Regulation of phospholipase C-beta isozymes by G-proteins.

Phospholipase C (PLC) isozymes are known to be regulated, in part, by heterotrimeric GTP-binding protein (G-protein) subunits, including Galpha subunits of the G(q) family and Gbetagamma subunits. New data show that PLC can also be regulated by a high molecular weight G-protein that doubles as a cellular transglutaminase. Furthermore, a soluble phosphatidylinositol transfer protein (PITP) has been implicated in sustaining the activity of PLC by delivering substrate to the plasma membrane. Such diverse regulatory mechanisms imply that the PLC isozymes are precisely controlled and have specific roles in generating second messengers in response to various external stimuli.     

NMR analysis of G-protein βγ subunit complexes reveals a dynamic Gα-Gβγ subunit interface and multiple protein recognition modes

G-protein βγ (Gβγ) subunits interact with a wide range of molecular partners including: Gα subunits, effectors, peptides, and small molecule inhibitors. The molecular mechanisms underlying the ability to accommodate this wide range of structurally distinct binding partners are not well understood. To uncover the role of protein flexibility and alterations in protein conformation in molecular recognition by Gβγ, a method for site-specific ¹⁵N-labeling of Gβ-Trp residue backbone and indole amines in insect cells was developed. Transverse Relaxation Optimized Spectroscopy-Heteronuclear Single-Quantum Coherence Nuclear Magnetic Resonance (TROSY-HSQC NMR) analysis of ¹⁵N-Trp Gβγ identified well-dispersed signals for the individual Trp residue side chain and amide positions. Surprisingly, a wide range of signal intensities was observed in the spectrum, likely representing a range of backbone and side chain mobilities. The signal for GβW99 indole was very intense, suggesting a high level of mobility on the protein surface and molecular dynamics simulations indicate that GβW99 is highly mobile on the nanosecond timescale in comparison with other Gβ tryptophans. Binding of peptides and phosducin dramatically altered the mobility of GβW99 and GβW332 in the binding site and the chemical shifts at sites distant from the direct binding surface in distinct ways. In contrast, binding of Gα_i1-GDP to Gβγ had relatively little effect on the spectrum and, most surprisingly, did not significantly alter Trp mobility at the subunit interface. This suggests the inactive heterotrimer in solution adopts a conformation with an open subunit interface a large percentage of the time. Overall, these data show that Gβγ subunits explore a range of conformations that can be exploited during molecular recognition by diverse binding partners.     

Evidence that the nucleotide exchange and hydrolysis cycle of G proteins causes acute desensitization of G-protein gated inward rectifier K+ channels

The G-protein gated inward rectifier K⁺ channel (GIRK) is activated in vivo by the Gβγ subunits liberated upon G_i-coupled receptor activation. We have recapitulated the acute desensitization of receptor-activated GIRK currents in heterologous systems and shown that it is a membrane-delimited process. Its kinetics depends on the guanine nucleotide species available and could be accounted for by the nucleotide exchange and hydrolysis cycle of G proteins. Indeed, acute desensitization is abolished by nonhydrolyzable GTP analogues. Whereas regulators of G-protein signaling (RGS) proteins by their GTPase-activating protein activities are regarded as negative regulators, a positive regulatory function of RGS4 is uncovered in our study; the opposing effects allow RGS4 to potentiate acute desensitization without compromising GIRK activation.     

Structures of the Gβ-CCT and PhLP1–Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly

G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gβ and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1–Gβ-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ fold, disrupting interactions with CCT and releasing a PhLP1–Gβ dimer for assembly with Gγ. This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate.Cells detect and respond to a myriad of extracellular signals via G-protein signaling pathways. G proteins form complexes consisting of Gα, Gβ, and Gγ subunits that play a key role in propagating signals between activated receptors and downstream effectors (1). To perform this role, the G-protein heterotrimer must be assembled from its nascent polypeptides. A critical step in this process is the formation of the Gβγ dimer (2). Gβγ is an obligate dimer in which the individual subunits cannot fold into a stable structure on their own, but require the molecular chaperone cytosolic chaperonin containing tailless complex polypeptide 1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1) (3, 4).CCT is a member of the group II chaperonin family found in eukaryotes. It is a large protein-folding machine, made up of eight homologous subunits that assemble to form a double-ring structure, with each ring encompassing a central cavity. Nascent polypeptides and denatured proteins bind these cavities and are thereby sequestered from the other proteins in the cytosol and protected from aggregation (5). Each of the eight CCT subunits is an ATPase, and the binding and hydrolysis of ATP generates a conformational change in CCT that encapsulates the protein and assists in its folding (6–8). An important class of CCT substrates is WD40 repeat proteins that form β-propeller structures (9). One of these WD40 substrates is Gβ, which forms a seven-bladed β-propeller (10) with the assistance of CCT (3). However, unlike other CCT substrates, Gβ cannot achieve its native structure and release from CCT on its own, but requires the help of PhLP1 (3, 4). PhLP1 triggers the release of Gβ from CCT, allowing Gβ to interact with Gγ and form the Gβγ dimer (3, 4, 11).Given their vital roles in Gβγ assembly, it is important to understand at the molecular level how CCT and PhLP1 mediate Gβ folding. To achieve this objective, we have isolated two key intermediates in the Gβγ assembly process, the Gβ-CCT complex and the PhLP1–Gβ-CCT complex, and examined their structures by cryo-electron microscopy (cryo-EM), site-specific chemical cross-linking using unnatural amino acids, and chemical cross-linking coupled with mass-spectrometric identification of the cross-links (XL-MS). This analysis reveals a complex molecular mechanism by which CCT and PhLP1 fold Gβ and assist in Gβγ assembly.     

GAIP is membrane-anchored by palmitoylation and interacts with the activated (GTP-bound) form of Gαi subunits

GAIP (G Alpha Interacting Protein) is a member of the recently described RGS (Regulators of G-protein Signaling) family that was isolated by interaction cloning with the heterotrimeric G-protein Gα_i3 and was recently shown to be a GTPase-activating protein (GAP). In AtT-20 cells stably expressing GAIP, we found that GAIP is membrane-anchored and faces the cytoplasm, because it was not released by sodium carbonate treatment but was digested by proteinase K. When Cos cells were transiently transfected with GAIP and metabolically labeled with [³⁵S]methionine, two pools of GAIP—a soluble and a membrane-anchored pool—were found. Since the N terminus of GAIP contains a cysteine string motif and cysteine string proteins are heavily palmitoylated, we investigated the possibility that membrane-anchored GAIP might be palmitoylated. We found that after labeling with [³H]palmitic acid, the membrane-anchored pool but not the soluble pool was palmitoylated. In the yeast two-hybrid system, GAIP was found to interact specifically with members of the Gα_i subfamily, Gα_i1, Gα_i2, Gα_i3, Gα_z, and Gα_o, but not with members of other Gα subfamilies, Gα_s, Gα_q, and Gα_12/13. The C terminus of Gα_i3 is important for binding because a 10-aa C-terminal truncation and a point mutant of Gα_i3 showed significantly diminished interaction. GAIP interacted preferentially with the activated (GTP) form of Gα_i3, which is in keeping with its GAP activity. We conclude that GAIP is a membrane-anchored GAP with a cysteine string motif. This motif, present in cysteine string proteins found on synaptic vesicles, pancreatic zymogen granules, and chromaffin granules, suggests GAIP’s possible involvement in membrane trafficking.     

Modification of ghrelin receptor signaling by somatostatin receptor-5 regulates insulin release

Both ghrelin and somatostatin (SST) inhibit glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells, but how these independent actions are regulated has been unclear. The mechanism must accommodate noncanonical ghrelin receptor (GHS-R1a)–G-protein coupling to Gα_i/o instead of Gα_q11 and dependence on energy balance. Here we present evidence for an equilibrium model of receptor heteromerization that fulfills these criteria. We show that GHS-R1a coupling to Gα_i/o rather than Gα_q11 requires interactions between GHS-R1a and SST receptor subtype 5 (SST5) and that in the absence of SST5 ghrelin enhances GSIS. At concentrations of GHS-R1a and SST5 expressed in islets, time-resolved FRET and bioluminescence resonance energy transfer assays illustrate constitutive formation of GHS-R1a:SST5 heteromers in which ghrelin, but not SST, suppresses GSIS and cAMP accumulation. GHS-R1a–G-protein coupling and the formation of GHS-R1a:SST5 heteromers is dependent on the ratio of ghrelin to SST. A high ratio enhances heteromer formation and Gα_i/o coupling, whereas a low ratio destabilizes heteromer conformation, restoring GHS-R1a–Gα_q11 coupling. The [ghrelin]/[SST] ratio is dependent on energy balance: Ghrelin levels peak during acute fasting, whereas postprandially ghrelin is at a nadir, and islet SST concentrations increase. Hence, under conditions of low energy balance our model predicts that endogenous ghrelin rather than SST establishes inhibitory tone on the β-cell. Collectively, our data are consistent with physiologically relevant GHS-R1a:SST5 heteromerization that explains differential regulation of islet function by ghrelin and SST. These findings reinforce the concept that signaling by the G-protein receptor is dynamic and dependent on protomer interactions and physiological context.     

The interaction of nucleoside diphosphate kinase B with Gβγ dimers controls heterotrimeric G protein function

Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein βγ dimer (Gβγ) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in Gβ₁γ₂ dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating Gα_s and Gα_i subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish Gβ₁ orthologs, Gβ₁ and Gβ_1like, causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of Gβ₁/Gβ_1like is associated with a down-regulation in caveolins, AC-regulating Gα-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with Gβγ dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.     

Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel α1A subunit

Synaptic transmission is regulated by G protein-coupled receptors whose activation releases G protein βγ subunits that modulate presynaptic Ca²⁺ channels. The sequence motif QXXER has been proposed to be involved in the interaction between G protein βγ subunits and target proteins including adenylyl cyclase 2. This motif is present in the intracellular loop connecting domains I and II (L_I-II) of Ca²⁺ channel α_1A subunits, which are modulated by G proteins, but not in α_1C subunits, which are not modulated. Peptides containing the QXXER motif from adenylate cyclase 2 or from α_1A block G protein modulation but a mutant peptide containing the sequence AXXAA does not, suggesting that the QXXER-containing peptide from α_1A can competitively inhibit Gβγ modulation. Conversion of the R in the QQIER sequence of α_1A to E as in α_1C slows channel inactivation and shifts the voltage dependence of steady-state inactivation to more positive membrane potentials. Conversion of the final E in the QQLEE sequence of α_1C to R has opposite effects on voltage-dependent inactivation, although the changes are not as large as those for α_1A. Mutation of the QQIER sequence in α_1A to QQIEE enhanced G protein modulation, and mutation to QQLEE as in α_1C greatly reduced G protein modulation and increased the rate of reversal of G protein effects. These results indicate that the QXXER motif in L_I-II is an important determinant of both voltage-dependent inactivation and G protein modulation, and that the amino acid in the third position of this motif has an unexpectedly large influence on modulation by Gβγ. Overlap of this motif with the consensus sequence for binding of Ca²⁺ channel β subunits suggests that this region of L_I-II is important for three different modulatory influences on Ca²⁺ channel activity.     

Gαo is necessary for muscarinic regulation of Ca2+ channels in mouse heart

Heterotrimeric G proteins, composed of Gα and Gβγ subunits, transmit signals from cell surface receptors to cellular effector enzymes and ion channels. The Gα_o protein is the most abundant Gα subtype in the nervous system, but it is also found in the heart. Its function is not completely known, although it is required for regulation of N-type Ca²⁺ channels in GH₃ cells and also interacts with GAP43, a major protein in growth cones, suggesting a role in neuronal pathfinding. To analyze the function of Gα_o, we have generated mice lacking both isoforms of Gα_o by homologous recombination. Surprisingly, the nervous system is grossly intact, despite the fact that Gα_o makes up 0.2–0.5% of brain particulate protein and 10% of the growth cone membrane. The Gα_o−/− mice do suffer tremors and occasional seizures, but there is no obvious histologic abnormality in the nervous system. In contrast, Gα_o−/− mice have a clear and specific defect in ion channel regulation in the heart. Normal muscarinic regulation of L-type calcium channels in ventricular myocytes is absent in the mutant mice. The L-type calcium channel responds normally to isoproterenol, but there is no evident muscarinic inhibition. Muscarinic regulation of atrial K⁺ channels is normal, as is the electrocardiogram. The levels of other Gα subunits (Gα_s, Gα_q, and Gα_i) are unchanged in the hearts of Gα_o−/− mice, but the amount of Gβγ is decreased. Whichever subunit, Gα_o or Gβγ, carries the signal forward, these studies show that muscarinic inhibition of L-type Ca²⁺ channels requires coupling of the muscarinic receptor to Gα_o. Other cardiac Gα subunits cannot substitute.     

Regulators of G-protein Signaling accelerate GPCR signaling kinetics and govern sensitivity solely by accelerating GTPase activity

G-protein heterotrimers, composed of a guanine nucleotide-binding Gα subunit and an obligate Gβγ dimer, regulate signal transduction pathways by cycling between GDP- and GTP-bound states. Signal deactivation is achieved by Gα-mediated GTP hydrolysis (GTPase activity) which is enhanced by the GTPase-accelerating protein (GAP) activity of “regulator of G-protein signaling” (RGS) proteins. In a cellular context, RGS proteins have also been shown to speed up the onset of signaling, and to accelerate deactivation without changing amplitude or sensitivity of the signal. This latter paradoxical activity has been variably attributed to GAP/enzymatic or non-GAP/scaffolding functions of these proteins. Here, we validated and exploited a Gα switch-region point mutation, known to engender increased GTPase activity, to mimic in cis the GAP function of RGS proteins. While the transition-state, GDP·AlF₄ ⁻-bound conformation of the G202A mutant was found to be nearly identical to wild-type, Gα_i1(G202A)·GDP assumed a divergent conformation more closely resembling the GDP·AlF₄ ⁻-bound state. When placed within Saccharomyces cerevisiae Gα subunit Gpa1, the fast-hydrolysis mutation restored appropriate dose–response behaviors to pheromone signaling in the absence of RGS-mediated GAP activity. A bioluminescence resonance energy transfer (BRET) readout of heterotrimer activation with high temporal resolution revealed that fast intrinsic GTPase activity could recapitulate in cis the kinetic sharpening (increased onset and deactivation rates) and blunting of sensitivity also engendered by RGS protein action in trans. Thus Gα-directed GAP activity, the first biochemical function ascribed to RGS proteins, is sufficient to explain the activation kinetics and agonist sensitivity observed from G-protein–coupled receptor (GPCR) signaling in a cellular context.     

Analysis of C5a-mediated chemotaxis by lentiviral delivery of small interfering RNA

Immune cells respond to chemotactic signals by means of G protein-coupled receptors. Attempts to elucidate the function of specific G protein family members in these responses is complicated by redundancy among the different G protein isoforms. We have used lentiviral-based RNA interference to eliminate expression of specific G protein subunits selectively in J774A.1 mouse macrophages. The chemotactic response to the complement factors C5a and C3a is ablated in cells lacking Gβ₂ but is unaffected in cells lacking Gβ₁, Gαi₂, or Gαi₃. Similarly, the C5a-mediated calcium response of single cells is either absent or significantly delayed and weakened by Gβ₂ knockdown. Assessment of Akt1 phosphorylation levels in response to C5a shows rapid and sustained phosphorylation in both wild-type cells and cells lacking Gβ₁. Cells lacking Gβ₂ retain the rapid response but cannot sustain phospho-Akt1 levels. The phenotype of cells lacking Gβ₂ can be reversed by overexpression of either human Gβ₂ or mouse Gβ₁. These data demonstrate the usefulness of lentiviral-based RNA interference in the systematic analysis of a signaling pathway, and they suggest that in J774A.1 cells, Gβ₂-derived Gβγ is the most effective mediator of chemotaxis to C5a.     

Receptor and Gβγ isoform-specific interactions with G protein-coupled receptor kinases

The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and desensitize agonist-occupied GPCRs. GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free βγ subunits of heterotrimeric G proteins (Gβγ). Here we demonstrate that this mechanism operates in intact cells and that specificity is imparted by the selective interaction of discrete pools of Gβγ with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a β-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known Gβγ sequestrant. Furthermore, in cells overexpressing both GRK2 and Gβ₁γ₂, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/Gβγ complex. That Gβγ specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/Gβγ complex is formed after agonist occupancy of the lysophosphatidic acid and β-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a Gβγ complex after stimulation of all three GPCRs. This Gβγ binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds Gβ₁ and Gβ₂ but not Gβ₃, while the GRK3 fusion protein binds all three Gβ isoforms. This study provides a direct demonstration of a role for Gβγ in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components.     

Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits

We have shown that resistance to inhibitors of cholinesterase 8 (Ric-8) proteins regulate an early step of heterotrimeric G protein α (Gα) subunit biosynthesis. Here, mammalian and plant cell-free translation systems were used to study Ric-8A action during Gα subunit translation and protein folding. Gα translation rates and overall produced protein amounts were equivalent in mock and Ric-8A–immunodepleted rabbit reticulocyte lysate (RRL). GDP-AlF₄⁻–bound Gαi, Gαq, Gα13, and Gαs produced in mock-depleted RRL had characteristic resistance to limited trypsinolysis, showing that these G proteins were folded properly. Gαi, Gαq, and Gα13, but not Gαs produced from Ric-8A–depleted RRL were not protected from trypsinization and therefore not folded correctly. Addition of recombinant Ric-8A to the Ric-8A–depleted RRL enhanced GDP-AlF₄⁻–bound Gα subunit trypsin protection. Dramatic results were obtained in wheat germ extract (WGE) that has no endogenous Ric-8 component. WGE-translated Gαq was gel filtered and found to be an aggregate. Ric-8A supplementation of WGE allowed production of Gαq that gel filtered as a ∼100 kDa Ric-8A:Gαq heterodimer. Addition of GTPγS to Ric-8A–supplemented WGE Gαq translation resulted in dissociation of the Ric-8A:Gαq heterodimer and production of functional Gαq-GTPγS monomer. Excess Gβγ supplementation of WGE did not support functional Gαq production. The molecular chaperoning function of Ric-8 is to participate in the folding of nascent G protein α subunits.     

The helical domain of a G protein α subunit is a regulator of its effector

The α subunit (Gα) of heterotrimeric G proteins is a major determinant of signaling selectivity. The Gα structure essentially comprises a GTPase “Ras-like” domain (RasD) and a unique α-helical domain (HD). We used the vertebrate phototransduction model to test for potential functions of HD and found that the HD of the retinal transducin Gα (Gα_t) and the closely related gustducin (Gα_g), but not Gα_i1, Gα_s, or Gα_q synergistically enhance guanosine 5′-γ[-thio]triphosphate bound Gα_t (Gα_tGTPγS) activation of bovine rod cGMP phosphodiesterase (PDE). In addition, both HD_t and HD_g, but not HD_i1, HD_s, or HD_q attenuate the trypsin-activated PDE. Gα_tGDP and HD_t attenuation of trypsin-activated PDE saturate with similar affinities and to an identical 38% of initial activity. These data suggest that interaction of intact Gα_t with the PDE catalytic core may be caused by the HD moiety, and they indicate an independent site(s) for the HD moiety of Gα_t within the PDE catalytic core in addition to the sites for the inhibitory Pγ subunits. The HD moiety of Gα_tGDP is an attenuator of the activated catalytic core, whereas in the presence of activated Gα_tGTPγS the independently expressed HD_t is a potent synergist. Rhodopsin catalysis of Gα_t activation enhances the PDE activation produced by subsaturating levels of Gα_t, suggesting a HD-moiety synergism from a transient conformation of Gα_t. These results establish HD-selective regulations of vertebrate retinal PDE, and they provide evidence demonstrating that the HD is a modulatory domain. We suggest that the HD works in concert with the RasD, enhancing the efficiency of G protein signaling.     

Structural basis for the specific inhibition of heterotrimeric Gq protein by a small molecule

Heterotrimeric GTP-binding proteins (G proteins) transmit extracellular stimuli perceived by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. Hundreds of GPCRs exist in humans and are the targets of a large percentage of the pharmaceutical drugs used today. Because G proteins are regulated by GPCRs, small molecules that directly modulate G proteins have the potential to become therapeutic agents. However, strategies to develop modulators have been hampered by a lack of structural knowledge of targeting sites for specific modulator binding. Here we present the mechanism of action of the cyclic depsipeptide YM-254890, which is a recently discovered G_q-selective inhibitor. YM-254890 specifically inhibits the GDP/GTP exchange reaction of α subunit of G_q protein (Gα_q) by inhibiting the GDP release from Gα_q. X-ray crystal structure analysis of the Gα_qβγ–YM-254890 complex shows that YM-254890 binds the hydrophobic cleft between two interdomain linkers connecting the GTPase and helical domains of the Gα_q. The binding stabilizes an inactive GDP-bound form through direct interactions with switch I and impairs the linker flexibility. Our studies provide a novel targeting site for the development of small molecules that selectively inhibit each Gα subunit and an insight into the molecular mechanism of G protein activation.     

Protein kinase C-theta (PKCθ) phosphorylates and inhibits the guanine exchange factor,GIV/Girdin

Gα-interacting, vesicle-associated protein (GIV/Girdin) is a multidomain signal transducer that enhances PI3K-Akt signals downstream of both G-protein–coupled receptors and growth factor receptor tyrosine kinases during diverse biological processes and cancer metastasis. Mechanistically, GIV serves as a non-receptor guanine nucleotide exchange factor (GEF) that enhances PI3K signals by activating trimeric G proteins, Gαi1/2/3. Site-directed mutations in GIV’s GEF motif disrupt its ability to bind or activate Gi and abrogate PI3K-Akt signals; however, nothing is known about how GIV’s GEF function is regulated. Here we report that PKCθ, a novel protein kinase C, down-regulates GIV’s GEF function by phosphorylating Ser(S)1689 located within GIV’s GEF motif. We demonstrate that PKCθ specifically binds and phosphorylates GIV at S1689, and this phosphoevent abolishes GIV’s ability to bind and activate Gαi. HeLa cells stably expressing the phosphomimetic mutant of GIV, GIV-S1689→D, are phenotypically identical to those expressing the GEF-deficient F1685A mutant: Actin stress fibers are decreased and cell migration is inhibited whereas cell proliferation is triggered, and Akt (a.k.a. protein kinase B, PKB) activation is impaired downstream of both the lysophosphatidic acid receptor, a G-protein–coupled receptor, and the insulin receptor, a receptor tyrosine kinase. These findings indicate that phosphorylation of GIV by PKCθ inhibits GIV''s GEF function and generates a unique negative feedback loop for downregulating the GIV-Gi axis of prometastatic signaling downstream of multiple ligand-activated receptors. This phosphoevent constitutes the only regulatory pathway described for terminating signaling by any of the growing family of nonreceptor GEFs that modulate G-protein activity.     

Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

Agonist binding to G-protein–coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of α_i1 or α_sL and all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β₅. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gα_i1βγ combinations containing γ₁ and γ₁₁. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.G-protein–coupled receptors (GPCRs) are a large class of eukaryotic seven-transmembrane receptors encoded by >800 genes in the human genome. After stimulation by a vast variety of chemically diverse ligands, GPCRs regulate many cellular responses by the activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) (1, 2). The heterotrimeric G-protein complex is assembled from a pool of 16 α-subunits, 5 β-subunits, and 12 γ-subunits (3–5). Extensive analyses of the βγ-dimer formation potential had indicated unrestricted dimer formation for β₁ and β₄ (i.e., dimers with all γ-subunits are found), restricted dimer formation of β₂ and β₃ (e.g., no dimers with γ₁ or γ₁₁), and no or only weak dimer formation for β₅ (6, 7). Although a comprehensive analysis of Gαβγ complex formation is missing, it is likely that most of the Gαβγ combinations are capable of forming a functional complex (8–10). Taking into account the βγ-dimers believed to be nonexistent, this restriction still results in a number of ∼700 potential Gαβγ combinations.The enormous number of potential interactions between the >800 GPCRs and several hundred G-protein combinations quickly raised the question of how the interaction between GPCR and G protein is determined. Besides tissue-specific expression (4), it has quickly become clear that GPCRs display specificity for G-protein coupling and biased agonism (9, 11, 12). Although a variety of structures have been solved for G proteins and, more recently, for GPCRs (13–15), the only structural snapshot of the interaction between a GPCR and a G protein is provided by the structure of the complex between β₂ adrenergic receptor and Gα_sSβ₁γ₂ (16). This structure reveals—as many previous studies had suggested—that the α-subunit is the main interaction partner of the GPCR. Nevertheless, how the GPCR discriminates between the different α-subunits and how the βγ-dimer influences this interaction has not been definitively answered yet. To this end, additional structures of GPCR/G-protein complexes are needed that could shed more light on these questions. However, the crystallization of GPCR/G-protein complexes poses a big challenge because, on the one hand, GPCRs tend to show low expression levels and low stability in detergent (17), and, on the other hand, the Gα protein gains flexibility in complex with a GPCR (16, 18–20).In the past years, we developed strategies based on directed evolution to generate GPCRs that not only exhibit higher expression levels, but also higher stability in detergents (21–24). Recently, these efforts have led to the determination of several structures of evolved mutants of neurotensin receptor 1 (NTR1), which were solved from crystals obtained by vapor diffusion in short-chain detergents (25). Many of the evolved NTR1 mutants, besides displaying better expression and better stability, still showed functional coupling to G proteins (23, 25). Signaling is especially improved if one of the persistently selected mutations that increases stability—replacing wild-type (WT) arginine at position 167 by leucine—is reversed to the WT amino acid arginine. This result is unsurprising, because this arginine is part of the signaling-relevant E/DR¹⁶⁷Y motif (21, 23).With optimized GPCRs at hand, we set out to find those G-protein combinations that show the most efficient interaction with our NTR1 mutants. For this purpose, we screened the natural pool of G proteins composed of α_i1- or α_sL-subunits and all possible βγ-dimers for their formation of a heterotrimeric G-protein complex and their interaction with solubilized NTR1 mutants in detergent.Here, we present the results of, to our knowledge, the first comprehensive analysis of heterotrimeric G-protein complex diversity and GPCR interactions. This analysis reveals that combinations like α_i1β₂γ₁, which were previously believed to be nonexistent (6), indeed exist and can be purified. Moreover, those newly identified combinations are among the combinations that performed best in forming a complex between NTR1 and heterotrimeric G protein. We also present data indicating that GPCR mutants, which exhibit modest functional coupling with G protein, still form a GPCR/G-protein complex and may be stabilizing this complex. Our study suggests that the combination of stable GPCR mutants and carefully selected G-protein combinations may be a promising way of stabilizing this intrinsically dynamic signaling complex for detailed structural and functional studies.     

G-protein signaling modulator 1 deficiency accelerates cystic disease in an orthologous mouse model of autosomal dominant polycystic kidney disease

Polycystic kidney diseases are the most common genetic diseases that affect the kidney. There remains a paucity of information regarding mechanisms by which G proteins are regulated in the context of polycystic kidney disease to promote abnormal epithelial cell expansion and cystogenesis. In this study, we describe a functional role for the accessory protein, G-protein signaling modulator 1 (GPSM1), also known as activator of G-protein signaling 3, to act as a modulator of cyst progression in an orthologous mouse model of autosomal dominant polycystic kidney disease (ADPKD). A complete loss of Gpsm1 in the Pkd1^V/V mouse model of ADPKD, which displays a hypomorphic phenotype of polycystin-1, demonstrated increased cyst progression and reduced renal function compared with age-matched cystic Gpsm1^+/+ and Gpsm1^+/− mice. Electrophysiological studies identified a role by which GPSM1 increased heteromeric polycystin-1/polycystin-2 ion channel activity via Gβγ subunits. In summary, the present study demonstrates an important role for GPSM1 in controlling the dynamics of cyst progression in an orthologous mouse model of ADPKD and presents a therapeutic target for drug development in the treatment of this costly disease.