Specificity of G protein beta and gamma subunit interactions.

Multiple heterotrimeric guanine nucleotide binding protein (G protein) subunits have evolved to couple a large variety of receptors to intracellular effectors. G protein beta gamma subunits are essential for efficient coupling of alpha subunits to receptors, and they are also important for modulation of effectors. Several different beta and gamma subunits exist, but it is not known whether all possible combinations of beta and gamma can form functional dimers. To answer this question, we have compared the ability of in vitro translated beta 1, beta 2, and beta 3 to form dimers with either gamma 1 or gamma 2. Dimerization was monitored by gel filtration, resistance to tryptic digestion, and chemical cross-linking. The results indicate that beta 1 binds both gamma subunits, beta 2 binds only gamma 2, and beta 3 will bind neither gamma 1 or gamma 2. Hence, the occurrence of beta gamma dimers may be partially regulated by the ability of the subunits to associate. Specificity of dimerization might allow cells to co-express multiple beta and gamma subunits while maintaining efficient and specific signal transduction.     

In vitro synthesis of G protein beta gamma dimers

The guanine nucleotide-binding proteins (G proteins), which play a central role in coupling membrane-bound receptors to intracellular effectors, are heterotrimers composed of alpha, beta, and gamma subunits. The beta and gamma subunits form a functional monomer that does not appear to separate under physiological conditions. This has made it difficult to differentiate the individual roles of beta and gamma subunits in signal transduction. To characterize the individual subunits, the 36-kDa beta subunit (beta 1), brain gamma (gamma 2), and transducin gamma (gamma t) were translated in vitro in a rabbit reticulocyte lysate system. Hydrodynamic studies and tryptic proteolysis were used to compare the physical properties of the in vitro translation products with those of beta gamma dimers purified from bovine brain. The hydrodynamic studies indicate that, without gamma subunits, the beta subunits are not stable but tend to aggregate into high molecular weight complexes. When beta and gamma subunits were co-translated, stable beta gamma dimers formed that bound alpha 0 in a guanine nucleotide-dependent manner. The beta gamma dimers were less hydrophobic than those purified from bovine brain. This may reflect a lack of post-translational modification in the reticulocyte lysate or other differences between the in vitro translation products and the purified beta gamma. When beta and gamma were translated separately and then mixed, beta gamma dimers also formed. Analysis of in vitro translated beta gamma subunits will provide ways to assess the function of these subunits and to determine the structural requirements for beta gamma formation.     

Chromosomal mapping of five mouse G protein gamma subunits

Heterotrimeric G proteins, composed of alpha, beta, and gamma subunits, transduce signals from transmembrane receptors to a wide range of intracellular effectors. The G protein gamma subunits, which play an indispensible role in this communication, constitute a large and diverse multigene family. Using an interspecific backcross panel, we have determined the mouse chromosomal locations of five gamma subunit genes: gamma2, gamma8, gamma10, gamma12, and gammaCone. Combined with previous mapping studies, these data indicate that, with the possible exception of gamma1 and gamma11, the G protein gamma subunit genes are well dispersed within the mouse and human genomes.     

Role of the gamma subunit prenyl moiety in G protein beta gamma complex interaction with phospholipase Cbeta

The G protein betagamma complex regulates a wide range of effectors, including the phospholipase Cbeta isozymes (PLCbetas). Prenyl modification of the gamma subunit is necessary for this activity. Evidence presented here supports a direct interaction between the G protein gamma subunit prenyl group and PLCbeta isozymes. A geranylgeranylated peptide corresponding to the C-terminal region of the gamma subunit type, gamma2, strongly inhibits stimulation of PLCbeta2 and PLCbeta3 activity by the betagamma complex. This effect is specific because the same peptide has no effect on stimulation of PLCbeta by an alpha subunit type, alphaq. Prenylation of the gamma peptide is required for its inhibitory effect. When interaction of prenylated gamma subunit peptide to fluorophore-tagged PLCbeta2 was examined by fluorescence spectroscopy, prenylated but not unprenylated peptide increased PLCbeta2 fluorescence emission energy, indicating direct binding of the prenyl moiety to PLCbeta. In addition, fluorescence resonance energy transfer was detected between fluorophore tagged PLCbeta and wild type betagamma complex but not an unprenylated mutant betagamma complex. We conclude that a major function of the gamma subunit prenyl group is to facilitate direct protein-protein interaction between the betagamma complex and an effector, phospholipase Cbeta.     

Mice with IFN-gamma receptor deficiency are less susceptible to experimental autoimmune myasthenia gravis

IFN-gamma can either adversely or beneficially affect certain experimental autoimmune diseases. To study the role of IFN-gamma in the autoantibody-mediated experimental autoimmune myasthenia gravis (EAMG), an animal model of myasthenia gravis in humans, IFN-gammaR-deficient (IFN-gammaR-/-) mutant C57BL/6 mice and congenic wild-type mice were immunized with Torpedo acetylcholine receptor (AChR) plus CFA. IFN-gammaR-/- mice exhibited significantly lower incidence and severity of muscle weakness, lower anti-AChR IgG Ab levels, and lower Ab affinity to AChR compared with wild-type mice. Passive transfer of serum from IFN-gammaR-/- mice induced less muscular weakness compared with serum from wild-type mice. In contrast, numbers of lymph node cells secreting IFN-gamma and of those expressing IFN-gamma mRNA were strongly augmented in the IFN-gammaR-/- mice, reflecting a failure of negative feedback circuits. Cytokine studies by in situ hybridization revealed lower levels of lymphoid cells expressing AChR-reactive IL-1beta and TNF-alpha mRNA in AChR + CFA-immunized IFN-gammaR-/- mice compared with wild-type mice. No differences were found for AChR-reactive cells expressing IL-4, IL-10, or TGF-beta mRNA. These results indicate that IFN-gamma promotes systemic humoral responses in EAMG by up-regulating the production and the affinity of anti-AChR autoantibodies, thereby contributing to susceptibility to EAMG in C57BL/6-type mice.     

G protein gamma subunit interaction with a receptor regulates receptor-stimulated nucleotide exchange

The surfaces of heterotrimeric G proteins (alphabetagamma) in contact with receptors and the molecular events at these sites, which lead to G protein activation, are largely unknown. We show here that a peptide from the C terminus of a G protein gamma subunit blocks muscarinic receptor-stimulated G protein activation in a sequence-dependent fashion. A G protein mutated at the same site on the gamma subunit shows enhanced receptor stimulated nucleotide exchange without affecting G protein heterotrimerization. Ineffective contact between the gamma subunit and receptor increases the rate of receptor-stimulated nucleotide exchange. Specific interaction of the G protein gamma subunit with the receptor thus helps the betagamma complex to act at a distance and control guanine nucleotide exchange in the alpha subunit.     

Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits

Members of the phospholipase C-beta (PLC-beta) family of proteins are activated either by G alpha or G beta gamma subunits of heterotrimeric G proteins. To define specific regions of PLC-beta 3 that are involved in binding and activation by G beta gamma, a series of fragments of PLC-beta 3 as glutathione-S-transferase (GST) fusion proteins were produced. A fragment encompassing the N-terminal pleckstrin homology (PH) domain and downstream sequence (GST-N) bound to G protein beta 1 gamma 2 in an in vitro binding assay, and binding was inhibited by G protein alpha subunit, G alpha i1. This PLC-beta 3 fragment also inhibited G beta gamma-stimulated PLC-beta activity in a reconstitution system, while having no significant effect on G alpha q-stimulated PLC-beta 3 activity. The N-terminal G beta gamma binding region was delineated further to the first 180 amino acids, and the sequence Asn150-Ser180, just distal to the PH domain, was found to be required for the interaction. Mutation of basic residues 154Arg, 155Lys, 159Lys, and 161Lys to Glu within this region reduced G beta gamma binding affinity and specifically reduced the EC50 for G beta gamma-dependent activation of the mutant enzyme 3-fold. Basal activity and G alpha q-dependent activation of the enzyme were unaffected by the mutations. While these basic residues may not directly mediate the interaction with G beta gamma, the data provide evidence for an N-terminal G beta gamma binding region of PLC-beta 3 that is involved in activation of the enzyme.     

Selective role of G protein gamma subunits in receptor interaction

Receptor stimulation of nucleotide exchange in a heterotrimeric G protein (alphabetagamma) is the primary event-modulating signaling by G proteins. The molecular mechanisms at the basis of this event and the role of the G protein subunits, especially the betagamma complex, in receptor activation are unclear. In a reconstituted system, a purified muscarinic receptor, M2, activates G protein heterotrimers alphai2beta1gamma5 and alphai2beta1gamma7 with equal efficacy. However, when the alpha subunit type is substituted with alphao, alphaobeta1gamma7 shows a 100% increase in M2-stimulated GTP hydrolysis compared with alphaobeta1gamma5. Using a sensitive assay based on betagamma complex stimulation of phospholipase C activity, we show that both beta1gamma5 and beta1gamma7 form heterotrimers equally well with alphao and alphai. These results indicate that the gamma subunit interaction with a receptor is critical for modulating nucleotide exchange and is influenced by the subunit-type composition of the heterotrimer.     

Differential sensitivity of phosphatidylinositol 3-kinase p110gamma to isoforms of G protein betagamma dimers

The ability of G protein alpha and betagamma subunits to activate the p110gamma isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110gamma form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTP-activated Gs, Gi, Gq, or Go alpha subunits were unable to activate PtdIns 3-kinase. Dimers containing Gbeta(1-4) complexed with gamma2-stimulated PtdIns 3-kinase activity about 26-fold with EC50 values ranging from 4 to 7 nm. Gbeta5gamma2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase Cbeta. A series of dimers with beta subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of betagamma with phosducin (beta1H311Agamma2, beta1R314Agamma2, and beta1W332Agamma2) was tested, and only beta1W332Agamma2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the beta1 subunit complexed with a panel of different Ggamma subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The beta1gamma2, beta1gamma10, beta1gamma12, and beta1gamma13 dimers all activated PtdIns 3-kinase about 26-fold with 4-25 nm EC50 values. The beta1gamma11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110gamma form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the gamma subunit in determining the activation of PtdIns 3-kinase was examined using gamma subunits with altered CAAX boxes directing the addition of farnesyl to the gamma2 subunit and geranylgeranyl to the gamma1 and gamma11 subunits. Replacement of the geranylgeranyl group of the gamma2 subunit with farnesyl inhibited the activity of beta1gamma2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the gamma1 and gamma11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all beta subunits, with the exception of beta5, interact equally well with PtdIns 3-kinase. In contrast, the composition of the gamma subunit and its prenyl group markedly affects the ability of the betagamma dimer to stimulate PtdIns 3-kinase.     

The bombesin receptor subtypes have distinct G protein specificities

We used an in situ reconstitution assay to examine the receptor coupling to purified G protein alpha subunits by the bombesin receptor family, including gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin receptor subtype 3 (BRS-3). Cells expressing GRP-R or NMB-R catalyzed the activation of squid retinal Galphaq and mouse Galphaq but not bovine retinal Galphat or bovine brain Galphai/o. The GRP-R- and NMB-R-catalyzed activations of Galphaq were dependent upon and enhanced by different betagamma dimers in the same rank order as follows: bovine brain betagamma > beta1gamma2 > beta1gamma1. Despite these qualitative similarities, GRP-R and NMB-R had distinct kinetic properties in receptor-G protein coupling. GRP-R had higher affinities for bovine brain betagamma, beta1gamma1, and beta1gamma2 and squid retinal Galphaq. In addition, GRP-R showed higher catalytic activity on squid Galphaq. Like GRP-R and NMB-R, BRS-3 did not catalyze GTPgammaS binding to Galphai/o or Galphat. However, BRS-3 showed little, if any, coupling with squid Galphaq but clearly activated mouse Galphaq. GRP-R and NMB-R catalyzed GTPgammaS binding to both squid and mouse Galphaq, with GRP-R activating squid Galphaq more effectively, and NMB-R also showed slight preference for squid Galphaq. These studies reveal that the structurally similar bombesin receptor subtypes, in particular BRS-3, possess distinct coupling preferences among members of the Galphaq family.     

Mice expressing only monosialoganglioside GM3 exhibit lethal audiogenic seizures

Gangliosides are a family of glycosphingolipids that contain sialic acid. Although they are abundant on neuronal cell membranes, their precise functions and importance in the central nervous system (CNS) remain largely undefined. We have disrupted the gene encoding GD3 synthase (GD3S), a sialyltransferase expressed in the CNS that is responsible for the synthesis of b-series gangliosides. GD3S-/- mice, even with an absence of b-series gangliosides, appear to undergo normal development and have a normal life span. To further restrict the expression of gangliosides, the GD3S mutant mice were crossbred with mice carrying a disrupted GalNAcT gene encoding beta1,4-N-acetylgalactosaminyltransferase. These double mutant mice expressed GM3 as their major ganglioside. In contrast to the single mutant mice, the double mutants displayed a sudden death phenotype and were extremely susceptible to induction of lethal seizures by sound stimulus. These results demonstrate unequivocally that gangliosides play an essential role in the proper functioning of the CNS.     

Characterization of the cDNA and genomic sequence of a G protein gamma subunit (gamma 5).

A cDNA from human placenta and liver tissues that contained both sequence for the lysosomal glycosidase di-N-acetylchitobiase and sequence homologous to the gamma subunit of GTP-binding proteins was previously isolated. Here we have shown that the gamma-subunit-homologous portion of this unusual cDNA is derived from a member of the gamma-subunit multigene family. The partial human gamma-subunit sequence was used to isolate the corresponding full-length cDNA clones from bovine and rat livers. The two cDNAs encode identical 68-amino-acid proteins (7.3 kDa) homologous to previously cloned G protein gamma subunits. The bovine gene sequence encoding this new gamma-subunit isoform (gamma 5) was determined and found to have an intron-exon structure consistent with the original human chitobiase-gamma 5-subunit hybrid mRNA being a product of alternative splicing. Genomic cloning also resulted in the isolation of a human gamma 5 pseudogene.     

A G protein gamma subunit-specific peptide inhibits muscarinic receptor signaling

Muscarinic acetylcholine receptors modulate the function of a variety of effectors through heterotrimeric G proteins. A prenylated peptide specific to the G protein gamma5 subunit type inhibits G protein activation by the M2 muscarinic receptor in a reconstitution assay. Scrambling the amino acid sequence of the peptide significantly reduces the efficacy of the peptide. The peptide does not disrupt the G protein heterotrimer. In cultured sympathetic neurons, the gamma5 peptide inhibits modulation of Ca(2+) current by the M4 receptor. Peptide activity is specific, the scrambled peptide and peptides specific to two other members of the G protein gamma subunit family are significantly less effective. The gamma5 peptide has no effect on Ca(2+) current modulation by the alpha2-adrenergic and somatostatin receptors. In addition, the gamma5 peptide inhibits muscarinic receptor signaling in spinal cord slices with specificity. These results support a specific role for G protein gamma subunit types in signal transduction, most likely at the receptor-G protein interface.     

Mice with selective elimination of striatal acetylcholine release are lean,show altered energy homeostasis and changed sleep/wake cycle

Cholinergic neurons are known to regulate striatal circuits; however, striatal‐dependent physiological outcomes influenced by acetylcholine (ACh) are still poorly under;?>stood. Here, we used vesicular acetylcholine transporter (VAChT)^{D2‐Cre‐flox/flox} mice, in which we selectively ablated the vesicular acetylcholine transporter in the striatum to dissect the specific roles of striatal ACh in metabolic homeostasis. We report that VAChT^{D2‐Cre‐flox/flox} mice are lean at a young age and maintain this lean phenotype with time. The reduced body weight observed in these mutant mice is not attributable to reduced food intake or to a decrease in growth rate. In addition, changed activity could not completely explain the lean phenotype, as only young VAChT^{D2‐Cre‐flox/flox} mice showed increased physical activity. Interestingly, VAChT^{D2‐Cre‐flox/flox} mice show several metabolic changes, including increased plasma levels of insulin and leptin. They also show increased periods of wakefulness when compared with littermate controls. Taken together, our data suggest that striatal ACh has an important role in the modulation of metabolism and highlight the importance of striatum cholinergic tone in the regulation of energy expenditure. These new insights on how cholinergic neurons influence homeostasis open new avenues for the search of drug targets to treat obesity.     

Role of the G protein gamma subunit in beta gamma complex modulation of phospholipase Cbeta function

The G protein betagamma complex regulates a wide range of effectors, including the phospholipase C isozymes (PLCbetas). Different domains on the beta subunit are known to contact phospholipase Cbeta and affect its regulation. In contrast, the role of the gamma subunit in Gbetagamma modulation of PLCbeta function is not known. Results here show that the gamma subunit C-terminal domain is involved in mediating Gbetagamma interactions with phospholipase Cbeta. Mutations were introduced to alter the position of the post-translational prenyl modification at the C terminus of the gamma subunit with reference to the beta subunit. These mutants were appropriately post-translationally modified with the geranylgeranyl moiety. A deletion that shortened the C-terminal domain, insertions that extended this domain, and a point mutation, F59A, that disrupted the interaction of this domain with the beta subunit were all affected in their ability to activate PLCbeta to varying degrees. All mutants, however, interacted equally effectively with the G(o)alpha subunit. The results indicate that the G protein gamma subunit plays a direct role in the modulation of effector function by the betagamma complex.     

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.     

Phosphorylation of F-actin-associating G protein gamma12 subunit enhances fibroblast motility

Eleven isoforms of G protein gamma subunit have been found thus far, but the precise roles of individual gamma subunits are not known. The gamma12 subunit has two unique properties: phosphorylation by protein kinase C and association with F-actin. To elucidate the role of gamma12, we overexpressed gamma12 and other gamma subunits in NIH 3T3 cells together with the beta1 subunit. The overexpressed gamma12 as well as endogenous gamma12, but not gamma2, gamma5, and gamma7 subunits, associated with cytoskeletal components. Expression of gamma12 induced remarkable changes including cell rounding, disruption of stress fibers, and enhancement of cell migration, but expression of other gamma subunits did not induce significant changes. Deletion of the N-terminal region of gamma12 decreased the abilities of gamma12 to associate with cytoskeletal fractions, to induce cell rounding, and to increase cell motility. Replacement by alanine of Ser2 of gamma12 (Ser1 of a mature gamma12 protein), a phosphorylation site for protein kinase C, eliminated these effects of gamma12, whereas a mutant in which Ser2 was replaced with glutamic acid showed effects equivalent to wild-type gamma12. These results indicate that phosphorylation of gamma12 at Ser2 enhances the motility of cells.     

Proteomic analysis of bovine brain G protein gamma subunit processing heterogeneity

We characterized the variable processing of the G protein gamma subunit isoforms associated with bovine brain G proteins, a primary mediator of cellular communication. Ggamma subunits were isolated from purified brain G proteins and characterized by Edman sequencing, by MALDI MS, by chemical and/or enzymatic fragmentation assayed by MALDI MS, and by MS/MS fragmentation and sequencing. Multiple forms of six different Ggamma isoforms were detected. Significant variation in processing was found at both the amino termini and particularly the carboxyl termini of the proteins. All Ggamma isoforms contain a carboxyl-terminal CAAX motif for prenylation, carboxyl-terminal proteolysis, and carboxymethylation. Characterization of these proteins indicates significant variability in the normal processing of all of these steps in the prenylation reaction, including a new variation of prenyl processing resulting from cysteinylation of the carboxyl terminus. These results have multiple implications for intracellular signaling mechanisms by G proteins, for the role of prenyl processing variation in cell signaling, and for the site of action and consequences of drugs that target the prenylation modification.     

G蛋白βγ亚单位介导的信号转导途径

跨膜信息传递有关的Ｇ蛋白由α、β和γ亚单位所组成,受体激动后,引起ＧＴＰ与α亚单位结合,导致Ｇα与Ｇβγ分离。近年来发现Ｇα、受体本射和许多效应分子如Ｋ＾＋通道、Ｇａ＾２＋通道、磷脂酶Ｃ－β、腺苷酸环化酶、酷氨酸、ＭＡＰＫ和受体激酶等都受Ｇβγ的调节,Ｇβγ同Ｇα一样均可引起效应蛋白的激活,在细胞信号转导中起同样重要作用,共同介导一系列的生物学效应。