of the disrupted chemosensory behavior characteristic of Caenorhabditis elegans grk-2 mutants to discern the interactions required for proper in vivo Ce-GRK-2 function. Informed by mammalian crystallographic and biochemical data, we introduced amino acid substitutions into the Ce-grk-2 coding sequence that are predicted to selectively disrupt GPCR phosphorylation, Gα(q/11) binding, Gβγ binding, or phospholipid binding. Changing the most amino-terminal residues, which have been shown in mammalian systems to be required specifically for GPCR phosphorylation but not phosphorylation of alternative substrates or recruitment to activated GPCRs, eliminated the ability of Ce-GRK-2 to restore chemosensory signaling. Disrupting interaction between the predicted Ce-GRK-2 amino-terminal α-helix and kinase domain, posited to stabilize GRKs in their active ATP- and GPCR-bound conformation, also eliminated Ce-GRK-2 chemosensory function. Finally, although changing residues within the RH domain, predicted to disrupt interaction with Gα(q/11), did not affect Ce-GRK-2 chemosensory function, disruption of the predicted PH domain-mediated interactions with Gβγ and phospholipids revealed that both contribute to Ce-GRK-2 function in vivo. Combined, we have demonstrated functional roles for broadly conserved GRK2/3 structural domains in the in vivo regulation of organismal behavior.     

GRK2 Targeted Knock-down Results in Spontaneous Hypertension,and Altered Vascular GPCR Signaling

Hypertension, elevated arterial pressure, occurs as the consequence of increased peripheral resistance. G protein-coupled receptors (GPCRs) contribute to the regulation of vasodilator and vasoconstrictor responses, and their activity is regulated by a family of GPCR kinases (GRKs). GRK2 expression is increased in hypertension and this facilitates the development of the hypertensive state by increasing the desensitization of GPCRs important for vasodilation. We demonstrate here, that genetic knockdown of GRK2 using a small hairpin (sh) RNA results in altered vascular reactivity and the development of hypertension between 8–12 weeks of age in shGRK2 mice due to enhanced Gα_q/11 signaling. Vascular smooth muscle cells (VSMCs) cultured from shGRK2 knockdown mice show increases in GPCR-mediated Gα_s and Gα_q/11 signaling, as the consequence of reduced GRK2-mediated desensitization. In addition, agonists and biased agonists exhibited age-dependent alterations in ERK1/2 and Akt signaling, as well as cell proliferation and migration responses in shGRK2 knockdown VSMCs when cultured from mice that are either 3 months or 6 months of age. Changes in angiotensin II-stimulated ERK1/2 phosphorylation are observed in VSMCs derived from 6-week-old shGRK2 mice prior to the development of the hypertensive phenotype. Thus, our findings indicate that the balance between mechanisms regulating vascular tone are shifted to favor vasoconstriction in the absence of GRK2 expression and that this leads to the age-dependent development of hypertension, as a consequence of global alterations in GPCR signaling. Consequently, therapeutic strategies that target GRK2 activity, not expression, may be more effective for the treatment of hypertension.     

β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent 5‐HT4 receptor signalling

G protein‐coupled receptors (GPCRs) have been found to trigger G protein‐independent signalling. However, the regulation of G protein‐independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein‐independent 5‐HT₄ receptor (5‐HT₄R)‐operated Src/ERK (extracellular signal‐regulated kinase) pathway, but not the G_s pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor’ C‐terminus in both human embryonic kidney (HEK)‐293 cells and colliculi neurons. This inhibition required two sequences of events: the association of β–arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C‐t domain and the phosphorylation, by GRK5, of β–arrestin1 (at Ser⁴¹²) bound to the receptor. Phosphorylated β‐arrestin1 in turn prevented activation of Src constitutively bound to 5‐HT₄Rs, a necessary step in receptor‐stimulated ERK signalling. This is the first demonstration that β‐arrestin1 phosphorylation by GRK5 regulates G protein‐independent signalling.     

The G protein Gαs acts as a tumor suppressor in sonic hedgehog signaling-driven tumorigenesis

G protein-coupled receptors (GPCRs) are critical players in tumor growth and progression. The redundant roles of GPCRs in tumor development confound effective treatment; therefore, targeting a single common signaling component downstream of these receptors may be efficacious. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gβγ subunits. Hyperactive Gα_s signaling can mediate tumor progression in some tissues; however, recent work in medulloblastoma and basal cell carcinoma revealed that Gα_s can also function as a tumor suppressor in neoplasms derived from ectoderm cells including neural and epidermal stem/progenitor cells. In these stem-cell compartments, signaling through Gα_s suppresses self-renewal by inhibiting the Sonic Hedgehog (SHH) and Hippo pathways. The loss of GNAS, which encodes Gα_s, leads to activation of these pathways, over-proliferation of progenitor cells, and tumor formation. Gα_s activates the cAMP-dependent protein kinase A (PKA) signaling pathway and inhibits activation of SHH effectors Smoothened-Gli. In addition, Gα_s-cAMP-PKA activation negatively regulates the Hippo pathway by blocking the NF2-LATS1/2-Yap signaling. In this review, we will address the novel function of the signaling network regulated by Gα_s in suppression of SHH-driven tumorigenesis and the therapeutic approaches that can be envisioned to harness this pathway to inhibit tumor growth and progression.     

Gβγ interacts with mTOR and promotes its activation

Diverse G protein-coupled receptors depend on Gβγ heterodimers to promote cell polarization and survival via direct activation of PI3Kγ and potentially other effectors. These events involve full activation of AKT via its phosphorylation at Ser473, suggesting that mTORC2, the kinase that phosphorylates AKT at Ser473, is activated downstream of Gβγ. Thus, we tested the hypothesis that Gβγ directly contributes to mTOR signaling. Here, we demonstrate that endogenous mTOR interacts with Gβγ. Cell stimulation with serum modulates Gβγ interaction with mTOR. The carboxyl terminal region of mTOR, expressed as a GST-fusion protein, including the serine/threonine kinase domain, binds Gβγ heterodimers containing different Gβ subunits, except Gβ₄. Both, mTORC1 and mTORC2 complexes interact with Gβ₁γ₂ which promotes phosphorylation of their respective substrates, p70S6K and AKT. In addition, chronic treatment with rapamycin, a condition known to interfere with assembly of mTORC2, reduces the interaction between Gβγ and mTOR and the phosphorylation of AKT; whereas overexpression of Gαi interfered with the effect of Gβγ as promoter of p70S6K and AKT phosphorylation. Altogether, our results suggest that Gβγ positively regulates mTOR signaling via direct interactions and provide further support to emerging strategies based on the therapeutical potential of inhibiting different Gβγ signaling interfaces.     

Molecular modelling of human 5-hydroxytryptamine receptor (5-HT2A) and virtual screening studies towards the identification of agonist and antagonist molecules

The serotonin receptors, also known as 5-hydroxytryptamine (5-HT) receptors, are a group of G protein-coupled receptors (GPCRs) and ligand-gated ion channels found in the central and peripheral nervous systems. GPCRs have a characteristic feature of activating different signalling pathways upon ligand binding and these ligands display several efficacy levels to differentially activate the receptor. GPCRs are primary drug targets due to their central role in several signal transduction pathways. Drug design for GPCRs is also most challenging due to their inherent promiscuity in ligand recognition, which gives rise to several side effects of existing drugs. Here, we have performed the ligand interaction study using the two prominent states of GPCR, namely the active and inactive state of the 5-HT_2A receptor. Active state of 5-HT_2A receptor model enhances the understanding of conformational difference which influences the ligand-binding site. A 5-HT_2A receptor active state model was constructed by homology modelling using active state β₂-adrenergic receptor (β₂-AR). In addition, virtual screening and docking studies with both active and inactive state models reveal potential small molecule hits which could be considered as agonist-like and antagonist-like molecules. The results from the all-atom molecular dynamics simulations further confirmed that agonists and antagonists interact in different modes with the receptor.     

Sensing and transduction of nutritional and chemical signals in filamentous fungi: Impact on cell development and secondary metabolites biosynthesis

Filamentous fungi respond to hundreds of nutritional, chemical and environmental signals that affect expression of primary metabolism and biosynthesis of secondary metabolites. These signals are sensed at the membrane level by G protein coupled receptors (GPCRs). GPCRs contain usually seven transmembrane domains, an external amino terminal fragment that interacts with the ligand, and an internal carboxy terminal end interacting with the intracellular G protein. There is a great variety of GPCRs in filamentous fungi involved in sensing of sugars, amino acids, cellulose, cell-wall components, sex pheromones, oxylipins, calcium ions and other ligands. Mechanisms of signal transduction at the membrane level by GPCRs are discussed, including the internalization and compartmentalisation of these sensor proteins. We have identified and analysed the GPCRs in the genome of Penicillium chrysogenum and compared them with GPCRs of several other filamentous fungi. We have found 66 GPCRs classified into 14 classes, depending on the ligand recognized by these proteins, including most previously proposed classes of GPCRs. We have found 66 putative GPCRs, representatives of twelve of the fourteen previously proposed classes of GPCRs, depending on the ligand recognized by these proteins. A staggering fortytwo putative members of the new GPCR class XIV, the so-called Pth11 sensors of cellulosic material as reported for Neurospora crassa and some other fungi, were identified. Several GPCRs sensing sex pheromones, known in yeast and in several fungi, were also identified in P. chrysogenum, confirming the recent unravelling of the hidden sexual capacity of this species. Other sensing mechanisms do not involve GPCRs, including the two-component systems (HKRR), the HOG signalling system and the PalH mediated pH transduction sensor. GPCR sensor proteins transmit their signals by interacting with intracellular heterotrimeric G proteins, that are well known in several fungi, including P. chrysogenum. These G proteins are inactive in the GDP containing heterotrimeric state, and become active by nucleotide exchange, allowing the separation of the heterotrimeric protein in active Gα and Gβγ dimer subunits. The conversion of GTP in GDP is mediated by the endogenous GTPase activity of the G proteins. Downstream of the ligand interaction, the activated Gα protein and also the Gβ/Gγ dimer, transduce the signals through at least three different cascades: adenylate cyclase/cAMP, MAPK kinase, and phospholipase C mediated pathways.     

Phosphatidic acid potentiates Gαq stimulation of phospholipase C-β1 signaling

Phosphatidic acid (PA) is interactive with Gα_q-linked agonists to stimulate GPCR signaling via phospholipase C-β₁ (PLC-β₁). Phorbol 12-myristate 13-acetate (PMA) increases cellular levels of PA and phospholipase D activity (PLD). This study evaluated whether PMA can stimulate PLC-β₁ activity via PA, independent of GPCR input in transfected COS 7 cells. PMA alone had little effect on PLC activity in cells co-transfected with PLC-β₁ and Gα_q. Activated Gα_q, induced by co-transfecting muscarinic cholinergic receptor (m1R), was necessary for stimulation of PLC-β₁ activity by PMA. Stimulation by PMA was dependent on the PA-regulatory motif of PLC-β₁ implicating PA in this mechanism. PLD1 knockdown by antisense decreased responsiveness of PLC-β₁ to both PMA and carbachol. PA alone thus has little effect on PLC-β₁ activity, but PA and PLD1 synergize with activated Gα_q to stimulate PLC-β₁ signaling. Coordinate interaction with activated Gα_q may serve as an important mechanism to fine tune response to ligands while preventing spurious initiation of PLC-β signaling by PA in cells.     

POTENTIATION OF GPCR-SIGNALING VIA MEMBRANE TARGETING OF G PROTEIN α SUBUNITS

ABSTRACT

Different assay technologies are available that allow ligand occupancy of G protein coupled receptors to be converted into robust functional assay signals. Of particular interest are universal screening systems such that activation of any GPCR can be detected with a common assay end point. The promiscuous G protein Gα₁₆ and chimeric G proteins are broadly used tools for setting up almost universal assay systems. Many efforts focused on making G proteins more promiscuous, however no attempts have been made to make promiscuos G proteins more sensitive by interfering with their cellular protein distribution. As a model system, we used a promiscuous G protein α_q subunit, that lacks the highly conserved six amino acid N-terminal extension and bears four residues of α_i sequence at its C-terminus replacing the corresponding α_q sequence (referred to as Δ6qi4). When expressed in COS7 cells, Δ6qi4 undergoes palmitoylation at its N-terminus. Cell fractionation and immunoblotting analysis indicated localization in the particulate and cytosolic fraction. Interestingly, introduction of a consensus site for N-terminal myristoylation (the resulting mutant referred to as Δ6qi4myr) created a protein that was dually acylated and exclusively located in the particulate fraction. As a measure of G protein activation Δ6qi4 and Δ6qi4myr were coexpressed (in CHO cells) with a series of different Gi/o coupled receptors and ligand induced increases in intracellular Ca²⁺ release were determined with the FLIPR? technology (Fluorescence plate imaging reader from Molecular Devices Corp.). All of the receptors interacted more efficiently with Δ6qi4myr as compared with Δ6qi4. It could be shown that increased functional responses of agonist activated GPCRs are due to the higher content of Δ6qi4myr in the plasma membrane. Our results indicate that manipulation of subcellular localization of G protein α subunits—moving them from the cytosol to the plasma membrane-potentiates signaling of agonist activated GPCRs. It is concluded that addition of myristoylation sites into otherwise exclusively palmitoylated G proteins is a new and sensitive approach and may be applicable when functional assays are expected to yield weak signals as is the case when screening extracts of tissues for biologically active GPCR ligands.     

The Epstein-Barr Virus-encoded G Protein-coupled Receptor BILF1 Hetero-oligomerizes with Human CXCR4, Scavenges Gαi Proteins,and Constitutively Impairs CXCR4 Functioning

Cells express distinct G protein-coupled receptor (GPCR) subtypes on their surface, allowing them to react to a corresponding variety of extracellular stimuli. Cross-regulation between different ligand-GPCR pairs is essential to generate appropriate physiological responses. GPCRs can physically affect each other''s functioning by forming heteromeric complexes, whereas cross-regulation between activated GPCRs also occurs through integration of shared intracellular signaling networks. Human herpesviruses utilize virally encoded GPCRs to hijack cellular signaling networks for their own benefit. Previously, we demonstrated that the Epstein-Barr virus-encoded GPCR BILF1 forms heterodimeric complexes with human chemokine receptors. Using a combination of bimolecular complementation and bioluminescence resonance energy transfer approaches, we now show the formation of hetero-oligomeric complexes between this viral GPCR and human CXCR4. BILF1 impaired CXCL12 binding to CXCR4 and, consequently, also CXCL12-induced signaling. In contrast, the G protein uncoupled mutant BILF1-K^3.50A affected CXCL12-induced CXCR4 signaling to a much lesser extent, indicating that BILF1-mediated CXCR4 inhibition is a consequence of its constitutive activity. Co-expression of Gα_i1 with BILF1 and CXCR4 restored CXCL12-induced signaling. Likewise, BILF1 formed heteromers with the human histamine H₄ receptor (H₄R). BILF1 inhibited histamine-induced Gα_i-mediated signaling by H₄R, however, without affecting histamine binding to this receptor. These data indicate that functional cross-regulation of Gα_i-coupled GPCRs by BILF1 is at the level of G proteins, even though these GPCRs are assembled in hetero-oligomeric complexes.     

G蛋白偶联受体激酶5在有丝分裂中的作用

G蛋白偶联受体激酶（GRK）是G蛋白偶联受体（GPCR）信号通路的负性调节因子。近来的研究发现,GRK除了磷酸化G蛋白偶联受体使其脱敏外,还能与其他非受体底物结合,功能呈现多样性。GRK5是GRK家族成员之一,该研究探索了GRK5在细胞周期和有丝分裂中的作用,结果显示：在细胞内干扰GRK5的表达导致分裂中期的细胞数目增多和细胞凋亡。进一步的研究发现,干扰GRK5的表达导致有丝分裂中期的染色体不能正常排列到赤道板,而对分裂后期染色质分离以及胞质分裂没有影响。在细胞内干扰GRK蛋白家族的另一个成员GRK2对有丝分裂则没有明显影响。该研究提示GRK5是细胞有丝分裂的重要调控蛋白。     

A yeast screening method to decipher the interaction between the adenosine A2B receptor and the C-terminus of different G protein α-subunits

The expression of human G protein-coupled receptors (GPCRs) in Saccharomyces cerevisiae containing chimeric yeast/mammalian G_α subunits provides a useful tool for the study of GPCR activation. In this study, we used a one-GPCR-one-G protein yeast screening method in combination with molecular modeling and mutagenesis studies to decipher the interaction between GPCRs and the C-terminus of different α-subunits of G proteins. We chose the human adenosine A_2B receptor (hA_2BR) as a paradigm, a typical class A GPCR that shows promiscuous behavior in G protein coupling in this yeast system. The wild-type hA_2BR and five mutant receptors were expressed in 8 yeast strains with different humanized G proteins, covering the four major classes: G_αi, G_αs, G_αq, and G_α12. Our experiments showed that a tyrosine residue (Y) at the C-terminus of the G_α subunit plays an important role in controlling the activation of GPCRs. Receptor residues R103^3.50 and I107^3.54 are vital too in G protein-coupling and the activation of the hA_2BR, whereas L213^IL3 is more important in G protein inactivation. Substitution of S235^6.36 to alanine provided the most divergent G protein-coupling profile. Finally, L236^6.37 substitution decreased receptor activation in all G protein pathways, although to a different extent. In conclusion, our findings shed light on the selectivity of receptor/G protein coupling, which may help in further understanding GPCR signaling.     

β-Arrestin2 directly or through GRK2 inhibits PKCβII activation in a ubiquitination-dependent manner

The GRK/β-arrestin and PKC/PKA mediate the homologous and heterologous regulation of G protein-coupled receptors (GPCRs), respectively. Interaction between the two pathways is one of the most important issues in understanding the regulation of GPCRs. The present study investigated the regulatory effect of GRK2 and β-arrestins on PKC activation. The roles of GRK2 and β-arrestins in the functional regulation of PKC were assessed by determining their influence on PKC autophosphorylation and intracellular translocation. Radioligand binding assay was utilized to characterize intracellular trafficking of dopamine D₂R, D₃R, and β₂ adrenergic receptor (β₂AR). The subdomains involved in the mutual interactions among GRK2, β-arrestin2, and PKCβII were determined by in vitro binding assay. Various point mutants of key regulatory players were combined with knockdown cells of GRK2, β-arrestins, and Mdm2 to functionally correlate the biochemical changes with functional outcomes. GRK2 and β-arrestin2 mutually inhibited the PKCβII autophosphorylation, a hallmark of PKCβII activation. β-Arrestin2 ubiquitination was required for the inhibitory activities of GRK2 as well as β-arrestin2. Furthermore, GRK2 facilitated β-arrestin2 ubiquitination, thus to enhance the inhibitory actions of β-arrestin2 on PKCβII activity. Aforementioned processes were also involved in the GRK2/β-arrestin2-mediated inhibition of the D₂R, D₃R, and β₂AR endocytosis. The present study provides new insights into the intricate interactions between the homologous and heterologous GPCR regulation pathways. In addition, a novel regulatory role of GRK2 was proposed for the ubiquitination of β-arrestin in the context of the PKC-mediated heterologous regulation of GPCRs.     

TGFβ-induced GRK2 expression attenuates AngII-regulated vascular smooth muscle cell proliferation and migration

Through diametric actions, the transforming growth factor β (TGFβ) and Angiotensin II (AngII) play important roles in regulating various biological responses such as cell proliferation and migration. Signaling initiated by TGFβ and AngII occurs through two structurally and functionally distinct receptor super families, the serine/threonine kinase and G protein-coupled receptors (GPCRs). Previously, we identified the G protein-coupled receptor kinase-2 (GRK2), a key regulatory factor in the desensitization of GPCRs, as a direct downstream target of the TGFβ signaling cascade. GRK2 acts through a negative feed-back loop mechanism to terminate TGFβ-induced smad signaling. To investigate the impact of TGFβ-induced GRK2 expression on GPCR signaling, we examined its effect on AngII signaling in vascular smooth muscle cells (VSMCs). In this study, we show that activation of the TGFβ signaling cascade in VSMCs results in increased GRK2 expression levels, which consequently inhibits AngII-induced ERK phosphorylation and antagonizes AngII-induced VSMC proliferation and migration. Moreover, the inhibitory effect of TGFβ on AngII signaling occurs at the Mek-Erk interface and is abrogated when an anti-sense oligonucleotide directed against GRK2 is used. Thus, we conclude that TGFβ signaling antagonizes AngII-induced VSMC proliferation and migration through the inhibition of ERK phosphorylation and that GRK2 is a key factor mediating the cross-talk between these two receptor super families.     

The WD40 repeat protein,WDR36, orchestrates sphingosine kinase-1 recruitment and phospholipase C-β activation by Gq-coupled receptors

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that G_q-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed G_q regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gα_q to phospholipase C (PLC)-β and the thromboxane A₂ receptor-β (TPβ receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPβ receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M₃ and bradykinin B₂ receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca²⁺]_i increases. Surprisingly, WDR36 also augmented M₃ receptor-induced [Ca²⁺]_i increases, which was due to increased Ca²⁺ mobilization while the Ca²⁺ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by G_q-coupled M₃, B₂ and H₁ receptors in HEK-293 cells, while TPβ receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B₂ receptors. It is concluded that WDR36 reduces signalling of G_q-coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gα_q and PLCβ. Our results support a role of WDR36 in orchestration of G_q signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.     

Subtype-dependent regulation of Gβγ signalling

G protein-coupled receptors (GPCRs) transmit information to the cell interior by transducing external signals to heterotrimeric G protein subunits, Gα and Gβγ subunits, localized on the inner leaflet of the plasma membrane. Though the initial focus was mainly on Gα-mediated events, Gβγ subunits were later identified as major contributors to GPCR-G protein signalling. A broad functional array of Gβγ signalling has recently been attributed to Gβ and Gγ subtype diversity, comprising 5 Gβ and 12 Gγ subtypes, respectively. In addition to displaying selectivity towards each other to form the Gβγ dimer, numerous studies have identified preferences of distinct Gβγ combinations for specific GPCRs, Gα subtypes and effector molecules. Importantly, Gβ and Gγ subtype-dependent regulation of downstream effectors, representing a diverse range of signalling pathways and physiological functions have been found. Here, we review the literature on the repercussions of Gβ and Gγ subtype diversity on direct and indirect regulation of GPCR/G protein signalling events and their physiological outcomes. Our discussion additionally provides perspective in understanding the intricacies underlying molecular regulation of subtype-specific roles of Gβγ signalling and associated diseases.