G蛋白亚单位基因家族研究进展

G蛋白由α、β、γ三个亚单位组成异源三聚体。目前已发现16个α、6个β和12个γ基因。G蛋白亚单位基因家族相当保守并且原始,几乎所有G蛋白基因外显子-内含子连接均遵从GT-AG规则,并且各亚单位基因编码区内含子结构和位置显示出很高的保守性。多数G蛋白基因具有持家基因的特点。G蛋白基因在基因组中的分布存在着丛集的倾向,有5对α基因呈二联串连排列。     

The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein

The STE4 and STE18 genes are required for haploid yeast cell mating. Sequencing of the cloned genes revealed that the STE4 polypeptide shows extensive homology to the beta subunits of mammalian G proteins, while the STE18 polypeptide shows weak similarity to the gamma subunit of transducin. Null mutations in either gene can suppress the haploid-specific cell-cycle arrest caused by mutations in the SCG1 gene (previously shown to encode a protein with similarity to the alpha subunit of G proteins). We propose that the products of the STE4 and STE18 genes comprise the beta and gamma subunits of a G protein complex coupled to the mating pheromone receptors. The genetic data suggest pheromone-receptor binding leads to the dissociation of the alpha subunit from beta gamma (as shown for mammalian G proteins), and the free beta gamma element initiates the pheromone response.     

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.     

Members of the Gq alpha subunit gene family activate phospholipase C beta isozymes.

The relative specificities of members of the G alpha q family of GTP-binding proteins were tested for their ability to activate different phosphoinositide-specific phospholipase C (PI-PLC) beta isozymes. Cos-7 cells were transfected with cDNA corresponding to G alpha q, G alpha 11, G alpha 14, and G alpha 16. Most of the recombinant protein was bound to the cell membrane and these membranes were washed to elute endogenous PI-PLC activity. The membrane preparation was reconstituted with purified preparations of the PI-PLC beta isozymes and guanosine 5'-O-thiotriphosphate (GTP gamma S)-stimulated enzyme activity was measured. All four proteins of the G alpha q family were found to stimulate PI-PLC beta 1, with G alpha q and G alpha 11 being most efficient. On the other hand, G alpha 16 was found to most effectively activate PI-PLC beta 2, while G alpha q, G alpha 11, and G alpha 14 showed less stimulation. Specific anti- G alpha 16 antibody blocked the stimulation of both PI-PLC beta 1 and PI-PLC beta 2 in the enriched membrane fraction. We conclude that there is specificity in the interaction of different members of the Gq family with different PI-PLC beta effectors. This specificity may be important in generating tissue- or receptor-specific responses in vivo.     

Chromosomal localization of the genes encoding two forms of the G protein beta polypeptide, beta 1 and beta 3, in man

The signal-transducing G proteins are heterotrimers composed of three subunits, alpha, beta, and gamma. Multiple distinctive forms of the alpha, beta, and gamma subunits, each encoded by a distinct gene, have been described. To investigate further the structural diversity of the beta subunits, we recently cloned and characterized a novel cDNA encoding a third form of the G protein beta subunit, which we have termed beta 3. The protein corresponding to beta 3 has not yet been identified. The three forms of the beta subunit show 81-90% amino acid sequence identity. Previous studies had localized the human genes for the beta 1 and beta 2 subunits to chromosomes 1 and 7, respectively. The present studies were designed to determine whether the gene encoding beta 3 is linked to either the beta 1 or the beta 2 gene. Genomic DNA was isolated from a panel of rodent-human hybrid cell lines and analyzed by hybridization to cDNAs for beta 1 and beta 3. Discordancy analysis allowed assignment of the beta 3 gene to chromosome 12 and confirmed the previous assignment of the beta 1 gene to chromosome 1. These results were confirmed and extended by using in situ chromosome hybridization, which permitted the regional localization of the beta 1 gene to 1pter----p31.2 and the beta 3 gene to 12pter----p12.3. Digestion of human genomic DNA with 10 restriction enzymes failed to disclose a restriction fragment length polymorphism for the beta 3 gene. These data indicate that there is considerable diversity in the genomic organization of the beta subunit family.     

Diversity among the beta subunits of heterotrimeric GTP-binding proteins: characterization of a novel beta-subunit cDNA.

Heterotrimeric guanine nucleotide binding proteins transduce signals from cell surface receptors to intracellular effectors. The alpha subunit is believed to confer receptor and effector specificity on the G protein. This role is reflected in the diversity of genes that encode these subunits. The beta and gamma subunits are thought to have a more passive role in G protein function; biochemical data suggests that beta-gamma dimers are shared among the alpha subunits. However, there is growing evidence for active participation of beta-gamma dimers in some G protein mediated signaling systems. To further investigate this role, we examined the diversity of the beta subunit family in mouse. Using the polymerase chain reaction, we uncovered a new member of this family, G beta 4, which is expressed at widely varying levels in a variety of tissues. The predicted amino acid sequence of G beta 4 is 79% to 89% identical to the three previously known beta subunits. The diversity of beta gene products may be an important corollary to the functional diversity of G proteins.     

Cloning, characterization, and mapping of the gene encoding the human G protein gamma 2 subunit

G proteins play vital roles in cellular responses to external signals. The specificity of G protein-receptor interaction is mediated mostly by the gamma-subunit and the individual members of the gamma-subunit multigene family would hence be expected to each have a particular expression profile. In an experiment designed to isolate genes expressed predominantly in human testis we identified a cDNA fragment corresponding to the gamma2 gene. Although the protein sequence of the gamma2 subunit has previously been published, the cDNA sequence, expression pattern, genomic structure, and localisation of the human GNG2 gene have not been described. We report the complete sequence of the GNG2 cDNA which is 1066 bp long and contains an open reading frame encoding a protein of 71 amino acids. This protein is 100% homologous to the bovine, mouse, and rat G protein gamma2 subunit. The gene structure is very similar to that of other Ggamma-subunit genes in that there are two introns, one located in the 5' UTR and the other within the ORF. We show that this gene is expressed in a range of foetal tissues as well as adult testis, adrenal gland, brain, white blood cells and lung but not in adult liver, muscle, sperm, prostate gland nor in the testes of two different infertile patients. There is evidence that GNG2 is expressed in malignant tissues. Using two independent methods, we have mapped the human GNG2 gene to chromosome 14q21.     

Gz, a guanine nucleotide-binding protein with unique biochemical properties

Cloning of a complementary DNA (cDNA) for Gz alpha, a newly appreciated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the beta gamma subunit complex common to other G proteins; another 26-kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant Gz alpha (rGz alpha) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rGz alpha has several properties that distinguish it from other G protein alpha subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min-1), which is reduced greater than 20-fold in the presence of mM concentrations of Mg2+. In addition, the rate of the intrinsic GTPase activity of Gz alpha is extremely slow. The hydrolysis rate (kcat) for rGz alpha at 30 degrees C is 0.05 min-1, or 200-fold slower than that determined for other G protein alpha subunits. rGz alpha can interact with bovine brain beta gamma but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family.     

G protein betagamma11 complex translocation is induced by Gi, Gq and Gs coupling receptors and is regulated by the alpha subunit type

G protein activation by Gi/Go coupling M2 muscarinic receptors, Gq coupling M3 receptors and Gs coupling beta2 adrenergic receptors causes rapid reversible translocation of the G protein gamma11 subunit from the plasma membrane to the Golgi complex. Co-translocation of the beta1 subunit suggests that gamma11 translocates as a betagamma complex. Pertussis toxin ADP ribosylation of the alphai subunit type or substitution of the C terminal domain of alphao with the corresponding region of alphas inhibits gamma11 translocation demonstrating that alpha subunit interaction with a receptor and its activation are requirements for the translocation. The rate of gamma11 translocation is sensitive to the rate of activation of the G protein alpha subunit. alpha subunit types that show high receptor activated rates of guanine nucleotide exchange in vitro support high rates of gamma11 translocation compared to alpha subunit types that have a relatively lower rate of guanine nucleotide exchange. The results suggest that the receptor induced translocation of gamma11 is controlled by the rate of cycling of the G protein through active and inactive forms. They also demonstrate that imaging of gamma11 translocation can be used as a non-invasive tool to measure the relative activities of wild type or mutant receptor and alpha subunit types in a live cell.     

Structural and functional studies of cross-linked Go protein subunits

The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit.     

Receptor activation of G proteins

G proteins are a highly conserved family of membrane-associated proteins composed of alpha, beta, and gamma subunits. The alpha subunit, which is unique for each G protein, binds GDP or GTP. Receptors such as those for beta- and alpha-adrenergic catecholamines, muscarinic agonists, and the retinal photoreceptor rhodopsin, catalyze the exchange of GDP for GTP binding to the alpha subunit of a specific G protein. G alpha.GTP regulates appropriate effector enzymes such as adenylyl cyclase or the cyclic GMP phosphodiesterase. The beta gamma-subunit complex of G proteins is required for efficient receptor-catalyzed alpha subunit guanine nucleotide exchange and also functions as an attenuator of alpha subunit activation of effector enzymes. Recent elucidation of both receptor and G protein primary sequence has allowed structural predictions and new experimental approaches to study the mechanism of receptor-catalyzed G protein regulation of specific effector systems and the control of cell function including metabolism, secretion, and growth.     

Identification and isolation of common and tissue-specific geranylgeranylated gamma subunits of guanine-nucleotide-binding regulatory proteins in various tissues.

Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their alpha subunits, though a notable multiplicity of gamma subunits has also been demonstrated. To investigate whether each subtype of alpha subunit is associated with a particular gamma subunit, various oligomeric G proteins, purified from bovine tissues, were subjected to gel electrophoresis in a Tricine buffer system. All G proteins examined were shown to have more than two kinds of gamma subunit. Of the brain G proteins, GoA, GoB, and Gi1 contain the same set of three gamma subunits, but Gi2 contains only two of these subunits. Lung Gi1 and Gi2 and spleen Gi2 and Gi3 had similar sets of two gamma subunits, one of which was distinct from the gamma subunits of brain G proteins. These observations indicate that each subtype of alpha subunit is associated with a variety of beta gamma subunits, and that the combinations differ among cells. For analyses of the structural diversity of the gamma subunits, beta gamma subunits were purified from the total G proteins of each tissue and subjected to reverse-phase HPLC under denaturing conditions, where none of the beta subunits were eluted from the column. Three distinct gamma subunits were isolated in this way from brain beta gamma subunits. In contrast, lung and spleen beta gamma subunits contained at least five gamma subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several gamma subunits, two subspecies appeared to be common to the three tissues. In fact, in each case, the partial amino acid sequence of the most abundant gamma subunit in each tissue was identical, and the sequences coincided exactly with that of 'gamma 6' [Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758-15761]. Fast-atom-bombardment mass spectrometry analysis indicated that this abundant gamma subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C-terminus, as was 'gamma 6' from brain. In addition to abundant gamma subunits, other tissue-specific gamma subunits were also shown to be geranylgeranylated by gas-chromatography-coupled mass spectrometry analysis of Raney nickel-treated gamma subunits. These results suggest that most gamma subunits associated with many different subtypes of alpha subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated gamma subunit.     

Characterization of heterotrimeric G protein complexes in rice plasma membrane

Two genes in the rice genome were identified as those encoding the gamma subunits, gamma1 and gamma2, of heterotrimeric G proteins. Using antibodies against the recombinant proteins for the alpha, beta, gamma1, and gamma2 subunits of the G protein complexes, all of the subunits were proven to be localized in the plasma membrane in rice. Gel filtration of solubilized plasma membrane proteins showed that all of the alpha subunits were present in large protein complexes (about 400 kDa) containing the other subunits, beta, gamma1, and gamma2, and probably also some other proteins, whereas large amounts of the beta and gamma (gamma1 and gamma2) subunits were freed from the large complexes and took a 60-kDa form. A yeast two-hybrid assay and co-immunoprecipitation experiments showed that the beta subunit interacted tightly with the gamma1 and gamma2 subunits, and so the beta and gamma subunits appeared to form dimers in rice cells. Some dimers were associated with the alpha subunit, because few beta, gamma1, and gamma2 subunits were present in the 400-kDa complexes in a rice mutant, d1, which was lacking in the alpha subunit. When a constitutively active form of the alpha subunit was prepared by the exchange of one amino acid residue and introduced into d1, the mutagenized subunit was localized in the plasma membrane of the transformants and took a free, and not the 400-kDa, form.     

The 35- and 36-kDa beta subunits of GTP-binding regulatory proteins are products of separate genes

The wide range of functions attributed to GTP-binding regulatory proteins (G proteins) is reflected in the structural diversity which exists among the alpha, beta, and gamma subunits of G proteins. Recently two cDNA clones encoding beta subunits, beta 1 and beta 2, were isolated from bovine and human cDNA libraries. We report here that the beta 2 gene encodes the 35-kilodalton (kDa) component of the beta 35/beta 36 subunit of G proteins and that the beta 1 gene encodes the 36-kilodalton component. The in vitro translation product of the beta 2 cDNA co-migrates with the 35-kDa beta subunit (beta 35), while the in vitro product of the beta 1 cDNA co-migrates with the 36-kDa beta subunit (beta 36) on denaturing polyacrylamide gels. In addition, antisera generated against synthetic beta 2 peptides bind specifically to the beta 35 component of isolated G proteins and to a 35-kDa protein in myeloid cell membranes. Our results suggest that the two beta subunits could serve distinct functions, as they are derived from separate genes which have been highly conserved in evolution.     

Identification of a region in G protein γ subunits conserved across species but hypervariable among subunit isoforms

The heterotrimeric GTP binding proteins, G proteins, consist of three distinct subunits: alpha, beta, and gamma. There are 12 known mammalian gamma subunit genes whose products are the smallest and most variable of the G protein subunits. Sequencing of the bovine brain gamma(10) protein by electrospray mass spectrometry revealed that it differs from the human protein by an Ala to Val substitution near the N-terminus. Comparison of gamma isoform subunit sequences indicated that they vary substantially more at the N-terminus than at other parts of the protein. Thus, species variation of this region might reflect the lack of conservation of a functionally unimportant part of the protein. Analysis of 38 gamma subunit sequences from four different species shows that the N-terminus of a given gamma subunit isoform is as conserved between different species as any other part of the protein, including highly conserved regions. These data suggest that the N-terminus of gamma is a functionally important part of the protein exhibiting substantial isoform-specific variation.     

G Protein beta subunit types differentially interact with a muscarinic receptor but not adenylyl cyclase type II or phospholipase C-beta 2/3

In comparison with the alpha subunit of G proteins, the role of the beta subunit in signaling is less well understood. During the regulation of effectors by the betagamma complex, it is known that the beta subunit contacts effectors directly, whereas the role of the beta subunit is undefined in receptor-G protein interaction. Among the five G protein beta subunits known, the beta(4) subunit type is the least studied. We compared the ability of betagamma complexes containing beta(4) and the well characterized beta(1) to stimulate three different effectors: phospholipase C-beta2, phospholipase C-beta3, and adenylyl cyclase type II. beta(4)gamma(2) and beta(1)gamma(2) activated all three of these effectors with equal efficacy. However, nucleotide exchange in a G protein constituting alpha(o)beta(4)gamma(2) was stimulated significantly more by the M2 muscarinic receptor compared with alpha(o)beta(1)gamma(2). Because alpha(o) forms heterotrimers with beta(4)gamma(2) and beta(1)gamma(2) equally well, these results show that the beta subunit type plays a direct role in the receptor activation of a G protein.     

Conserved structural features are found upstream from the three co-ordinately regulated discoidin I genes of Dictyostelium discoideum

The discoidin I genes of Dictyostelium form a small, co-ordinately regulated multigene family. We have sequenced and compared the upstream regions of the DiscI-alpha, -beta and -gamma genes. For the most part the upstream regions of the three genes are non-homologous. The upstream sequences of the beta and gamma genes are exceedingly A + T-rich, while those of the alpha gene are less so. All three genes have a relatively G + C-rich region 20 to 40 base-pairs in length, found approximately 200 base-pairs 5' to the messenger RNA start site. This G + C-rich region 5' to the beta and gamma genes is flanked by short inverted repeats. Within this region, there is an 11 base-pair exact homology between the alpha and gamma genes, and a less perfect homology between these genes and the beta gene. The homology is flanked at a short distance by interspersed G and T residues. The gamma gene is greater than 90% A + T for greater than 800 base-pairs upstream. Further upstream there is a G + C-rich region that is also found inverted approximately 3.5 X 10(3) base-pairs away. The gamma and beta genes are tandemly linked, and the entire approximately 500 base-pair intergene region between the 3' end of the gamma gene and the 5' end of the beta gene is A + T-rich (approximately 90%) with the exception of the homology region 5' to the gamma gene. We demonstrate also the presence of a discoidin I pseudogene fragment having only 139 base-pairs of discoidin homology with greater than 8% mismatch. It is flanked upstream by five 39 base-pair G + C-rich repeats, and downstream by sequences that are extremely A + T-rich. We discuss the possible significance of the conserved G + C-rich structures on discoidin I gene expression.     

Selective tissue distribution of G protein gamma subunits, including a new form of the gamma subunits identified by cDNA cloning.

The GTP-binding regulatory proteins (G proteins) that transduce signals from receptors to effectors are composed of alpha, beta, and gamma subunits. Whereas the role of alpha subunits in directly regulating effector activity is widely accepted, it has recently been demonstrated that beta gamma subunits may also directly regulate effector activity. This has made clear the importance of identifying and characterizing beta and gamma subunits. We have isolated a cDNA clone encoding a new gamma subunit, referred to here as the gamma 7 subunit, using probes based on peptide sequences of a gamma subunit previously purified from bovine brain. The clone contains a 1.47-kilobase cDNA insert, which includes an open reading frame of 204 base pairs that predicts a 68-amino acid polypeptide with a calculated M(r) of 7553. The predicted protein shares amino acid identities with the other known gamma subunits, ranging from 38 to 68%. Also characteristic of gamma subunits is a carboxyl-terminal CAAX motif. The expression of the gamma 7 subunit as well as the gamma 2, gamma 3, and gamma 5 subunits was examined in several bovine tissues at both the mRNA and protein levels. Whereas the gamma 2 and gamma 3 subunits were selectively expressed in brain, the gamma 5 and gamma 7 subunits were expressed in a variety of tissues. Thus, the gamma 5 and gamma 7 subunits are the first G protein gamma subunits known that could participate in the regulation of widely distributed signal transduction pathways.     

Interaction between cGMP-phosphodiesterase and transducin alpha-subunit in retinal rods. A cross-linking study.

Cross-linking of the different subunits of the retinal cGMP-phosphodiesterase (PDE) with its activator G alpha GTP gamma S (alpha subunit of the retinal G-protein transducin with GTP gamma S (guanosine 5'-O-(3-thiotriphosphate) bound) has been investigated using purified proteins, with a N-hydroxysuccinimide homobifunctional cross-linker, bis(sulfosuccinimidyl)suberate (BS3) and its cleavable analog 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). Interaction of purified G-protein and PDE is achieved in the presence of lecithin vesicles, at protein concentrations sufficient for full PDE activation. Protein subunits linked with DTSSP are separated by cleavage of the disulfide bridge and identified by electrophoresis. Complexes of PDE alpha (PDE beta) with 1 and 2 molecules of activator G alpha GTP gamma S are observed, providing direct evidence for an interaction or at least a close proximity between 2 molecules of activator G alpha and each of the catalytic PDE subunits in the activated state of PDE. The results also reveal symmetrical roles of PDE alpha and PDE beta, with the existence of one site for PDE gamma and one site for G alpha on each catalytic subunit.