The amino terminus of G protein alpha subunits is required for interaction with beta gamma

The guanine nucleotide-binding proteins (G proteins), which transduce hormonal and light signals across the plasma membrane, are heterotrimers composed of alpha, beta, and gamma subunits. Activation of G proteins by guanine nucleotides is accompanied by dissociation of the heterotrimer: G + alpha.beta.gamma in equilibrium alpha G + beta.gamma. Brain contains several G proteins of which the most abundant are alpha 39.beta.gamma and alpha 41.beta.gamma. We have used proteolysis by trypsin to study the functional domains of the alpha subunits. In the presence of guanosine 5'-(3-O-thio)triphosphate, trypsin removes a 2-kDa peptide from the amino terminus of these proteins (Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D., and Gilman, A. G. (1984) Science 226, 860-862; Winslow, J. W., Van Amsterdam, J. R., and Neer, E. J. (1986) J. Biol. Chem. 261, 7571-7579). Tryptic cleavage does not affect the GTPase activity of the truncated molecule nor the apparent Km for GTP. However, removal of the 2-kDa amino-terminal peptide prevents association of the alpha subunits with beta.gamma. Since the apparent substrate for pertussis toxin-catalyzed ADP-ribosylation is the alpha.beta.gamma heterotrimer, the trypsin-cleaved alpha subunit is not a substrate for the toxin. Digestion of the carboxyl terminus of alpha 39 with carboxypeptidase A prevents ADP-ribosylation by pertussis toxin but does not interfere with the formation of alpha 39.beta.gamma heterotrimers. We do not yet know whether the amino-terminal region of alpha 39 interacts with beta gamma directly or whether it is necessary to maintain a conformation of alpha 39 which is required for heterotrimer formation. Further studies are needed to define the nature of the contracts between alpha and beta gamma subunits since understanding the structural basis for their reversible interaction is fundamental to understanding their function.     

Lipid modifications of G protein subunits. Myristoylation of Go alpha increases its affinity for beta gamma

Myristoylated recombinant proteins can be synthesized in Escherichia coli by concurrent expression of the enzyme myristoyl-CoA:protein N-myristoyl-transferase with its protein substrates (Duronio, R.J., Jackson-Machelski, E., Heuckeroth, R.O., Olins, P. O., Devine, C.S., Yonemoto, W., Slice, L. W., Taylor, S. S., and Gordon, J. I. (1990) Proc. Natl. Acad. Sci. U. S.A. 87, 1506-1510). Expression of the G protein subunit Go alpha in this system results in the synthesis of two forms of the protein; these were separated on a column of heptylamine-Sepharose. Purification of the more abundant form of Go alpha yielded a product that has a blocked amino terminus. Chemical analysis of the fatty acids released by acid hydrolysis of the protein revealed myristic acid. The second form of the protein was not myristoylated. Myristoylated and nonmyristoylated recombinant Go alpha were compared with brain Go alpha (which is myristoylated) for their ability to interact with G protein beta gamma subunits. The nonmyristoylated recombinant protein clearly had a reduced affinity for beta gamma, while the myristoylated recombinant protein was indistinguishable from native Go alpha in its subunit interactions. Thus, myristoylation increases the affinity of alpha subunits for beta gamma. We propose that the function of myristoylation of G protein alpha subunits is, at least in part, to facilitate formation of the heterotrimer and the localization of alpha to the plasma membrane.     

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.     

Promotion of the GTP-liganded state of the Go alpha protein by deletion of the C terminus.

G proteins are active as long as GTP is bound to the alpha subunit. Activation ends when GTP is cleaved to GDP that then stays bound to the active site. Agonist-liganded receptors allow formation of the active state by decreasing the affinity of alpha subunits for GDP allowing exchange of GDP for GTP. Since receptors interact with the C terminus of the alpha subunits, we tested whether deletion of the C terminus could mimic activation by receptors. Three deletions and one point mutation at the C terminus of alpha o were engineered in alpha o cDNA by the polymerase chain reaction, transcribed into RNA, and translated in a rabbit reticulocyte lysate. The ability of in vitro synthesized protein to bind guanine nucleotide was inferred from analysis of native tryptic cleavage patterns, while the ability of the proteins to associate with beta gamma was measured by sucrose density gradient centrifugation. Deletion of 14 amino acids, alpha oD[341], from the C terminus causes a large decrease in GDP affinity, with little or no change in guanosine 5'-3-O-(thio)triphosphate affinity. When GTP is present, alpha oD[341] remains in the activated conformation because exchange of GTP for GDP is rapid. Deletion of 10 amino acids, alpha oD[345], lowers GDP affinity, but less dramatically than in alpha oD[341]. Deletion of 5 amino acids, alpha oD[350], or mutation of Arg-349 to proline alpha oR[349P] has no detectable effects on GDP affinity. Deletion of up to 10 amino acids from the C terminus does not prevent formation of alpha beta gamma heterotrimers. We propose that the C terminus of the alpha subunit is a mobile region that blocks dissociation of GDP. Agonist-liganded receptors may move it aside to allow release of GDP, exchange for GTP, and activation of the alpha subunit.     

Inhibition of the catalytic subunit of phosphorylase kinase by its alpha/beta subunits

The subunits of phosphorylase kinase are separated and isolated in high yield by gel filtration chromatography in pH 3.3 phosphate buffer containing 8 M urea. Three protein peaks are obtained: the alpha and beta subunits coelute in the first, whereas the gamma and delta subunits are separate peaks. Upon dilution of the denaturant, catalytic activity reappears, associated only with the gamma subunit. As has been previously observed (Kee, S.M., and Graves, D.J. (1986) J. Biol. Chem. 261, 4732-4737), addition of calmodulin dramatically stimulates the reactivation of gamma. Inclusion of increasing amounts of the alpha/beta subunit mixture in the renaturation progressively decreases the activity of the renatured gamma or gamma-calmodulin. This inhibition by alpha/beta is likely due to specific interactions with the gamma subunit because the inhibition is less at pH 8.2 than at pH 6.8 and less when equivalent amounts of phosphorylated alpha/beta subunits are used (both alkaline pH and phosphorylation are known to stimulate the activity of the holoenzyme). These results suggest that the role of either the alpha or beta subunits, or perhaps both, in the nonactivated (alpha 2 beta 2 gamma 2 delta 2)2 complex of phosphorylase kinase is to suppress the activity of the gamma subunit and that activation of the enzyme, by phosphorylation for instance, is due to deinhibition caused by release of this quaternary constraint by alpha and/or beta upon gamma.     

The carboxy-terminal domain of Gs alpha is necessary for anchorage of the activated form in the plasma membrane

GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the alpha beta gamma-type. In the activated state, the alpha subunit dissociates from the beta gamma complex but remains anchored in the membrane. The alpha subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the alpha subunit of Gs (Gs alpha), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gs alpha, we used a reconstitution model in which posttranslational addition of in vitro-translated Gs alpha to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gs alpha) restores the coupling between the beta-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated alpha chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gs alpha whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated alpha subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gs alpha are required for association of this protein with the membrane.     

A conserved sequence immediately N-terminal to the Bateman domains in AMP-activated protein kinase gamma subunits is required for the interaction with the beta subunits

Mammalian AMP-activated protein kinase is a serine/threonine protein kinase that acts as a sensor of cellular energy status. AMP-activated protein kinase is a heterotrimer of three different subunits, i.e. alpha, beta, and gamma, with alpha being the catalytic subunit and beta and gamma having regulatory roles. Although several studies have defined different domains in alpha and beta involved in the interaction with the other subunits of the complex, little is known about the regions of the gamma subunits involved in these interactions. To study this, we have made sequential deletions from the N termini of the gamma subunit isoforms and studied the interactions with alpha and beta subunits, both by two-hybrid analysis and by co-immunoprecipitation. Our results suggest that a conserved region of 20-25 amino acids in gamma1, gamma2, and gamma3, immediately N-terminal to the Bateman domains, is required for the formation of a functional, active alphabetagamma complex. This region is required for the interaction with the beta subunits. The interaction between the alpha and gamma subunits does not require this region and occurs instead within the Bateman domains of the gamma subunit, although the alpha-gamma interaction does appear to stabilize the beta-gamma interaction. In addition, sequential deletions from the C termini of the gamma subunits indicate that deletion of any of the CBS (cystathionine beta-synthase) motifs prevents the formation of a functional complex with the alpha and beta subunits.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

Two forms of the bovine brain Go that stimulate the inositol trisphosphate-mediated Cl- currents in Xenopus oocytes. Distinct guanine nucleotide binding properties.

Heterotrimeric GTP-binding proteins from bovine brain were resolved by fast protein liquid chromatography chromatography using Mono Q columns. Two distinct forms of the protein Go were identified. Both forms had stochiometric amounts of alpha- and beta gamma-subunits. The a-subunits of both forms were recognized by an alpha o-specific antiserum, but not by any of the alpha i-specific antisera. The two forms showed distinct migration patterns on 9% sodium dodecyl sulfate-polyacrylamide gels containing 4-8 M urea gradients. Neither form comigrated with the recombinant alpha o1. Both the recombinant alpha o1 and the most abundant form of Go were recognized by an antiserum, H-660, against a peptide encoding amino acids 3-17 of alpha i2. H-660 has been shown previously to recognize alpha o and alpha i (Mumby, S. M., Pang, I. K., Gilman, A. G., and Sternweis, P. C. (1988) J. Biol. Chem. 263, 2020-2026). This more abundant form is called Go A most likely corresponds to the cloned alpha o1. The less abundant form, Go B, was not recognized by H-660. However, both forms of bovine brain Go were recognized by GC/2, an antiserum against the N-terminal region of alpha o1. Hence alpha oA and alpha oB may be different in their N terminus regions. Neither form of bovine brain Go was recognized by an antisera made to a peptide encoding the unique regions of the cloned alpha o2 from HIT cells (Hsu W. H., Rudolph, U., Sanford, J., Bertrand, P., Olate, J., Nelson, C., Moss, L.E., Boyd, A. E., III, Codina, J., and Birnbaumer, L. (1990) J. Biol. Chem. 265, 11220-11226). Go A and Go B have similar guanine nucleotide binding and release properties. Both release GDP within 1 min in the absence of added Mg2+. Both bind guanosine (GTP gamma S) rapidly as well. However Go A binds GTP gamma S about 2.5-fold faster than Go B, in the absence of added Mg2+ ion. Both forms of Go as well as the recombinant alpha o (alpha o1) can increase muscarinic stimulation of inositol trisphosphate-mediated Cl- current in Xenopus oocytes. These data indicate that we have identified two structurally distinct forms of Go that have different guanine nucleotide binding properties and are capable of functioning in the receptor-regulated phospholipase C pathway in Xenopus oocytes.     

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.     

The intrinsic fluorescence of the alpha subunit of transducin. Measurement of receptor-dependent guanine nucleotide exchange

We have made use of the enhancement of the intrinsic fluorescence of the alpha subunit of transducin (alpha T), which accompanies guanine nucleotide exchange, to follow the reconstituted interactions between pure rhodopsin and pure transducin in phospholipid vesicles. When the pure alpha T.GDP complex is added to lipid vesicles containing rhodopsin and the beta gamma T complex, a light- and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-dependent enhancement of the fluorescence emission of alpha T is observed. When GTP is substituted for GTP gamma S, a similar enhancement of the intrinsic fluorescence of alpha T occurs; however, this enhancement is transient and precedes a fluorescence decay which is complete in 2-5 min. The fact that the fluorescence decay is specifically induced by GTP and is not observed either with nonhydrolyzable GTP analogs or with NaF (plus AlCl3) indicates that the decay represents GTP hydrolysis in alpha T. The dose-response profiles for the effects of the beta gamma T complex on the rate and extent of the GTP gamma S-stimulated fluorescence enhancement of alpha T have also been examined. The addition of relatively low levels of beta gamma T to these reconstituted systems can promote the GTP gamma S-stimulated enhancement of the fluorescence of multiple alpha T subunits with half-maximal enhancement occurring at alpha T:beta gamma T ratios of 150:1. These findings are consistent with earlier suggestions (Fung, B. K.-K. (1983) J. Biol. Chem. 258, 10495-10502) that the beta gamma T subunit dissociates from alpha T as a result of the GDP-GTP exchange reaction and thus can act catalytically to promote the activation of a number of inactive alpha T species. However, the dependence of the rate of the GTP gamma S-stimulated fluorescence enhancement on beta gamma T is complex and cannot be explained adequately by simple models where alpha T-beta gamma T interactions (or rhodopsin-transducin interactions) are rate-limiting for the rhodopsin-stimulated activation of the alpha T subunits. Overall, the results reported here demonstrate that fluorescence spectroscopy can be used to monitor directly a receptor-catalyzed activation-deactivation cycle of a GTP-binding protein within a lipid milieu.     

Expression of cDNAs for G proteins in Escherichia coli. Two forms of Gs alpha stimulate adenylate cyclase

Complementary DNAs that encode two forms of the alpha subunit (Gs alpha) of the guanine nucleotide-binding protein responsible for stimulation of adenylate cyclase (Gs) have been inserted into plasmid vectors for expression in Escherichia coli. Following transformation of either of these plasmids into E. coli K38, Gs alpha accumulates to 0.4-0.8 mg/liter (approximately 0.1% of total protein), as judged by immunoblot analysis with specific antisera. Based on deduced amino acid sequence, the two cDNAs should encode proteins with molecular weights of 44,500 and 46,000, respectively (Robishaw, J.D., Smigel, M. D., and Gilman, A. G. (1986) J. Biol. Chem. 261, 9587-9590). Expression of these cDNAs in E. coli yields proteins that co-migrate on sodium dodecyl sulfate-polyacrylamide gels with the Gs alpha subunits from S49 lymphoma cell membranes, with apparent molecular weights of 45,000 and 52,000, respectively. Low levels of activity are detected in the 100,000 X g supernatant after lysis and fractionation of E. coli expressing either form of Gs alpha. Partial purification of Gs alpha from E. coli lysates yields preparations in which significant and stable activity can be assayed. Both forms of Gs alpha migrate through sucrose gradients as soluble, monodisperse species in the absence of detergent. As expressed in E. coli, both forms of Gs alpha can reconstitute isoproterenol-, guanine nucleotide-, and fluoride-stimulated adenylate cyclase activity in S49 cyc-cell membranes to approximately the same degree and can be ADP-ribosylated with [32P]NAD+ and cholera toxin. However, based on the specific activity of purified rabbit liver Gs, only 1-2% of the Gs alpha expressed in E. coli appears to be active. Incubation of partially purified fractions of recombinant Gs alpha with guanosine 5'-(3-O-thio)triphosphate and resolved beta gamma subunits isolated from purified bovine brain G proteins results in a 7-10-fold increase in Gs activity. Incubation of bovine brain beta gamma with recombinant Gs alpha also leads to a dramatic increase in observed levels of cholera toxin-catalyzed [32P]ADP-ribosylation.     

Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.     

Reactive sulfhydryl groups of alpha 39, a guanine nucleotide-binding protein from brain. Location and function

The guanine nucleotide-binding proteins which mediate hormonal inhibition of adenylate cyclase as well as hormonal regulation of other membrane functions are alpha, beta, and gamma heterotrimers which are structurally homologous to each other. In brain, the predominant guanine nucleotide-binding component is a 39-kDa protein whose physiological role is as yet unknown. We have used N-ethylmaleimide to define functionally important sulfhydryl groups on alpha 39. Three cysteine residues in the molecule are reactive in unliganded alpha 39. Alkylation of two of these is reduced when guanosine 5'-(3'-O-thio)triphosphate (GTP gamma S) is bound. We have isolated and sequenced tryptic peptides containing the three reactive cysteines. The octapeptide containing the GTP gamma S-insensitive cysteine is at a position equivalent to amino acids 106-113 of the transducin alpha subunit (Lochrie, M. A., Hurley, J. B., and Simon, M. I. (1985) Science 228, 96-99). However, the equivalent peptide in transducin does not contain a cysteine residue. Alkylation of this cysteine blocks ADP-ribosylation of cysteine 351 by pertussis toxin. However, alkylation does not prevent association of alpha with the beta X gamma subunits nor does it inhibit GTPase activity. The two GTP gamma S-sensitive cysteines are at positions equivalent to cysteines 139 and 286 of the transducin alpha subunit. Alkylation of these residues inhibits GTPase activity. Neither of these GTP gamma S-sensitive cysteines are in those regions of alpha 39 which are highly homologous to the GTP-binding site of elongation factor Tu (Jurnak, F. (1985) Science 230, 32-36). However, both are present in the brain 41-kDa guanine nucleotide-binding protein and in the two transducins. The conservation of these cysteine residues suggests that they are important for the function of the subunits.     

Family of G protein alpha chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulation of enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase. Each G protein contains an alpha subunit that binds and hydrolyzes guanine nucleotides and interacts with beta gamma subunits and specific receptor and effector proteins. Amphipathic and secondary structure analysis of the primary sequences of five different alpha chains (bovine alpha s, alpha t1 and alpha t2, mouse alpha i, and rat alpha o) predicted the secondary structure of a composite alpha chain (alpha avg). The alpha chains contain four short regions of sequence homologous to regions in the GDP binding domain of bacterial elongation factor Tu (EF-Tu). Similarities between the predicted secondary structures of these regions in alpha avg and the known secondary structure of EF-Tu allowed us to construct a three-dimensional model of the GDP binding domain of alpha avg. Identification of the GDP binding domain of alpha avg defined three additional domains in the composite polypeptide. The first includes the amino terminal 41 residues of alpha avg, with a predicted amphipathic alpha helical structure; this domain may control binding of the alpha chains to the beta gamma complex. The second domain, containing predicted beta strands and alpha helices, several of which are strongly amphipathic, probably contains sequences responsible for interaction of alpha chains with effector enzymes. The predicted structure of the third domain, containing the carboxy terminal 100 amino acids, is predominantly beta sheet with an amphipathic alpha helix at the carboxy terminus. We propose that this domain is responsible for receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of Gs alpha in Escherichia coli. Purification and properties of two forms of the protein

Cloning of complementary DNAs that encode either of two forms of the alpha subunit of the guanine nucleotide-binding regulatory protein (Gs) that stimulates adenylyl cyclase into appropriate plasmid vectors has allowed these proteins to be synthesized in Escherichia coli (Graziano, M.P., Casey, P.J., and Gilman, A.G. (1987) J. Biol. Chem. 262, 11375-11381). A rapid procedure for purification of milligram quantities of these proteins is described. As expressed in E. coli, both forms of Gs alpha (apparent molecular weights of 45,000 and 52,000) bind guanosine 5'-(3-O-thio)triphosphate stoichiometrically. The proteins also hydrolyze GTP, although at different rates (i.e. 0.13.min-1 and 0.34.min-1 at 20 degrees C for the 45- and the 52-kDa forms, respectively). These rates reflect differences in the rate of dissociation of GDP from the two proteins. Both forms of recombinant Gs alpha have essentially the same kcat for GTP hydrolysis, approximately 4.min-1. Recombinant Gs alpha interacts functionally with G protein beta gamma subunits and with beta-adrenergic receptors. The proteins can also be ADP-ribosylated stoichiometrically by cholera toxin. This reaction requires the addition of beta gamma subunits. Both forms of recombinant Gs alpha can reconstitute GTP-, isoproterenol + GTP-, guanosine 5'-(3-O-thio)triphosphate-, and fluoride-stimulated adenylyl cyclase activity in S49 cyc- membranes to maximal levels, although their specific activities for this reaction are lower than that observed for Gs purified from rabbit liver. Experiments with purified bovine brain adenylyl cyclase indicate that the affinity of recombinant Gs alpha for adenylyl cyclase is 5-10 times lower than that of liver Gs under these assay conditions; however, the intrinsic capacity of the recombinant protein to activate adenylyl cyclase is normal. These findings suggest that Gs alpha, when synthesized in E. coli, may fail to undergo a posttranslational modification that is crucial for high affinity interaction of the G protein with adenylyl cyclase.     

Physical and immunological characterization of a guanine nucleotide-binding protein purified from bovine cerebral cortex

ADP-ribosylation by pertussis toxin has been used to identify the alpha subunit of Ni, the guanine nucleotide-binding protein which mediates hormone and GTP inhibition of adenylate cyclase. Two proteins have been purified from bovine cerebral cortex which are substrates for ADP-ribosylation by pertussis toxin, a 41-kDa protein (alpha 41) and a 39-kDa protein (alpha 39). The 41-kDa protein is very similar to the subunit of Ni purified from other tissues while the function of the 39-kDa protein is unknown (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229; Sternweis, P. C., and Robishaw, J. D. (1984) J. Biol. Chem. 259, 13806-13813). We now show that the purified alpha 39 protein from bovine brain is a relatively hydrophilic protein which associates with a hydrophobic beta gamma component. The complex can be dissociated by guanosine 5'-(3-O-thio)triphosphate. The alpha 39 component binds guanosine 5'-(3-O-thio)triphosphate with a KD of 27 nM. We have developed polyclonal antibodies to alpha 39 and beta. The antibodies to alpha 39 cross-react weakly with alpha 41 in an immunoblot assay indicating some homology between the two proteins but making it unlikely that alpha 39 is derived from alpha 41. Using the antibodies for quantitation we found that alpha 39 is 0.5% and beta is 0.7% of membrane proteins. While the antibodies cross-react with alpha 39 and beta proteins in many different species, central nervous system tissues always have more immunoreactivity than membranes from peripheral organs. Anti-beta antibody recognizes the beta subunit when it is associated with alpha 39 or alpha 41 and can immunoprecipitate both alpha . beta gamma trimers. The guanine nucleotide-dependent dissociation of the alpha 39 . beta gamma trimer suggests that the complex could inhibit adenylate cyclase by liberating free beta gamma units. The function of alpha 39 may not, however, be exclusively to regulate adenylate cyclase but may include coupling hormone receptors to other effectors. Antibodies specific for alpha 39 and beta will be useful tools in determining the functions of alpha 39 and beta in hormone-responsive cells.     

Site-specific antibodies directed against G protein beta and gamma subunits: effects on alpha and beta gamma subunit interaction.

Little is known about the specific domains of G protein beta and gamma subunits which interact with each other and with the alpha subunit. We used site-specific anti-peptide antibodies directed against beta and gamma subunits to investigate domains on beta and gamma subunits involved in alpha subunit interaction. Antibodies included four against the transducin (Gt) beta subunit (residues 1-10 = MS, 127-136 = KT, 256-265 = RA, and 330-340 = SW) and two against the gamma subunit (residues 2-12 = PV and 58-68 = PE). All antisera, when affinity-purified on peptide columns, yielded antibodies capable of recognizing the denatured cognate subunit on immunoblots, but only RA, SW, PV, and PE recognized native beta gamma t subunits. Affinity purification of MS and KT antisera on columns of immobilized native Gt yielded antibodies capable of recognizing native beta gamma t subunits. The functional effects of each antibody preparation on alpha t-beta gamma t interaction were assessed by assaying the ability of the preparations to immunoprecipitate beta gamma t subunits in the presence of excess alpha subunits and by testing the inhibition of beta gamma t-dependent ADP-ribosylation of alpha t-subunits catalyzed by pertussis toxin. On the basis of the results, we conclude that the domains on beta gamma t which may be directly involved in alpha t-beta gamma t interaction include the extreme amino terminus, residues 127-136 and 256-265 of beta t, and the carboxyl terminus of gamma t.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of the catalyst of adenylate cyclase

The catalytic moiety of hormone-sensitive adenylate cyclase has been purified from bovine brain. It is isolated largely without its guanine nucleotide-binding regulatory protein, Gs, by affinity chromatography on 7-O-hemisuccinyldeacetylforskolin-agarose. It appears to be a single polypeptide which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of approximately 120,000. When subjected to electrophoresis on gradient (5-10%) sodium dodecyl sulfate-polyacrylamide gels, it displays a larger apparent Mr of 150,000. The adenylate cyclase activity of the preparation can be stimulated by the addition of Gs, forskolin, or calcium-calmodulin. The preparation has been reconstituted with purified beta-adrenergic receptors and Gs to form a hormone-stimulated adenylate cyclase system (May, D., Ross, E.M., Gilman, A.G., and Smigel, M.D. (1985) J. Biol. Chem. 260, 15829-15833). In contrast to its stimulation by Gs, inhibition by the alpha subunits of Gi and Go, G proteins known to be coupled to inhibitory receptors (Sternweis, P., and Florio, V. (1985) J. Biol. Chem. 260, 3477-3483), is not seen. Preparations of adenylate cyclase show varying degrees of inhibition by added G protein beta . gamma subunit. This inhibition can be explained as reflecting a variable, small (under 5%) contamination of the preparation by Gs alpha which would be deactivated by complexing with the added beta . gamma subunit.