G-protein Beta Subunit Research Articles

Molecular characterization of the 77-kDa echinoderm microtubule-associated protein. Homology to the beta-transducin family.

The major microtubule-associated protein (MAP) of sea urchins and several other echinoderms is a polypeptide of M(r) 77,000. The echinoderm MAP (EMAP) is abundant in embryonic and differentiated cells, as well as in mitotic and interphase microtubule arrays. To characterize the molecular structure and function of the EMAP, we isolated a full-length cDNA clone, which has one open reading frame that predicts a polypeptide of 686 amino acids with a calculated M(r) of 75,488. On the basis of charge distribution, EMAP can be divided into two distinctive domains: The NH2-terminal basic region (amino acids 1-137, pI = 10.0) and a slightly acidic, COOH-terminal region (amino acids 138-686, pI = 5.8). This charge distribution is typical of many microtubule-binding proteins, but no significant sequence homology has been detected with any known MAPs. The EMAP, however, does show significant sequence similarity with the beta-subunit of the heterotrimeric G-protein, transducin. The homology lies in a series of 10 imperfect, 43-amino acid repeats (WD-40 repeats) that have been found in many proteins of diverse functions, including beta-transducins, Drosophila Enhancer of split, the yeast STE4, CDC4, CDC20, PRP4, and Tup1 gene products, and the dTAFII80 subunit of Drosophila TFIID. The function of these repeats still remains unknown. It is possible that these repeats are involved in protein-protein interactions, perhaps with the tetratricopeptide repeat-containing protein family. Alternatively, the EMAP may be an important link between signal transduction events and a change in microtubule organization during the cell cycle.

A polyadenylation factor subunit is the human homologue of the Drosophila suppressor of forked protein.

Polyadenylation of messenger RNA precursors is a complex process that requires multiple protein factors (for reviews, see refs 1, 2). Cleavage stimulation factor (CstF) is one of these, functioning together with cleavage-polyadenylation specificity factor, two cleavage factors, and poly(A)+ polymerase. CstF is composed of three subunits of M(r) 77, 64 and 50K. The 64K and 50K subunits contain, respectively, an RNP-type RNA-binding domain that contacts the pre-mRNA and transducin repeats characteristic of G-protein beta-subunits. Here we report the cloning and characterization of the 77K subunit of human CstF (referred to as 77K). We show that the 77K subunit is required for formation of active CstF and bridges the 64K and 50K subunits. Sequence analyses indicate that the 77K subunit is the homologue of the protein encoded by the Drosophila melanogaster suppressor of forked (su(f)) gene. Mutations in su(f) can enhance or suppress the effects of transposable element insertions, and our data indicate that this is due to changes in polyadenylation. Both the 77K subunit and the su(f) protein share homology with Saccharomyces cerevisiae RNA14, previously shown to be involved in mRNA metabolism. Our results thus also indicate that components of the complex polyadenylation machinery are conserved from yeast to man.

Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose.

We identified four polypeptides of 47, 44, 40, and 35 kD that bind to profilin-Sepharose and elute with high salt. When purified by conventional chromatography using an antibody to the 47-kD polypeptide, these four polypeptides copurified as a stoichiometric complex together with three additional polypeptides of 19, 18, and 13 kD that varied in their proportions to the other polypeptides. Partial protein sequences showed that the 47-kD polypeptide is a homologue of S. pombe act2 and the 44-kD polypeptide is a homologue of S. cerevisiae ACT2, both unconventional actins. The 40-kD polypeptide contains a sequence similar to the WD40 motif of the G beta subunit of a trimeric G-protein from Dictyostelium discoideum. From partial sequences, the 35-, 19-, and 18-kD polypeptides appear to be novel proteins. On gel filtration the complex of purified polypeptides cochromatograph with a Stokes' radius of 4.8 nm, a value consistent with a globular particle of 220 kD containing one copy of each polypeptide. Cell extracts also contain components of the complex that do not bind the profilin column. Affinity purified antibodies localize 47- and 18/19-kD polypeptides in the cortex and filopodia of Acanthamoeba. Antibodies to the 47-kD unconventional actin cross-react on immunoblots with polypeptides of similar size in Dictyostelium, rabbit muscle, and conventional preparations of rabbit muscle actin but do not react with actin.

The palmitoylation status of the G-protein G(o)1 alpha regulates its activity of interaction with the plasma membrane.

Plasmids containing cDNAs encoding either the wild-type guanine-nucleotide-binding protein G(o)1 alpha or the palmitoylation-negative cysteine-3-to-serine (C3S) mutant of G(o)1 alpha were transfected into Rat 1 cells, and clones stably expressing immunoreactivity corresponding to these polypeptides were isolated. Clones C5B (expressing wild-type G(o)1 alpha) and D3 (expressing the mutant form) were selected for detailed study. Immunoprecipitation of whole cell lysates of each clone labelled with either [3H]palmitate or [3H]myristate demonstrated incorporation of [3H]myristate into both wild-type and the C3S mutant of G(o)1 alpha, but that incorporation of hydroxylamine-sensitive [3H]palmitate was restricted to the wild type. When membrane and cytoplasmic fractions were prepared from cells of either the C5B or D3 clones, although immunodetection of wild-type G(o)1 alpha was observed only in the membrane fraction, the C3S mutant was present in both membrane and cytoplasmic fractions. Furthermore, a significant proportion of the C3S G(o)1 alpha immunoreactivity was also detected in the cytoplasmic fraction if immunoprecipitation of recently synthesized G(o)1 alpha was performed from fractions derived from cells pulse-labelled with [35S]Trans label. Pretreatment of cells of both clones C5B and D3 with pertussis toxin led to complete ADP-ribosylation of the cellular population of G(o)1 alpha in both cell types, irrespective of whether the polypeptide was subsequently found in the membrane or cytoplasmic fraction following cellular disruption. By contrast, separation of membrane and cytoplasmic fractions before pertussis-toxin-catalysed [32P]ADP-ribosylation allowed modification only of the membrane-associated G(o)1 alpha (whether wild-type or the C3S mutant). This labelling was decreased substantially by incubation of the membranes with guanosine 5'-[beta gamma-imido]triphosphate. No cytoplasmic G-protein beta subunit was detected immunologically, and the non-membrane-associated C3S G(o)1 alpha from D3 cells migrated as an apparently monomeric 40 kDa protein on a Superose 12 gel-filtration column. Membrane-associated wild-type and C3S G(o)1 alpha appeared to interact with guanine nucleotides with similar affinity, as no alteration in the dose-response curves for guanine-nucleotide-induced maintenance of a stable 37 kDa tryptic fragment was noted for the two forms of G(o)1 alpha. Chemical depalmitoylation of membranes of clone C5B with neutral 1 M hydroxylamine caused a release of some 25-30% of each of G(o)1 alpha, Gi2 alpha and Gq alpha/G11 alpha from the membranes. Equivalent treatment of D3 cells caused an equivalent release of Gi2 alpha and Gq alpha/G11 alpha, but was unable to cause any appreciable release of the CS3 form of G(o)1 alpha, which was membrane-bound.

A fifth member of the mammalian G-protein beta-subunit family. Expression in brain and activation of the beta 2 isotype of phospholipase C.

The beta-subunits of the heterotrimeric GTP-binding proteins (G-proteins) are important regulators of G-protein alpha-subunits as well as of certain signal transduction receptors and effectors. We have identified a fifth member of the G-protein beta-subunit family, G beta 5, by molecular cloning. Sequence analysis of recombinant clones revealed an open reading frame of 1,059 base pairs, potentially encoding a protein of 353 amino acids having 53% identity to previously identified G beta-subunit proteins. Northern blot analysis of poly(A)-selected mRNA prepared from different mouse tissues demonstrated that G beta 5 is predominantly expressed in adult brain as two transcripts of 2.3 and 1.75 kilobases. A G beta 5-specific antiserum detected a band of 39.3 kilodaltons on immunoblots of crude membrane proteins isolated from normal mouse brain but not from other tissues. Using a transient cotransfection assay, we showed that a plasmid construct expressing the G beta 5 open reading frame, when coexpressed with different G-protein gamma (G gamma)-subunits, can stimulate the activity of the beta 2 isoform of phosphoinositide-specific phospholipase C (PLC beta 2). The stimulation of PLC beta 2 was most pronounced when G beta 5 was co-expressed with G gamma 2, although activation was observed when G beta 5 was cotransfected with either G gamma 1, G gamma 3, G gamma 5, or G gamma cone. These results prove that G beta 5 can functionally associate with G gamma proteins and is therefore a bona fide member of the G beta protein family. The rather low level of identity between G beta 5 and the other mammalian G beta-subunits may shed light on structural/sequence elements necessary for G beta protein function.

1,25-Dihydroxyvitamin D3 attenuates adenylyl cyclase activity in rat thyroid cells: reduction of thyrotropin receptor number and increase in guanine nucleotide-binding protein Gi-2 alpha.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is the most potent of the naturally occurring vitamin D metabolites. In rat thyroid FRTL-5 cells, 1,25-(OH)2D3 attenuated the increase in TSH-stimulated adenylyl cyclase activity obtained by removing TSH from the culture medium. When cells were incubated with 1,25-(OH)2D3 (10 nmol/liter; 4 days), the binding capacity for specific [125I]TSH binding decreased from 20.1 +/- 1.8 to 8.8 +/- 1.6 fmol/10(6) cells (mean +/- SEM; n = 4; P < 0.01) compared to that in control cells. The Kd did not change (mean +/- SEM, 0.46 +/- 0.09 vs. 0.25 +/- 0.07 nmol/liter; n = 4; P = NS). Western blotting revealed no change in the membrane content of the adenylyl cyclase (AC) stimulatory guanine nucleotide-binding protein (G-protein) alpha-subunit (Gs alpha) during 1,25-(OH)2D3 treatment. Similarly, levels of the AC inhibitory G-protein Gi-3 alpha- and G-protein beta-subunits were not altered by 1,25-(OH)2D3. However, Western blotting with antibodies recognizing both Gi-1 alpha and Gi-2 alpha was augmented 4-fold, presumably representing an increase in Gi-2 alpha only, as Gi-1 alpha messenger RNA (mRNA) was not detected in FRTL-5 cells. 1,25-(OH)2D3 (10 nmol/liter; 4 days) reduced cholera toxin (10 nmol/liter)-stimulated AC activity to 85% of the control value (P < 0.05), whereas forskolin (100 mumol/liter)-stimulated direct activation of AC was inhibited by 39%. The TSH receptor mRNA level correlated to the beta-actin mRNA was 2-fold higher in control cells compared to that in 1,25-(OH)2D3-treated cells 12 h after TSH removal. Only minor alterations in the Gs alpha mRNA/beta-actin mRNA and Gi-3 alpha mRNA/beta-actin mRNA ratios were observed during 1,25-(OH)2D3 treatment, whereas Gi-2 alpha mRNA increased 3-fold compared to that in control cells. No change in the resting intracellular Ca2+ concentration could be detected after 4 days of 1,25-(OH)2D3 treatment. Our studies show that 1,25-(OH)2D3 attenuates AC activity by reducing the TSH receptor number and increasing the level of the AC inhibitory G-protein Gi-2 alpha in FRTL-5 cells.

An eye-specific G beta subunit essential for termination of the phototransduction cascade.

Heterotrimeric G proteins couple various receptors to intracellular effector molecules. Although the role of the G alpha subunit in effector activation, guanine nucleotide exchange and GTP hydrolysis has been well studied, the cellular functions of the G beta subunits are less well understood. G beta gamma dimers bind G alpha subunits and anchor them to the membrane for presentation to the receptor. In specific systems, the G beta subunits have also been implicated in direct coupling to ion channels and to effector molecules. We have isolated Drosophila melanogaster mutants defective in an eye-specific G-protein beta-subunit (G beta e), and show here that the beta-subunit is essential for G-protein-receptor coupling in vivo. Remarkably, G beta mutants are also severely defective in the deactivation of the light response, demonstrating an essential role for the G beta subunit in terminating the active state of this signalling cascade.

Molecular characterization of SIG1, a Saccharomyces cerevisiae gene involved in negative regulation of G-protein-mediated signal transduction.

Two recessive mutations in the Saccharomyces cerevisiae SIG1 (suppressor of inhibitory G-protein) gene have been identified by their ability to suppress the signalling defect of dominant-negative variants of the mating response G-protein beta-subunit. The mutations and deletion of SIG1 enhance the sensitivity of the cells to pheromone and stimulate the basal transcription of a mating specific gene, FUS1, suggesting that Sig1p plays a negatively regulatory role in G beta gamma-mediated signal transduction. An additional function of Sig1p in vegetatively growing cells is suggested by the finding that the mutations and deletion of SIG1 cause temperature-sensitive growth defects. The SIG1 gene encodes a protein with a molecular weight of 65 kDa that contains at the amino-terminus two zinc finger-like sequence motifs. Epistasis experiments localize the action of Sig1p within the pheromone signalling pathway at a position at or shortly after the G-protein. We propose that Sig1p represents a novel negative regulator of G beta gamma-mediated signal transduction.

Regional characterization of G-protein subunits in glomeruli, cortices and medullas of the rat kidney.

We examined the types of guanine nucleotide-binding regulatory (G) protein subunits in isolated glomeruli, cortices excluding glomeruli and medullas of rat kidneys using bacterial toxin-catalyzed adenosine 5'-diphosphate (ADP) ribosylation and specific immunoblots. ADP ribosylation catalyzed by cholera or pertussis toxin revealed the presence of stimulatory G (Gs) or inhibitory G (Gi) proteins in membranes of the 3 segments of the kidney. Immunoblots further demonstrated the existence of several G-protein subunits, two Gs-protein alpha-subunits (G alpha s: 45 and 52 kD), Gi-protein alpha 1, alpha 2 and alpha 3-subunits (G alpha i1, G alpha i2: 40-41 kD, G alpha i3: 40 kD), bacterial toxin-insensitive G-protein alpha q- and alpha 11-subunits (G alpha q/11: 42 kD) and G-protein beta-subunits (G beta: 35-36 kD), in membranes of the preparations. The predominant subspecies of G alpha s was a 52-kD protein in glomerular membranes and a 45-kD protein in membranes of cortices and medullas. All of the G-protein subunits examined, however, were not detected in cytosolic fractions of glomeruli, cortices and medullas. Thus, we conclude that detectable quantities of several G-protein subunits including the new G-protein subunit, G alpha q/11, are present in membranes of glomeruli, cortices not containing glomeruli and medullas from the rat kidney. Both the existence of G alpha i1 and/or G alpha i2 subunits in glomeruli and the presence of G alpha q/11 subunits in the 3 preparations are new evidence.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific enhancement of beta-adrenergic receptor kinase activity by defined G-protein beta and gamma subunits.

The beta and gamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) have recently been shown to play an active role in signal transduction. Among other effects they enable translocation of the beta-adrenergic receptor kinase (beta ARK) from the cytosol to the plasma membrane and thus permit phosphorylation and ultimately desensitization of beta-adrenergic receptors and other G-protein-coupled receptors. To investigate the specificity of this effect, we have purified various combinations of recombinant beta and gamma subunits expressed in Sf9 cells and measured their effects on beta ARK-catalyzed phosphorylation of beta 2-adrenergic receptors and of rhodopsin. The combinations tested were beta 1 gamma 2, beta 1 gamma 3, beta 2 gamma 2, beta 2 gamma 3, and transducin beta gamma (beta 1 gamma 1). There were clear differences in enhancement of rhodopsin phosphorylation, with an order of efficacy beta 2 gamma 2 > beta 1 gamma 2 >> beta 2 gamma 3 approximately beta 1 gamma 3 approximately beta 1 gamma 1. The first two combinations had larger effects than a mixed beta gamma preparation from bovine brain. In enhancing phosphorylation of beta 2-adrenergic receptors, beta 1 gamma 2 was more efficient and potent than all other combinations. These data suggest a twofold specificity of beta gamma complexes in enhancing beta ARK-catalyzed receptor phosphorylation: the gamma subunits may be important in interacting with beta ARK, with gamma 2 being more potent than gamma 3, whereas the beta subunits may determine coupling to the receptors, with beta 2 being more effective than beta 1 for rhodopsin and beta 1 being more effective than beta 2 for beta 2-adrenergic receptors.

Cloning of Saccharomyces cerevisiae STE5 as a suppressor of a Ste20 protein kinase mutant: structural and functional similarity of Ste5 to Farl

The beta and gamma subunits of the mating response G-protein in the yeast Saccharomyces cerevisiae have been shown to transmit the mating pheromone signal to downstream components of the pheromone response pathway. A protein kinase homologue encoded by the STE20 gene has recently been identified as a potential G beta gamma target. We have searched multicopy plasmid genomic DNA libraries for high gene dosage suppressors of the signal transduction defect of ste20 mutant cells. This screen identified the STE5 gene encoding an essential component of the pheromone signal transduction pathway. We provide genetic evidence for a functional interrelationship between the STE5 gene product and the Ste20 protein kinase. We have sequenced the STE5 gene, which encodes a predicted protein of 917 amino acids and is specifically transcribed in haploid cells. Transcription is slightly induced by treatment of cells with pheromone. Ste5 has homology with Far1, a yeast protein required for efficient mating and the pheromone-inducible inhibition of a G1 cyclin, Cln2. A STE5 multicopy plasmid is able to suppress the signal transduction defect of far1 null mutant cells suggesting that Ste5, at elevated levels, is able functionally to replace Far1. The genetically predicted point of function of Ste5 within the pheromone signalling pathway suggests that Ste5 is involved in the regulation of a G beta gamma-activated protein kinase cascade which links a G-protein coupled receptor to yeast homologues of mitogen-activated protein kinases.

Is Receptor Cross-Regulation in Human Heart Caused by Alterations in Cardiac Guanine Nucleotide-Binding Proteins?

1. Guanine nucleotide-binding proteins (G-proteins) play a central role in signal transduction between a wide variety of cell-surface receptors and intracellular second messenger systems. Recently, we and others have demonstrated that cross-regulation can occur between a variety of G-protein-linked receptors in human heart. Chronic beta 1-adrenoceptor blockade gives rise to sensitization of beta 2-adrenoceptor and of 5HT 4-receptor responses, both of which are mediated via stimulation of adenylate cyclase through stimulatory G-proteins (Gs), and also gives rise to desensitization of muscarinic M2-receptor responses, which inhibit adenylate cyclase through inhibitory G-proteins (Gi). 2. In order to investigate whether these effects are due to quantitative changes in cardiac G-protein isoforms, we measured their abundance in right atrial appendage from patients taking or not taking beta 1-adrenoceptor antagonists, by immunoblotting. 3. Samples of right atrial appendage homogenate were subjected to SDS/PAGE, and proteins were electroblotted on to nitrocellulose membranes. These were then probed with specific anti-G protein anti-sera, and binding was revealed by means of a secondary antibody labelled with alkaline phosphatase and using a chromogenic substrate. The resulting bands were quantified by laser densitometry. 4. No quantitative differences were detected, between these two groups of patients, in the amounts of alpha-subunit of 'long' or 'short' Gs isoforms (Gs alpha L and Gs alpha S), or in the amounts of Gi 1 + 2 alpha-subunit (Gi alpha 1 + 2). Nor was any difference found in the abundance of the beta-subunit of G-proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

A G-protein beta-subunit is essential for Dictyostelium development.

Recent studies have demonstrated that G-protein-linked signal transduction pathways play a significant role in the developmental program of the simple eukaryotic organism Dictyostelium. We have reported previously the isolation of a G-protein beta-subunit and present here a more complete analysis of this gene. Low-stringency Southern blots and RFLP mapping studies suggest that the beta-subunit is a unique gene found on linkage group II. Its deduced amino acid sequence of 347 residues is approximately 60% identical to those of the human, Drosophila, and Caenorhabditis elegans beta-subunits. The carboxy-terminal 300 residues are about 70% identical; the amino-terminal 50 residues are quite divergent, containing only 10 identities. At all stages of growth and development, a single 1.9-kb beta-subunit mRNA is present at a high level, and a specific antibody detects a single 37-kD protein. We propose that G-protein heterotrimers are formed when this beta-subunit couples with each of the eight distinct G-protein alpha-subunits that are transiently expressed during development. Targeted disruption of the beta-subunit gene had no effect on the viability of haploid cells, but resulted in the inability of cells to aggregate.

A U3 snoRNP protein with homology to splicing factor PRP4 and G beta domains is required for ribosomal RNA processing.

Yeast fibrillarin (NOP1) is an evolutionarily conserved, nucleolar protein necessary for multiple steps in ribosome biogenesis. Yeast mutants lacking a functional NOP1 gene can be complemented by human fibrillarin but are temperature sensitive for growth and impaired in pre-rRNA processing. In order to identify components which interact functionally with human fibrillarin in yeast, we isolated extragenic suppressors of this phenotype. One dominant suppressor, sof1-56, which is allele-specific for human fibrillarin and restores growth and pre-RNA processing at 35 degrees C, was cloned by in vivo complementation. The wild-type allele of SOF1 is essential for cell growth and encodes a novel 56 kDa protein. In its central domain, SOF1 contains a repeated sequence also found in beta-subunits of trimeric G-proteins and the splicing factor PRP4. A single amino acid exchange in the G beta-like repeat domain is responsible for the suppressing activity of sof1-56. Indirect immunofluorescence shows that SOF1 is located within the yeast nucleolus. Co-immunoprecipitation demonstrates the physical association of SOF1 with U3 small nucleolar RNA and NOP1. In vivo depletion of SOF1 leads to impaired pre-rRNA processing and inhibition of 18S rRNA production. Thus, SOF1 is a new component of the nucleolar rRNA processing machinery.

Agonist activation of transfected human M1 muscarinic acetylcholine receptors in CHO cells results in down-regulation of both the receptor and the alpha subunit of the G-protein Gq.

CHO cells stably transfected with cDNA encoding the human M1 muscarinic acetylcholine (HM1) receptor were treated with the cholinergic agonist carbachol at various concentrations for differing times. Levels of the HM1 receptor and of a range of G-proteins were subsequently measured. Carbachol treatment of the transfected cells caused a substantial down-regulation of cellular levels of the alpha subunit of Gq (Gq alpha), but did not significantly alter cellular levels of the alpha subunits of Gs or Gi2. A small decrease in levels of G-protein beta-subunit was also produced. Parallel assessment of agonist-induced down-regulation of the HM1 receptor demonstrated that it was lost in concert with the G-protein. Similar concentrations of carbachol (5 microM) were required to produce half-maximal stimulation of inositol phosphate generation and loss of each of the HM1 receptor and Gq alpha, and half-maximal losses of both receptor and Gq alpha were produced by 3 h of treatment with 1 mM-carbachol. By contrast, treatment of the non-transfected parental CHO cells, which do not express detectable levels of the receptor, with carbachol had no effect on cellular Gq alpha levels. Concurrent treatment of the HM1-expressing CHO cells with carbachol and cycloheximide indicated that suppression of protein synthesis de novo did not mimic the effect of carbachol, and hence even complete inhibition of transcription of the Gq alpha gene and/or translation of pre-existing Gq alpha mRNA could not account for the agonist-induced effect. We have previously noted that cellular levels of both Gs alpha [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093] and the alpha subunits of the pertussis-toxin-sensitive G-proteins Gi1, Gi2 and Gi3 [Green, Johnson and Milligan (1990) J. Biol. Chem. 265, 5206-5210] can be regulated in certain cell systems by agonist activation of receptors expected to interact with these G-proteins. These results demonstrate that the same is true of Gq alpha and suggest that agonist-induced co-ordinate loss of receptors and associated G-proteins may be a more common feature than has been appreciated to date.

Dominant-negative mutants of a yeast G-protein beta subunit identify two functional regions involved in pheromone signalling.

The STE4 gene, which encodes the beta subunit of the mating response G-protein in the yeast Saccharomyces cerevisiae, was subjected to a saturation mutagenesis using 'doped' oligodeoxynucleotides. We employed a genetic screen to select dominant-negative STE4 mutants, which when overexpressed from the GAL1 promoter, interfered with the signalling function of the wild type protein. The identified inhibitory amino acid alterations define two small regions that are crucially involved in transmitting the mating signal from G beta to downstream components of the signalling pathway. These results underline the positive signalling role of yeast G beta and assign for the first time the positive signalling function of a G-protein beta subunit to specific structural features.

Interaction between G-protein beta and gamma subunit types is selective.

Signal-transducing guanine nucleotide-binding proteins (G proteins) are made up of three subunits, alpha, beta, and gamma. Each of these subunits comprises a family of proteins. The rules for association between members of one family with members of another to form a multimer are not known; it is not clear whether associations are specific or nonspecific. Other than transducin (Gt), the G protein in rod photoreceptors, most purified G proteins contain more than one subtype of beta or gamma subunits. The Gt alpha subunit is associated only with beta 1 and gamma 1. It is not known whether this specificity is due to the differential expression of these subunit types in a cell type or due to intrinsically different affinities between different beta and gamma subunit types. We have used a transfected cell assay system to examine the association of the beta 1, beta 2, and beta 3 proteins with the gamma 1 and gamma 2 proteins. Results show that gamma 1 does not associate with beta 2 and that beta 3 does not associate with gamma 1 or gamma 2. Differences in affinities between types of G protein subunits will impose restrictions on the formation of certain heterotrimers and determine which G protein will be active in a cell. A chimeric molecule of beta 1 and beta 2 was used to broadly map the regions on these subunits that determine specificity of association.

Characterization of GTP-binding proteins in Golgi-associated membrane vesicles from rat adipocytes.

We have previously reported that guanine nucleotides inhibit glucose transport activity reconstituted from adipocyte membrane fractions. In order to further investigate the hypothetical involvement of guanine-nucleotide-binding proteins (GTP-binding proteins) in the regulation of insulin-sensitive glucose transport activity, we studied their subcellular distribution in adipocytes treated or not with insulin. Adipocytes were homogenized and fractionated to yield plasma membranes (PM) and a Golgi-enriched fraction of intracellular membranes (low-density microsomes, LDM). In these membrane fractions, total guanosine 5'-[gamma-[35S]thio]triphosphate ([35S]GTP[S]) binding, alpha- and beta-subunits of heterotrimeric G-proteins, proto-oncogenes Ha-ras and K-ras, and 23-28 kDa GTP-binding proteins were assayed. The levels of alpha s and alpha i (the alpha-subunits of Gs and Gi) were approx. 8-fold lower in LDM than in PM; beta-subunits, Ha-ras and K-ras were not detectable in LDM. Total GTP[S]-binding sites and 23-28 kDa GTP-binding proteins were present in LDM in approximately the same concentrations as in PM. Insulin gave rise to the characteristic translocation of glucose transporters, but failed to alter the subcellular distribution of any of the GTP-binding proteins. Fractionation of the LDM on a discontinuous sucrose gradient revealed that alpha s and alpha i, as detected with antiserum against a common peptide sequence (alpha common), and the bulk of the 23-28 kDa G-proteins sedimented at different sucrose densities. None of the GTP-binding proteins co-sedimented with glucose transporters. Furthermore, the inhibitory effect of GTP[S] on the reconstituted transport activity was lost in the peak fractions of glucose transporters partially purified on the sucrose gradient. These data indicate that LDM from adipocytes contain several GTP-binding proteins in discrete vesicle populations. However, the intracellular GTP-binding proteins are not tightly associated with the vesicles containing the glucose transporter.

Distribution and expression of G-protein in rat cerebral cortical cells. I. Intact tissue

Localization and developmental expression of guanine nucleotide-binding regulatory protein (G-protein) were investigated in cerebral cortical tissue of rat. Immunohistochemical techniques using a rabbit polyclonal antibody were applied to reveal the pattern of G-protein distribution. Fluorescent staining in cortical sections was localized mainly to neurons; cell bodies were stained along with the proximal region of processes, including apical dendrites. All neurons appeared to be equally stained, and thus the staining activity reflected the laminar pattern of these cells in cortex. Moreover, areas of neuropil, white matter, and layer I were immunonegative, although synaptic labelling could not be directly eliminated. Most glial cells also lacked staining; however, moderately intense labelling was observed in the choroid plexus, ependymal cells and a subpopulation of microglia. Using Western immunoblot analysis, the antigens recognized in rat cerebral cortex were shown to co-electrophorese with the alpha- and beta-subunits of bovine brain G-protein, thus supporting the specificity of the antibody preparation utilized. In contrast to the adult tissue, in fetal and neonatal cortex faint to moderate staining was uniformly exhibited by essentially all cells. As determined by the immunoblot procedure, the overall level of G-protein increased from the fetal stage through the neonatal and adolescent periods to reach a maximum concentration in young adult tissue. Based on cellular distribution, G-protein may function in the overall regulation of neuronal activity within cerebral cortex. Further, these results suggest that there is a selective developmental increase in expression of G-protein in specific cell populations, particularly neurons.

Yeast α-mating factor receptor and G-protein-linked adenylyl cyclase inhibition requires RAS2 and GPA2 activities

Saccharomyces cerevisiae expresses two RAS gene products (RAS1 and RAS2) highly homologous to mammalian p21ras which mediate glucose-stimulated cyclic-AMP formation. Mating pheromone inhibits RAS-linked adenylyl cyclase activation and this is dependent upon the alpha-factor receptor (STE2) and its associated G-protein beta-subunit (STE4). We now show that this pheromone effect is independent of mating pathway signalling components "downstream" of STE4 but displays an absolute requirement for an additional G-protein alpha-subunit encoded by GPA2. alpha-mating factor effects also involve a specific suppression of normal RAS2 activity as the constitutively activated mutant RAS2vall9 as well as wild type. RAS1 are insensitive to inhibition. Interaction between GPA2, STE4-STE18, RAS2 and adenylyl cyclase in yeast could give important insight into signalling pathways controlling normal and oncogenic p21ras activity in man.