Phosducin-like protein acts as a molecular chaperone for G protein betagamma dimer assembly

Phosducin-like protein (PhLP) is a widely expressed binding partner of the G protein betagamma subunit dimer (Gbetagamma). However, its physiological role is poorly understood. To investigate PhLP function, its cellular expression was blocked using RNA interference, resulting in inhibition of Gbetagamma expression and G protein signaling. This inhibition was caused by an inability of nascent Gbetagamma to form dimers. Phosphorylation of PhLP at serines 18-20 by protein kinase CK2 was required for Gbetagamma formation, while a high-affinity interaction of PhLP with the cytosolic chaperonin complex appeared unnecessary. PhLP bound nascent Gbeta in the absence of Ggamma, and S18-20 phosphorylation was required for Ggamma to associate with the PhLP-Gbeta complex. Once Ggamma bound, PhLP was released. These results suggest a mechanism for Gbetagamma assembly in which PhLP stabilizes the nascent Gbeta polypeptide until Ggamma can associate, resulting in membrane binding of Gbetagamma and release of PhLP to catalyze another round of assembly.     

Phosducin-like protein regulates G-protein betagamma folding by interaction with tailless complex polypeptide-1alpha: dephosphorylation or splicing of PhLP turns the switch toward regulation of Gbetagamma folding

Phosducin-like protein (PhLP) exists in two splice variants PhLP(LONG) (PhLP(L)) and PhLP(SHORT) (PhLP(S)). Whereas PhLP(L) directly inhibits Gbetagamma-stimulated signaling, the G betagamma-inhibitory mechanism of PhLP(S) is not understood. We report here that inhibition of Gbetagamma signaling in intact HEK cells by PhLP(S) was independent of direct Gbetagamma binding; however, PhLP(S) caused down-regulation of Gbeta and Ggamma proteins. The down-regulation was partially suppressed by lactacystine, indicating the involvement of proteasomal degradation. N-terminal fusion of Gbeta or Ggamma with a dye-labeling protein resulted in their stabilization against down-regulation by PhLP(S) but did not lead to a functional rescue. Moreover, in the presence of PhLP(S), stabilized Ggamma subunits did not coprecipitate with stabilized Gbeta subunits, suggesting that PhLP(S) might interfere with Gbetagamma folding. PhLP(S) and several truncated mutants of PhLP(S) interacted with the subunit tailless complex polypeptide-1alpha (TCP-1alpha) of the CCT chaperonin complex, which is involved in protein folding. Knock-down of TCP-1alpha in HEK cells by small interfering RNA also led to down-regulation of Gbetagamma. We therefore conclude that the strong inhibitory action of PhLP(S) on Gbetagamma signaling is the result of a previously unrecognized mechanism of Gbetagamma-regulation, inhibition of Gbetagamma-folding by interference with TCP-1alpha.     

Dopamine receptor-interacting protein 78 acts as a molecular chaperone for Ggamma subunits before assembly with Gbeta

Heterotrimeric G proteins play a central role in intracellular communication mediated by extracellular signals, and both Galpha and Gbetagamma subunits regulate effectors downstream of activated receptors. The particular constituents of the G protein heterotrimer affect both specificity and efficiency of signal transduction. However, little is known about mechanistic aspects of G protein assembly in the cell that would certainly contribute to formation of heterotrimers of specific composition. It was recently shown that phosducin-like protein (PhLP) modulated both Gbetagamma expression and subsequent signaling by chaperoning nascent Gbeta and facilitating heterodimer formation with Ggamma subunits (Lukov, G. L., Hu, T., McLaughlin, J. N., Hamm, H. E., and Willardson, B. M. (2005) EMBO J. 24, 1965-1975; Humrich, J., Bermel, C., Bunemann, M., Harmark, L., Frost, R., Quitterer, U., and Lohse, M. J. (2005) J. Biol. Chem. 280, 20042-20050). Here we demonstrate using a variety of techniques that DRiP78, an endoplasmic reticulum resident protein known to regulate the trafficking of several seven transmembrane receptors, interacts specifically with the Ggamma subunit but not Gbeta or Galpha subunits. Furthermore, we demonstrate that DRiP78 and the Gbeta subunit can compete for the Ggamma subunit. DRiP78 also protects Ggamma from degradation until a stable partner such as Gbeta is provided. Furthermore, DRiP78 interaction may represent a mechanism for assembly of specific Gbetagamma heterodimers, as selectivity was observed among Ggamma isoforms for interaction with DRiP78 depending on the presence of particular Gbeta subunits. Interestingly, we could detect an interaction between DRiP78 and PhLP, suggesting a role of DRiP78 in the assembly of Gbetagamma by linking Ggamma to PhLP.Gbeta complexes. Our results, therefore, suggest a role of DRiP78 as a chaperone in the assembly of Gbetagamma subunits of the G protein.     

Role of the chaperonin CCT/TRiC complex in G protein betagamma-dimer assembly

Gbetagamma dimer formation occurs early in the assembly of heterotrimeric G proteins. On nondenaturing (native) gels, in vitro translated, (35)S-labeled Ggamma subunits traveled primarily according to their pI and apparently were not associated with other proteins. In contrast, in vitro translated, (35)S-labeled Gbeta subunits traveled at a high apparent molecular mass (approximately 700 kDa) and co-migrated with the chaperonin CCT complex (also called TRiC). Different FLAG-Gbeta isoforms coprecipitated CCT/TRiC to a variable extent, and this correlated with the ability of the different Gbeta subunits to efficiently form dimers with Ggamma. When translated Ggamma was added to translated Gbeta, a new band of low apparent molecular mass (approximately 50 kDa) was observed, which was labeled by either (35)S-labeled Gbeta or Ggamma, indicating that it is a dimer. Formation of the Gbetagamma dimer was ATP-dependent and inhibited by either adenosine 5'-O-(thiotriphosphate) or aluminum fluoride in the presence of Mg(2+). This inhibition led to increased association of Gbeta with CCT/TRiC. Although Ggamma did not bind CCT/TRiC, addition of Ggamma to previously synthesized Gbeta caused its release from the CCT/TRiC complex. We conclude that the chaperonin CCT/TRiC complex binds to and folds Gbeta subunits and that CCT/TRiC mediates Gbetagamma dimer formation by an ATP-dependent reaction.     

The phosducin-like protein PhLP1 is essential for G{beta}{gamma} dimer formation in Dictyostelium discoideum

Phosducin proteins are known to inhibit G protein-mediated signaling by sequestering Gbetagamma subunits. However, Dictyostelium discoideum cells lacking the phosducin-like protein PhLP1 display defective rather than enhanced G protein signaling. Here we show that green fluorescent protein (GFP)-tagged Gbeta (GFP-Gbeta) and GFP-Ggamma subunits exhibit drastically reduced steady-state levels and are absent from the plasma membrane in phlp1(-) cells. Triton X-114 partitioning suggests that lipid attachment to GFP-Ggamma occurs in wild-type cells but not in phlp1(-) and gbeta(-) cells. Moreover, Gbetagamma dimers could not be detected in vitro in coimmunoprecipitation assays with phlp1(-) cell lysates. Accordingly, in vivo diffusion measurements using fluorescence correlation spectroscopy showed that while GFP-Ggamma proteins are present in a complex in wild-type cells, they are free in phlp1(-) and gbeta(-) cells. Collectively, our data strongly suggest the absence of Gbetagamma dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a cochaperone assisting the assembly of Gbeta and Ggamma into a functional Gbetagamma complex. Thus, phosducin family proteins may fulfill hitherto unsuspected biosynthetic functions.     

G Protein betagamma dimer formation: Gbeta and Ggamma differentially determine efficiency of in vitro dimer formation

The Gbeta and Ggamma subunit of the heterotrimeric G proteins form a functional dimer that is stable once assembled in vivo or in vitro. The requirements, mechanism, and specificity of dimer formation are still incompletely understood, but represent important biochemical processes involved in the specificity of cellular signaling through G proteins. Here, seven Gbeta and 12 FLAG-epitope-tagged Ggamma subunits were separately synthesized in vitro using a rabbit reticulocyte lysate expression system. The translation products were combined and dimers isolated by immunoprecipitation. Gbeta1 and Gbeta4 formed dimers with all Ggamma subunit isoforms, generally with Gbeta/Ggamma stoichiometries between 0.2:1 and 0.5:1. Gbeta5, Gbeta5L, and Gbeta3s did not form significant amounts of dimer with any of the gamma subunit isoforms. Gbeta2 and Gbeta3 formed dimers with selected Ggamma isoforms to levels intermediate between that of Gbeta1/Gbeta4 and Gbeta3s/Gbeta5/Gbeta5L. We also expressed selected Gbetagamma in HEK293 cells and measured PLCbeta2 activity. Gbetagamma dimer-dependent increases in IP3 production were seen with most Gbeta1, Gbeta2, and Gbeta5 combinations, indicating functional dimer expression in intact cells. These results define the complete set of G protein betagamma dimers that are formed using a single biochemical assay method and suggest that there are Gbeta isoform-specific factors in rabbit reticulocyte lysates that determine the efficacy of Gbetagamma dimer formation.     

Critical determinants of the G protein gamma subunits in the Gbetagamma stimulation of G protein-activated inwardly rectifying potassium (GIRK) channel activity

The betagamma subunits of G proteins modulate inwardly rectifying potassium (GIRK) channels through direct interactions. Although GIRK currents are stimulated by mammalian Gbetagamma subunits, we show that they were inhibited by the yeast Gbetagamma (Ste4/Ste18) subunits. A chimera between the yeast and the mammalian Gbeta1 subunits (ymbeta) stimulated or inhibited GIRK currents, depending on whether it was co-expressed with mammalian or yeast Ggamma subunits, respectively. This result underscores the critical functional influence of the Ggamma subunits on the effectiveness of the Gbetagamma complex. A series of chimeras between Ggamma2 and the yeast Ggamma revealed that the C-terminal half of the Ggamma2 subunit is required for channel activation by the Gbetagamma complex. Point mutations of Ggamma2 to the corresponding yeast Ggamma residues identified several amino acids that reduced significantly the ability of Gbetagamma to stimulate channel activity, an effect that was not due to improper association with Gbeta. Most of the identified critical Ggamma residues clustered together, forming an intricate network of interactions with the Gbeta subunit, defining an interaction surface of the Gbetagamma complex with GIRK channels. These results show for the first time a functional role for Ggamma in the effector role of Gbetagamma.     

Role of the isoprenyl pocket of the G protein beta gamma subunit complex in the binding of phosducin and phosducin-like protein

Phosducin (Pdc) and phosducin-like protein (PhLP) regulate G protein-mediated signaling by binding to the betagamma subunit complex of heterotrimeric G proteins (Gbetagamma) and removing the dimer from cell membranes. The binding of Pdc induces a conformational change in the beta-propeller structure of Gbetagamma, creating a pocket between blades 6 and 7. It has been proposed that the isoprenyl group of Gbetagamma inserts into this pocket, stabilizing the Pdc.Gbetagamma structure and decreasing the affinity of the complex for the lipid bilayer. To test this hypothesis, the binding of Pdc and PhLP to several Gbetagamma dimers containing variants of the beta or gamma subunit was measured. These variants included modifications of the isoprenyl group (gamma), residues involved in the conformational change (beta), and residues lining the proposed prenyl pocket (beta). Switching prenyl groups from farnesyl to geranylgeranyl or vice versa had little effect on binding. However, alanine substitution of one residue in the beta subunit involved in the conformational change (W332) decreased binding 5-fold. Alanine substitution of certain residues within the prenyl pocket caused only minor decreases in binding, while a lysine substitution of T329 within the pocket inhibited binding 10-fold. Molecular modeling of the binding energy of the Pdc.Gbeta(1)gamma(2) complex required insertion of the geranylgeranyl group into the prenyl pocket in order to accurately predict the effects of prenyl pocket amino acid substitutions. Finally, a dimer containing a gamma subunit with no prenyl group (gamma(2)-C68S) decreased binding by nearly 20-fold. These results support the structural model in which the prenyl group escapes contact with the aqueous milieu by inserting into the prenyl pocket and stabilizing the Pdc-binding conformation of Gbetagamma.     

Regulation of Xenopus oocyte meiosis arrest by G protein betagamma subunits

BACKGROUND: Progesterone induces the resumption of meiosis (maturation) in Xenopus oocytes through a nongenomic mechanism involving inhibition of an oocyte adenylyl cyclase and reduction of intracellular cAMP. However, progesterone action in Xenopus oocytes is not blocked by pertussis toxin, and this finding indicates that the inhibition of the oocyte adenylyl cyclase is not mediated by the alpha subunits of classical G(i)-type G proteins. RESULTS: To investigate the possibility that G protein betagamma subunits, rather than alpha subunits, play a key role in regulating oocyte maturation, we have employed two structurally distinct G protein betagamma scavengers (G(t)alpha and betaARK-C(CAAX)) to sequester free Gbetagamma dimers. We demonstrated that the injection of mRNA encoding either of these Gbetagamma scavengers induced oocyte maturation. The Gbetagamma scavengers bound an endogenous, membrane-associated Gbeta subunit, indistinguishable from Xenopus Gbeta1 derived from mRNA injection. The injection of Xenopus Gbeta1 mRNA, together with bovine Ggamma2 mRNA, elevated oocyte cAMP levels and inhibited progesterone-induced oocyte maturation. CONCLUSION: An endogenous G protein betagamma dimer, likely including Xenopus Gbeta1, is responsible for maintaining oocyte meiosis arrest. Resumption of meiosis is induced by Gbetagamma scavengers in vitro or, naturally, by progesterone via a mechanism that suppresses the release of Gbetagamma.     

Characterization of the Arabidopsis heterotrimeric G protein

We have used fluorescence resonance energy transfer and co-immunoprecipitation to analyze the interactions among the alpha, beta, and gamma1 subunits of the Arabidopsis heterotrimeric G protein. Using cyan and yellow fluorescent protein fusion constructs, we show that overexpressed Ggamma1 localizes to protoplast membranes, but Gbeta exhibits membrane localization only when the Ggamma1 protein is co-overexpressed. Overexpressed Galpha shows membrane localization unaccompanied by overexpression of either Gbeta or Ggamma1. We detect fluorescence resonance energy transfer between Gbeta and Ggamma1 in the absence of Galpha overexpression and between Galpha and Ggamma1 but only when all three subunits are co-overexpressed. Both Galpha and Gbeta are associated with large macromolecular complexes of approximately 700 kDa in the plasma membrane. Galpha is present in both large complexes and as free Galpha in plasma membranes from wild type plants. In plants homozygous for a null allele of the Gbeta gene, Galpha is associated with smaller complexes in the 200-400-kDa range, indicating that its presence in the large complex depends on association with Gbetagamma. Activation of the Galpha subunit with guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) results in partial dissociation of Galpha from the complex. Hydrogen peroxide (H2O2) promotes extensive dissociation of the Galpha complex but does not interfere with binding of GTPgammaS to purified recombinant Galpha, suggesting that reactive oxygen species affect the stability of the large complex but not the activity of Galpha itself.     

Interaction of Gbeta3s,a splice variant of the G-protein Gbeta3, with Ggamma- and Galpha-proteins

The T-allele of a polymorphism (C825T) in the gene of the G-protein beta3-subunit is associated with a complex phenotype (hypertension, obesity, altered drug responses) and the occurrence of a splice variant termed Gbeta3s which lacks one of the seven WD-domains that compose Gbeta-proteins. Here, we analysed Gbetagamma dimer formation and Galpha activation by Gbeta3s, key functional characteristics of Gbeta-proteins. Cleavage protection assays frequently used to analyse Gbeta1gamma and Gbeta2gamma dimer formation failed for Gbeta3 and Gbeta3s, while in coprecipitation assays, dimerization of Gbeta3 and Gbeta3s with Ggamma5, Ggamma8(c) and Ggamma12 could be demonstrated. Upon expression of Gbeta3s in COS-7 and Sf9 insect cells, binding of GTPgammaS to Galpha-proteins induced by mastoparan-7 and the M(2) muscarinic acetylcholine receptor was facilitated in comparison with cells overexpressing wildtype Gbeta3, as indicated by twofold reduced agonist EC(50) values. Together, these results indicate that Gbeta3s is a biologically active Gbeta-protein that may mediate the enhanced signal transduction observed in cells with the 825T-allele.     

Regulation of angiotensin II-induced G protein signaling by phosducin-like protein

Phosducin-like protein (PhLP) is a broadly expressed member of the phosducin (Pd) family of G protein betagamma subunit (Gbetagamma)-binding proteins. Though PhLP has been shown to bind Gbetagamma in vitro, little is known about its physiological function. In the present study, the effect of PhLP on angiotensin II (Ang II) signaling was measured in Chinese hamster ovary cells expressing the type 1 Ang II receptor and various amounts of PhLP. Up to 3.6-fold overexpression of PhLP had no effect on Ang II-stimulated inositol trisphosphate (IP(3)) formation, whereas further increases caused an abrupt decrease in IP(3) production with half-maximal inhibition occurring at 6-fold PhLP overexpression. This threshold level for inhibition corresponds to the cellular concentration of cytosolic chaperonin complex, a recently described binding partner that preferentially binds PhLP over Gbetagamma. Results of pertussis toxin sensitivity, GTPgammaS binding, and immunoprecipitation experiments suggest that PhLP inhibits phospholipase Cbeta activation by dual mechanisms: (i) steric blockage of Gbetagamma activation of PLCbeta and (ii) interference with Gbetagamma-dependent cycling of G(q)alpha by the receptor. These results suggest that G protein signaling may be regulated through controlling the cellular concentration of free PhLP by inducing its expression or by regulating its binding to the chaperonin.     

Heterotrimeric G protein gamma subunits provide functional selectivity in Gbetagamma dimer signaling in Arabidopsis

The Arabidopsis thaliana heterotrimeric G protein complex is encoded by single canonical Galpha and Gbeta subunit genes and two Ggamma subunit genes (AGG1 and AGG2), raising the possibility that the two potential G protein complexes mediate different cellular processes. Mutants with reduced expression of one or both Ggamma genes revealed specialized roles for each Ggamma subunit. AGG1-deficient mutants, but not AGG2-deficient mutants, showed impaired resistance against necrotrophic pathogens, reduced induction of the plant defensin gene PDF1.2, and decreased sensitivity to methyl jasmonate. By contrast, both AGG1- and AGG2-deficient mutants were hypersensitive to auxin-mediated induction of lateral roots, suggesting that Gbetagamma1 and Gbetagamma2 synergistically inhibit auxin-dependent lateral root initiation. However, the involvement of each Ggamma subunit in this root response differs, with Gbetagamma1 acting within the central cylinder, attenuating acropetally transported auxin signaling, while Gbetagamma2 affects the action of basipetal auxin and graviresponsiveness within the epidermis and/or cortex. This selectivity also operates in the hypocotyl. Selectivity in Gbetagamma signaling was also found in other known AGB1-mediated pathways. agg1 mutants were hypersensitive to glucose and the osmotic agent mannitol during seed germination, while agg2 mutants were only affected by glucose. We show that both Ggamma subunits form functional Gbetagamma dimers and that each provides functional selectivity to the plant heterotrimeric G proteins, revealing a mechanism underlying the complexity of G protein-mediated signaling in plants.     

Activation of phospholipase C-epsilon by heterotrimeric G protein betagamma-subunits

PLC-epsilon was identified recently as a phosphoinositide-hydrolyzing phospholipase C (PLC) containing catalytic domains (X, Y, and C2) common to all PLC isozymes as well as unique CDC25- and Ras-associating domains. Novel regulation of this PLC isozyme by the Ras oncoprotein and alpha-subunits (Galpha(12)) of heterotrimeric G proteins was illustrated. Sequence analyses of PLC-epsilon revealed previously unrecognized PH and EF-hand domains in the amino terminus. The known interaction of Gbetagamma subunits with the PH domains of other proteins led us to examine the capacity of Gbetagamma to activate PLC-epsilon. Co-expression of Gbeta(1)gamma(2) with PLC-epsilon in COS-7 cells resulted in marked stimulation of phospholipase C activity. Gbeta(2) and Gbeta(4) in combination with Ggamma(1), Ggamma(2), Ggamma(3), or Ggamma(13) also activated PLC-epsilon to levels similar to those observed with Gbeta(1)-containing dimers of these Ggamma-subunits. Gbeta(3) in combination with the same Ggamma-subunits was less active, and Gbeta(5)-containing dimers were essentially inactive. Gbetagamma-promoted activation of PLC-epsilon was blocked by cotransfection with either of two Gbetagamma-interacting proteins, Galpha(i1) or the carboxyl terminus of G protein receptor kinase 2. Pharmacological inhibition of PI3-kinase-gamma had no effect on Gbeta(1)gamma(2)-promoted activation of PLC-epsilon. Similarly, activation of Ras in the action of Gbetagamma is unlikely, because a mutation in the second RA domain of PLC-epsilon that blocks Ras activation of PLC failed to alter the stimulatory activity of Gbeta(1)gamma(2). Taken together, these results reveal the presence of additional functional domains in PLC-epsilon and add a new level of complexity in the regulation of this novel enzyme by heterotrimeric G proteins.     

MAU-8 is a Phosducin-like Protein required for G protein signaling in C. elegans

The mau-8(qm57) mutation inhibits the function of GPB-2, a heterotrimeric G protein beta subunit, and profoundly affects behavior through the Galphaq/Galphao signaling network in C. elegans. mau-8 encodes a nematode Phosducin-like Protein (PhLP), and the qm57 mutation leads to the loss of a predicted phosphorylation site in the C-terminal domain of PhLP that binds the Gbetagamma surface implicated in membrane interactions. In developing embryos, MAU-8/PhLP localizes to the cortical region, concentrates at the centrosomes of mitotic cells and remains associated with the germline blastomere. In adult animals, MAU-8/PhLP is ubiquitously expressed in somatic tissues and germline cells. MAU-8/PhLP interacts with the PAR-5/14.3.3 protein and with the Gbeta subunit GPB-1. In mau-8 mutants, the disruption of MAU-8/PhLP stabilizes the association of GPB-1 with the microtubules of centrosomes. Our results indicate that MAU-8/PhLP modulates G protein signaling, stability and subcellular location to regulate various physiological functions, and they suggest that MAU-8 might not be limited to the Galphaq/Galphao network.     

Gbeta residues that do not interact with Galpha underlie agonist-independent activity of K+ channels.

Gbetagamma subunits interact directly and activate G protein-gated Inwardly Rectifying K(+) (GIRK) channels. Little is known about the identity of functionally important interactions between Gbetagamma and GIRK channels. We tested the effects of all mammalian Gbeta subunits on channel activity and showed that whereas Gbeta1-4 subunits activate heteromeric GIRK channels independently of receptor activation, Gbeta5 does not. Gbeta1 and Gbeta5 both bind the N and C termini of the GIRK1 and GIRK4 channel subunits. Chimeric analysis between the Gbeta1 and Gbeta5 proteins revealed a 90-amino acid stretch that spans blades two and three of the seven-propeller structure and is required for channel activation. Within this region, eight non-conserved amino acids were critical for the activity of Gbeta1, as mutation of each residue to its counterpart in Gbeta5 significantly reduced the ability of Gbeta1 to stimulate channel activity. In particular, mutation of residues Ser-67 and Thr-128 to the corresponding Gbeta5 residues completely abolished Gbeta1 stimulation of GIRK channel activity. Mapping these functionally important residues on the three-dimensional structure of Gbeta1 shows that Ser-67, Ser-98, and Thr-128 are the only surface accessible residues. Galpha(i)1 interacts with Ser-98 but not with Ser-67 and Thr-128 in the heterotrimeric Galphabetagamma structure. Further characterization of the three mutant proteins showed that they fold properly and interact with Ggamma2. Of the three identified functionally important residues, the Ser-67 and Thr-128 Gbeta mutants significantly inhibited basal currents of a channel point mutant that displays Gbetagamma-mediated basal but not agonist-induced currents. Our findings indicate that the presence of Gbeta residues that do not interact with Galpha are involved in Gbetagamma interactions in the absence of agonist stimulation.