G protein {beta}{gamma} gating confers volatile anesthetic inhibition to Kir3 channels

G protein-activated inwardly rectifying potassium (GIRK or Kir3) channels are directly gated by the βγ subunits of G proteins and contribute to inhibitory neurotransmitter signaling pathways. Paradoxically, volatile anesthetics such as halothane inhibit these channels. We find that neuronal Kir3 currents are highly sensitive to inhibition by halothane. Given that Kir3 currents result from increased Gβγ available to the channels, we asked whether reducing available Gβγ to the channel would adversely affect halothane inhibition. Remarkably, scavenging Gβγ using the C-terminal domain of β-adrenergic receptor kinase (cβARK) resulted in channel activation by halothane. Consistent with this effect, channel mutants that impair Gβγ activation were also activated by halothane. A single residue, phenylalanine 192, occupies the putative Gβγ gate of neuronal Kir3.2 channels. Mutation of Phe-192 at the gate to other residues rendered the channel non-responsive, either activated or inhibited by halothane. These data indicated that halothane predominantly interferes with Gβγ-mediated Kir3 currents, such as those functioning during inhibitory synaptic activity. Our report identifies the molecular correlate for anesthetic inhibition of Kir3 channels and highlights the significance of these effects in modulating neurotransmitter-mediated inhibitory signaling.     

A Mechanism Regulating G Protein-coupled Receptor Signaling That Requires Cycles of Protein Palmitoylation and Depalmitoylation

Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of G_i/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from G_i/o-gated G protein-regulated inwardly rectifying K⁺ (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating G_i/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.     

The WD40 repeat protein WDR26 binds Gβγ and promotes Gβγ-dependent signal transduction and leukocyte migration

The Gβγ subunits of heterotrimeric G proteins transmit signals to control many cellular processes, including leukocyte migration. Gβγ signaling may regulate and be regulated by numerous signaling partners. Here, we reveal that WDR26, a member of the WD40 repeat protein family, directly bound free Gβγ in vitro, and formed a complex with endogenous Gβγ in Jurkat T cells stimulated by the chemokine SDF1α. Suppression of WDR26 by siRNAs selectively inhibited Gβγ-dependent phospholipase Cβ and PI3K activation, and attenuated chemotaxis in Jurkat T cells and differentiated HL60 cells in vitro and Jurkat T cell homing to lymphoid tissues in scid mice. Similarly, disruption of the WDR26/Gβγ interaction via expression of a WDR26 deletion mutant impaired Gβγ signaling and Jurkat T cell migration, indicating that the function of WDR26 depends on its binding to Gβγ. Additional data show that WDR26 also controlled RACK1, a negative regulator, in binding Gβγ and inhibiting leukocyte migration. Collectively, these experiments identify WDR26 as a novel Gβγ-binding protein that is required for the efficacy of Gβγ signaling and leukocyte migration.     

G protein-coupled receptor signaling via Src kinase induces endogenous human transient receptor potential vanilloid type 6 (TRPV6) channel activation

Ca(2+) homeostasis plays a critical role in a variety of cellular processes. We showed previously that stimulation of the prostate-specific G protein-coupled receptor (PSGR) enhances cytosolic Ca(2+) and inhibits proliferation of prostate cells. Here, we analyzed the signaling mechanisms underlying the PSGR-mediated Ca(2+) increase. Using complementary molecular, biochemical, electrophysiological, and live-cell imaging techniques, we found that endogenous Ca(2+)-selective transient receptor potential vanilloid type 6 (TRPV6) channels are critically involved in the PSGR-induced Ca(2+) signal. Biophysical characterization of the current activated by PSGR stimulation revealed characteristic properties of TRPV6. The molecular identity of the involved channel was confirmed using RNA interference targeting TrpV6. TRPV6-mediated Ca(2+) influx depended on Src kinase activity. Src kinase activation occurred independently of G protein activation, presumably by direct interaction with PSGR. Taken together, we report that endogenous TRPV6 channels are activated downstream of a G protein-coupled receptor and present the first physiological characterization of these channels in situ.     

Macromolecular Composition Dictates Receptor and G Protein Selectivity of Regulator of G Protein Signaling (RGS) 7 and 9-2 Protein Complexes in Living Cells

Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by μ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein β subunit Gβ5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.     

Identification of the orphan G protein-coupled receptor GPR31 as a receptor for 12-(S)-hydroxyeicosatetraenoic acid

Hydroxy fatty acids are critical lipid mediators involved in various pathophysiologic functions. We cloned and identified GPR31, a plasma membrane orphan G protein-coupled receptor that displays high affinity for the human 12-lipoxygenase-derived product 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE). Thus, GPR31 is named 12-(S)-HETE receptor (12-HETER) in this study. The cloned 12-HETER demonstrated high affinity binding for 12-(S)-[(3)H]HETE (K(d) = 4.8 ± 0.12 nm). Also, 12-(S)-HETE efficiently and selectively stimulated GTPγS coupling in the membranes of 12-HETER-transfected cells (EC(50) = 0.28 ± 1.26 nm). Activating GTPγS coupling with 12-(S)-HETE proved to be both regio- and stereospecific. Also, 12-(S)-HETE/12-HETER interactions lead to activation of ERK1/2, MEK, and NFκB. Moreover, knocking down 12-HRTER specifically inhibited 12-(S)-HETE-stimulated cell invasion. Thus, 12-HETER represents the first identified high affinity receptor for the 12-(S)-HETE hydroxyl fatty acids.     

Protease-activated Receptor 2 (PAR2) Protein and Transient Receptor Potential Vanilloid 4 (TRPV4) Protein Coupling Is Required for Sustained Inflammatory Signaling

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR₂), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR₂ regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR₂ agonist stimulated a transient increase in [Ca²⁺]_i. TRPV4 expression led to a markedly sustained increase in [Ca²⁺]_i. Removal of extracellular Ca²⁺ and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A₂ and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR₂ generates arachidonic acid-derived lipid mediators, such as 5′,6′-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR₂-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR₂ was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR₂ signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR₂-stimulated neurogenic inflammation. Thus, PAR₂ activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR₂.     

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.     

Do Caveolae Have a Role in the Fidelity and Dynamics of Receptor Activation of G-protein-gated Inwardly Rectifying Potassium Channels?

In atrial and nodal cardiac myocytes, M2 muscarinic receptors activate inhibitory G-proteins (G_i/o), which in turn stimulate G-protein-gated inwardly rectifying K⁺ channels through direct binding of the Gβγ subunit. Despite also releasing Gβγ, G_s-coupled receptors such as the β-adrenergic receptor are not able to prominently activate this current. An appealing hypothesis would be if components were sequestered in membrane domains such as caveolae/rafts. Using biochemical fractionation followed by Western blotting and/or radioligand binding experiments, we examined the distribution of the components in stable HEK293 and HL-1 cells, which natively express the transduction cascade. The channel, M2 muscarinic, and A1 adenosine receptors were located in noncaveolar/nonraft fractions. G_iα_1/2 was enriched in both caveolar/raft and noncaveolar/nonraft fractions. In contrast, G_sα was only enriched in caveolar/raft fractions. We constructed YFP-tagged caveolin-2 (YFP-Cav2) and chimeras with the M2 (M2-YFP-Cav2) and A1 (A1-YFP-Cav2) receptors. Analysis of gradient fractions showed that these receptor chimeras were now localized to caveolae-enriched fractions. Microscopy showed that M2-YFP and A1-YFP had a diffuse homogenous membrane signal. YFP-Cav2, M2-YFP-Cav2, and A1-YFP-Cav2 revealed a more punctuate pattern. Finally, we looked at the consequences for signaling. Activation via M2-YFP-Cav2 or A1-YFP-Cav2 revealed substantially slower kinetics compared with M2-YFP or A1-YFP and was reversed by the addition of methyl-β-cyclodextrin. Thus the localization of the channel signal transduction cascade in non-cholesterol rich domains substantially enhances the speed of signaling. The presence of G_sα solely in caveolae may account for signaling selectivity between G_i/o and G_s-coupled receptors.     

Vitamin C degradation products and pathways in the human lens

Vitamin C and its degradation products participate in chemical modifications of proteins in vivo through non-enzymatic glycation (Maillard reaction) and formation of different products called advanced glycation end products. Vitamin C levels are particularly high in selected tissues, such as lens, brain and adrenal gland, and its degradation products can inflict substantial protein damage via formation of advanced glycation end products. However, the pathways of in vivo vitamin C degradation are poorly understood. Here we have determined the levels of vitamin C oxidation and degradation products dehydroascorbic acid, 2,3-diketogulonic acid, 3-deoxythreosone, xylosone, and threosone in the human lens using o-phenylenediamine to trap both free and protein-bound adducts. In the protein-free fraction and water-soluble proteins (WSP), all five listed degradation products were identified. Dehydroascorbic acid, 2,3-diketogulonic acid, and 3-deoxythreosone were the major products in the protein-free fraction, whereas in the WSP, 3-deoxythreosone was the most abundant measured dicarbonyl. In addition, 3-deoxythreosone in WSP showed positive linear correlation with age (p < 0.05). In water-insoluble proteins, only 3-deoxythreosone and threosone were detected, whereby the level of 3-deoxythreosone was ～20 times higher than the level of threosone. The identification of 3-deoxythreosone as the major degradation product bound to human lens proteins provides in vivo evidence for the non-oxidative pathway of dehydroascorbate degradation into erythrulose as a major pathway for vitamin C degradation in vivo.     

The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity

GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of β-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with β-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.     

Extracellular Ionic Locks Determine Variation in Constitutive Activity and Ligand Potency between Species Orthologs of the Free Fatty Acid Receptors FFA2 and FFA3

Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency such that acetate (C2) has been described as FFA2-selective, whereas propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs, it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations, marked variation in ligand-independent constitutive activity was identified using a [³⁵S]GTPγS assay. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity in this assay, whereas the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the second extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity and in most cases also yielded corresponding changes in SCFA potency.     

Recognition in the face of diversity: interactions of heterotrimeric G proteins and G protein-coupled receptor (GPCR) kinases with activated GPCRs

G protein-coupled receptors (GPCRs) represent the largest class of integral membrane protein receptors in the human genome. Despite the great diversity of ligands that activate these GPCRs, they interact with a relatively small number of intracellular proteins to induce profound physiological change. Both heterotrimeric G proteins and GPCR kinases are well known for their ability to specifically recognize GPCRs in their active state. Recent structural studies now suggest that heterotrimeric G proteins and GPCR kinases identify activated receptors via a common molecular mechanism despite having completely different folds.     

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.     

Contribution of Kv1.2 voltage-gated potassium channel to D2 autoreceptor regulation of axonal dopamine overflow

Impairments in axonal dopamine release are associated with neurological disorders such as schizophrenia and attention deficit hyperactivity disorder and pathophysiological conditions promoting drug abuse and obesity. The D2 dopamine autoreceptor (D2-AR) exerts tight regulatory control of axonal dopamine (DA) release through a mechanism suggested to involve K(+) channels. To evaluate the contribution of Kv1 voltage-gated potassium channels of the Shaker gene family to the regulation of axonal DA release by the D2-AR, the present study employed expression analyses, real time measurements of striatal DA overflow, K(+) current measurements and immunoprecipitation assays. Kv1.1, -1.2, -1.3, and -1.6 mRNA and protein were detected in midbrain DA neurons purified by fluorescence-activated cell sorting and in primary DA neuron cultures. In addition, Kv1.1, -1.2, and -1.6 were localized to DA axonal processes in the dorsal striatum. By means of fast scan cyclic voltammetry in striatal slice preparations, we found that the inhibition of stimulation-evoked DA overflow by a D2 agonist was attenuated by Kv1.1, -1.2, and -1.6 toxin blockers. A particular role for the Kv1.2 subunit in the process whereby axonal D2-AR inhibits DA overflow was established with the use of a selective Kv1.2 blocker and Kv1.2 knock-out mice. Moreover, we demonstrate the ability of D2-AR activation to increase Kv1.2 currents in co-transfected cells and its reliance on Gβγ subunit signaling along with the physical coupling of D2-AR and Kv1.2-containing channels in striatal tissue. These findings underline the contribution of Kv1.2 in the regulation of nigrostriatal DA release by the D2-AR and thereby offer a novel mechanism by which DA release is regulated.     

Type 5 G protein beta subunit (Gbeta5) controls the interaction of regulator of G protein signaling 9 (RGS9) with membrane anchors

The R7 family of regulators of G protein signaling (RGS) proteins, comprising RGS6, RGS7, RGS9, and RGS11, regulate neuronal G protein signaling pathways. All members of the R7 RGS form trimeric complexes with the atypical G protein β subunit, Gβ5, and membrane anchor R7BP or R9AP. Association with Gβ5 and membrane anchors has been shown to be critical for maintaining proteolytic stability of the R7 RGS proteins. However, despite its functional importance, the mechanism of how R7 RGS forms complexes with Gβ5 and membrane anchors remains poorly understood. Here, we used protein-protein interaction, co-localization, and protein stability assays to show that association of RGS9 with membrane anchors requires Gβ5. We further establish that the recruitment of R7BP to the complex requires an intact interface between the N-terminal lobe of RGS9 and protein interaction surface of Gβ5. Site-directed mutational analysis reveals that distinct molecular determinants in the interface between Gβ5 and N-terminal Dishevelled, EGL-10, Pleckstrin/DEP Helical Extension (DEP/DHEY) domains are differentially involved in R7BP binding and proteolytic stabilization. On the basis of these findings, we conclude that Gβ5 contributes to the formation of the binding site to the membrane anchors and thus is playing a central role in the assembly of the proteolytically stable trimeric complex and its correct localization in the cell.     

Cell adhesion receptor GPR133 couples to Gs protein

Adhesion G protein-coupled receptors (GPCR), with their very large and complex N termini, are thought to participate in cell-cell and cell-matrix interactions and appear to be highly relevant in several developmental processes. Their intracellular signaling is still poorly understood. Here we demonstrate that GPR133, a member of the adhesion GPCR subfamily, activates the G(s) protein/adenylyl cyclase pathway. The presence of the N terminus and the cleavage at the GPCR proteolysis site are not required for G protein signaling. G(s) protein coupling was verified by Gα(s) knockdown with siRNA, overexpression of Gα(s), co-expression of the chimeric Gq(s4) protein that routes GPR133 activity to the phospholipase C/inositol phosphate pathway, and missense mutation within the transmembrane domain that abolished receptor activity without changing cell surface expression. It is likely that not only GPR133 but also other adhesion GPCR signal via classical receptor/G protein-interaction.     

Altered immune response in mice deficient for the G protein-coupled receptor GPR34

The X-chromosomal GPR34 gene encodes an orphan G(i) protein-coupled receptor that is highly conserved among vertebrates. To evaluate the physiological relevance of GPR34, we generated a GPR34-deficient mouse line. GPR34-deficient mice were vital, reproduced normally, and showed no gross abnormalities in anatomical, histological, laboratory chemistry, or behavioral investigations under standard housing. Because GPR34 is highly expressed in mononuclear cells of the immune system, mice were specifically tested for altered functions of these cell types. Following immunization with methylated BSA, the number of granulocytes and macrophages in spleens was significantly lower in GPR34-deficient mice as in wild-type mice. GPR34-deficient mice showed significantly increased paw swelling in the delayed type hypersensitivity test and higher pathogen burden in extrapulmonary tissues after pulmonary infection with Cryptococcus neoformans compared with wild-type mice. The findings in delayed type hypersensitivity and infection tests were accompanied by significantly different basal and stimulated TNF-α, GM-CSF, and IFN-γ levels in GPR34-deficient animals. Our data point toward a functional role of GPR34 in the cellular response to immunological challenges.     

Resistance to inhibitors of cholinesterase-8A (Ric-8A) is critical for growth factor receptor-induced actin cytoskeletal reorganization

Heterotrimeric G proteins are critical transducers of cellular signaling. In addition to their classic roles in relaying signals from G protein-coupled receptors (GPCRs), heterotrimeric G proteins also mediate physiological functions from non-GPCRs. Previously, we have shown that Gα(13), a member of the heterotrimeric G proteins, is essential for growth factor receptor-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. These Gα(13)-mediated dorsal ruffle turnover and cell migration by growth factors acting on their receptor tyrosine kinases (RTKs) are independent of GPCRs. However, the mechanism by which RTKs signal to Gα(13) is not known. Here, we show that cholinesterase-8A (Ric-8A), a nonreceptor guanine nucleotide exchange factor for some heterotrimeric G proteins, is critical for coupling RTKs to Gα(13). Down-regulation of Ric-8A protein levels in cells by RNA interference slowed down platelet-derived growth factor (PDGF)-induced dorsal ruffle turnover and inhibited PDGF-initiated cell migration. PDGF was able to increase the activity of Ric-8A in cells. Furthermore, purified Ric-8A proteins interact directly with purified Gα(13) protein in a nucleotide-dependent manner. Deficiency of Ric-8A prevented the translocation of Gα(13) to the cell cortex. Hence, Ric-8A is critical for growth factor receptor-induced actin cytoskeletal reorganization.