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.     

Effect of monoclonal antibody binding on alpha-beta gamma subunit interactions in the rod outer segment G protein, Gt

The guanyl nucleotide binding regulatory protein of retinal rod outer segments, called Gt, that couples the photon receptor rhodopsin with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, alpha t and beta gamma t. The effect of monoclonal antibody binding to the alpha t subunit of Gt on subunit association has been investigated in the present study. It was previously shown that this monoclonal antibody, mAb 4A, blocks interactions with rhodopsin and its epitope was located within the region Arg310-Phe350 at the COOH terminus of the alpha t subunit. In this paper, we show that mAb 4A disrupts the Gt complex. Gt migrates in 5-20% linear sucrose density gradients as a monomer, with a sedimentation coefficient of 4.1 +/- 0.07 S, while in the presence of mAb 4A, the alpha t and beta gamma t subunits show sedimentation coefficients of 7.7 +/- 0.2 and 3.7 +/- 0.1 S, respectively. The beta gamma t subunit migrates with the same sedimentation rate as pure beta gamma t. Nonimmune rabbit IgG does not modify the sedimentation behavior of Gt. The Fab fragment of mAb 4A also dissociates the Gt complex, as suggested by the change of the sedimentation rate of alpha t. This effect of mAb 4A on Gt subunit association was also confirmed by immunoprecipitation studies in the presence of detergent. In the presence of detergent, subunit association is not affected, but the formation of Gt oligomers and, therefore, the nonspecific precipitation of beta gamma t subunit are reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships within the family of GTP-binding proteins isolated from bovine central nervous system

Four members of a family of GTP-binding proteins (G-proteins) which translate stimulation of extracellular receptors into regulation of intracellular enzymes were isolated from the bovine central nervous system. These proteins were examined for functional similarities and cross-reactivity with antibodies to the G-protein (transducin, Gt) from the photoreceptor system. Two proteins, Gs and Gi, can be distinguished by their respective abilities to stimulate or inhibit adenylate cyclase. The activated alpha subunits of Gt and a fourth member of the family, Go, did not affect this enzyme. Gt was shown to be unique in its ability to stimulate cGMP-dependent phosphodiesterase. While functionally diverse, the G-proteins were shown to have some common antigenic properties. Antibodies directed against the beta subunit of Gt recognize the beta 36 subunits of all preparations but not a putative second beta 35 subunit. Antibodies specific for the alpha subunit of Gt did not recognize other alpha subunits when immune blots from sodium dodecyl sulfate gels were examined. However, Go alpha, but not Gs alpha or Gi alpha, reacted strongly with the antibodies when the native subunit was spotted directly. This suggests that Go alpha and Gt alpha have homologous structural determinants. An antiserum that recognized Gt gamma did not recognize gamma subunits from other sources. These data support the proposed diversity of function and similarity of structure among the four G-proteins. The alpha and potentially gamma subunits appear to be responsible for the specificity of function.     

G-protein beta gamma forms: identity of beta and diversity of gamma subunits

Signal-transducing G-proteins are heterotrimers composed of GTP-binding alpha subunits in association with a tightly bound complex of beta and gamma subunits. While the alpha subunits are recognized as a family of diverse structures, beta and gamma subunits have also been found as heterogeneous isoforms. To investigate the diversity and tissue specificity of the beta gamma complexes, we have examined homogeneous oligomeric G-proteins from a variety of sources. The beta and gamma subunits isolated from the major-abundance G-proteins from bovine brain, bovine retina, rabbit liver, human placenta, and human platelets were purified and subjected to biochemical and immunological analysis. Protease mapping and immune recognition revealed an identical profile for each of the two distinctly migrating beta isoforms (beta 36 and beta 35) regardless of tissue or G-protein origin. Digestion with V8 protease revealed four distinct, clearly resolved terminal fragments for beta 36 and two for beta 35. Trypsin and chymotrypsin digestion yielded numerous bands, but again each form had a unique profile with no tissue specificity. Tryptic digestion was found to be conformationally specific with the most resistant structure being the native beta gamma complex. With increasing trypsin, the complex was digested but in a pattern distinct from that for denatured beta. In contrast to the two highly homologous beta structures, examination of this set of proteins revealed at least six distinct gamma peptides. Two unique gamma peptides were found in bovine retinal Gt and three gamma peptides in samples of bovine brain derived Go/Gi. Human placental and platelet Gi samples each contained a unique gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

G protein beta gamma subunits from bovine brain and retina: equivalent catalytic support of ADP-ribosylation of alpha subunits by pertussis toxin but differential interactions with Gs alpha

We have examined the ability of the beta gamma subunits of guanine nucleotide binding regulatory proteins (G proteins) to support the pertussis toxin (PT) catalyzed ADP-ribosylation of G protein alpha subunits. Substoichiometric amounts of the beta gamma complex purified from either bovine brain G proteins or the bovine retinal G protein, Gt, are sufficient to support the ADP-ribosylation of the alpha subunits of Gi (the G protein that mediates inhibition of adenylyl cyclase) and Go (a G protein of unknown function) by PT. This observation indicates that ADP-ribosylated G protein oligomers can dissociate into their respective alpha and beta gamma subunits in the absence of activating regulatory ligands, i.e., nonhydrolyzable GTP analogues or fluoride. Additionally, the catalytic support of ADP-ribosylation by bovine brain beta gamma does not require Mg2+. Although the beta gamma subunit complexes purified from bovine brain G proteins and the beta gamma complex of Gt support equally the ADP-ribosylation of alpha subunits by PT, there is a marked difference in their abilities to interact with Gs alpha. The enhancement of deactivation of fluoride-activated Gs alpha requires 25-fold more beta gamma from Gt than from brain G proteins to produce a similar response. This difference in potency of beta gamma complexes from the two sources was also observed in the ability of beta gamma to produce an increase in the activity of recombinant Gs alpha produced in Escherichia coli.     

Unique assignment of inter-subunit association in GABA(A) alpha 1 beta 3 gamma 2 receptors determined by molecular modeling

Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA(A) receptor (GABA(A)R alpha 1 beta 3 gamma 2). There is strong evidence that the heteropentameric receptor contains two alpha 1, two beta 3, and one gamma 2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either gamma 2 beta 3 alpha 1 beta 3 alpha 1 or gamma 2 alpha 1 beta 3 alpha 1 beta 3 configurations. Here we use molecular modeling to thread the relevant GABA(A)R subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA(A) sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA(A) sequences were threaded onto the AChBP template in the gamma 2 beta 3 alpha 1 beta 3 alpha 1 or gamma 2 alpha 1 beta 3 alpha 1 beta 3 arrangements. Only the gamma 2 alpha 1 beta 3 alpha 1 beta 3 arrangement satisfied three known criteria: (1) alpha 1 His(102) binds at the gamma 2 subunit interface in proximity to gamma 2 residues Thr(142), Phe(77), and Met(130); (2) alpha 1 residues 80-100 bind near gamma 2 residues 91-104; and (3) alpha 1 residues 58-67 bind near the beta 3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.     

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.     

Activation and inhibition of phosphorylase kinase by monospecific antibodies against preparatively isolated alpha, beta and gamma subunits

Homogeneous alpha and beta subunits were isolated for the first time in preparative amounts in the presence of sodium dodecyl sulfate. Analysis by analytical polyacrylamide electrophoresis, sedimentation velocity, and immunoprecipitation with monospecific antibodies indicated homogeneity. The apparent molecular masses of the purified subunits as determined electrophoretically in the presence of dodecyl sulfate are: alpha = 140.2 +/- 2.1 kDa and beta = 123 +/- 1.8 kDa. Amino acid analyses show that per 100 mol amino acid the alpha-subunit has a higher serine content (Ser alpha/Ser beta = 1.32, Ser alpha/Ser gamma = 1.42) and a lower aspartic acid/asparagine (Asx) content (AsX alpha/Asx beta = 0.76, Asx alpha/Asx gamma = 0.90) than the beta and gamma subunits. Monospecific antibodies against the purified alpha, beta and gamma subunits were produced in sheep [J. Immunol. Methods (1984) 70, 193-209] and their action on the catalytic activity of non-activated phosphorylase kinase assayed. It can be shown that certain antibody fractions of anti-alpha, anti-beta and anti-gamma inhibit the Ca2+-dependent and Ca2+-independent activity at pH 6.8 as well as at pH 8.2. Other antibody fractions against the beta and gamma subunits however activate the Ca2+-dependent activity at pH 6.8 threefold to fourfold, although they inhibit the activity at pH 8.2. These antibodies lead to a ca. five fold increase in the pH 6.8/8.2 activity ratio. Activating anti-beta can even overcome the inhibitory action of anti-alpha at pH 6.8. A kinetic analysis shows that inhibition is the result of a mixed type mechanism whereas activation is due to a fivefold to tenfold increase in V for phosphorylase b. The results illustrate the importance of possibly large, concerted conformational changes of phosphorylase kinase. It appears that activation or inhibition can be triggered by the antibody binding to conformational determinants of a single subunit type leading to a structural alteration of the holoenzyme.     

The NH2-terminal alpha subunit attenuator domain confers regulation of G protein activation by beta gamma complexes.

Gs and Gi, respectively, activate and inhibit the enzyme adenylyl cyclase. Regulation of adenylyl cyclase by the heterotrimeric Gs and Gi proteins requires the dissociation of GDP and binding of GTP to the alpha s or alpha i subunit. The beta gamma subunit complex of Gs and Gi functions, in part, to inhibit GDP dissociation and alpha subunit activation by GTP. Multiple beta and gamma polypeptides are expressed in different cell types, but the functional significance for this heterogeneity is unclear. The beta gamma complex from retinal rod outer segments (beta gamma t) has been shown to discriminate between alpha i and alpha s subunits (Helman et al: Eur J Biochem 169:431-439, 1987). beta gamma t efficiently interacts with alpha i-like G protein subunits, but poorly recognizes the alpha s subunit. beta gamma t was, therefore, used to define regions of the alpha i subunit polypeptide that conferred selective regulation compared to the alpha s polypeptide. A series of alpha subunit chimeras having NH2-terminal alpha i and COOH-terminal alpha s sequences were characterized for their regulation by beta gamma t, measured by the kinetics of GTP gamma S activation of adenylyl cyclase. A 122 amino acid NH2-terminal region of the alpha i polypeptide encoded within an alpha i/alpha s chimera was sufficient for beta gamma t to discriminate the chimera from alpha s. A shorter 54 amino acid alpha i sequence substituted for the corresponding NH2-terminal region of alpha s was insufficient to support the alpha i-like interaction with beta gamma t. The findings are consistent with our previous observation (Osawa et al: Cell 63:697-706, 1990) that a region in the NH2-terminal moiety functions as an attenuator domain controlling GDP dissociation and GTP activation of the alpha subunit polypeptide and that the attenuator domain is involved in functional recognition and regulation by beta gamma complexes.     

G alpha i-G alpha s chimeras define the function of alpha chain domains in control of G protein activation and beta gamma subunit complex interactions

Gs and Gi2 are G proteins whose alpha subunits are 65% homologous. Within the 355 amino acid alpha i2 polypeptide, substitution of residues Ile213-Lys319 with the corresponding alpha s region (Ile235-Arg356) generated a chimera that activated adenylyl cyclase, indicating that the alpha s activation domain resides within this 122 amino acid alpha s sequence. Mutation within alpha s residues Glu15-Pro144 resulted in an alpha s polypeptide having an enhanced rate of GDP dissociation. Mutation within two regions of the N-terminus influenced the ability of pertussis toxin to ADP-ribosylate the alpha subunit polypeptide, a reaction controlled by the beta gamma subunit complex. The findings define the G protein alpha subunit N-terminus as a regulatory region controlling beta gamma subunit interactions and GDP dissociation independent of the GTPase and effector activation domains.     

G beta association and effector interaction selectivities of the divergent G gamma subunit G gamma(13).

G gamma(13) is a divergent member of the G gamma subunit family considered to be a component of the gustducin G-protein heterotrimer involved in bitter and sweet taste reception in taste bud cells. G gamma(13) contains a C-terminal asparagine-proline-tryptophan (NPW) tripeptide, a hallmark of RGS protein G gamma-like (GGL) domains which dimerize exclusively with G beta(5) subunits. In this study, we investigated the functional range of G gamma(13) assembly with G beta subunits using multiple assays of G beta association and G beta gamma effector modulation. G gamma(13) was observed to associate with all five G beta subunits (G beta(1-5)) upon co-translation in vitro, as well as function with all five G beta subunits in the modulation of Kir3.1/3.4 (GIRK1/4) potassium and N-type (alpha(1B)) calcium channels. Multiple G beta/G gamma(13) pairings were also functional in cellular assays of phospholipase C (PLC) beta 2 activation and inhibition of G alpha(q)-stimulated PLC beta 1 activity. However, upon cellular co-expression of G gamma(13) with different G beta subunits, only G beta(1)/G gamma(13), G beta(3)/G gamma(13), and G beta(4)/G gamma(13) pairings were found to form stable dimers detectable by co-immunoprecipitation under high-detergent cell lysis conditions. Collectively, these data indicate that G gamma(13) forms functional G beta gamma dimers with a range of G beta subunits. Coupled with our detection of G gamma(13) mRNA in mouse and human brain and retina, these results imply that this divergent G gamma subunit can act in signal transduction pathways other than that dedicated to taste reception in sensory lingual tissue.     

The beta subunit of E. coli glycyl-tRNA synthetase plays a major role in tRNA recognition.

The contributions made by the alpha and beta subunits of E. coli glycyl-tRNA synthetase to the recognition of tRNA have been investigated via binding and immunological methods. Using the nitrocellulose filter assay, we have shown that isolated beta subunit, but not the alpha subunit, binds [14C]glycyl-tRNA with an affinity comparable to that of the native enzyme. Further, the data indicate that the beta subunit possesses one binding site for glycyl-tRNA while the native or reconstituted enzyme (alpha 2 beta 2) has two sites. Rabbit antibodies directed at the beta subunit or the holoenzyme inhibit efficiently the ability of the enzyme to aminoacylate tRNA while alpha-subunit antibodies have a smaller effect. Since none of the antisera have an appreciable effect on the ATP-PPi exchange activity of the enzyme under these conditions, the beta-subunit (and holoenzyme) antisera evidently interfere with productive tRNA binding. Taken together, the data indicate that the larger, beta subunit of glycyl-tRNA synthetase plays a major role in tRNA recognition.     

The GppNHp-activated adenylyl cyclase complex from turkey erythrocyte membranes can be isolated with its beta gamma subunits.

The adenylyl cyclase complex, derived from turkey erythrocyte membranes, was activated using guanosine 5'-[beta, gamma-imido]triphosphate (Gpp[NH]p) and separated under low-detergent and low-salt conditions using conventional molecular-sieve chromatography followed by high-pressure ion-exchange and molecular-sieve chromatography. Although the complex remains activated with Gpp[NH]p throughout the isolation, the beta gamma subunits copurify with the cyclase. The stoichiometry of the cyclase to the alpha subunit of the stimulatory guanosine-nucleotide-binding regulatory protein (alpha s) to the beta subunit is close to unity, demonstrating that the beta gamma subunits do not dissociate from the Gs.cyclase complex (Gs, guanosine-nucleotide-binding regulatory protein) upon activation of the enzyme. If the final purification step was performed at high-salt concentrations, the beta gamma subunits could be separated from the alpha s.cyclase complex. Previously reported results on bovine brain cyclase also showed that the Gs.cyclase complex remains intact subsequent to activation by hormone and Gpp[NH]p [Marbach, I., Bar-Sinai, A., Minich, M. and Levitzki, A. (1990) J. Biol. Chem. 265, 9999-10,004]. These results, using adenylyl cyclase from two different sources, support our previous kinetic experiments which first suggested that beta gamma subunits are not released from Gs upon cyclase activation. We, therefore, argue that the mode of adenylyl cyclase inhibition by the inhibitory guanosine-nucleotide-binding regulatory protein cannot be via shifting the alpha s to beta gamma equilibrium as is commonly believed, and an alternate hypothesis is proposed.     

Calcium channel beta subunit promotes voltage-dependent modulation of alpha 1 B by G beta gamma

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.     

Stimulation of phospholipase C by guanine-nucleotide-binding protein beta gamma subunits.

We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'-[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743-748]. To investigate whether this stimulation was due to a soluble alpha subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein beta gamma dimers on the phospholipase-C-mediated formation of inositol phosphates by HL-60 cytosol. We found that beta gamma subunits, purified from bovine retinal transducin (beta gamma t), markedly stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by beta gamma t was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin alpha subunit and was additive to activation of phospholipase C by guanosine 5'-[3-O-thio]triphosphate. Beta gamma t also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL-60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to beta gamma t, but was also observed with highly purified beta gamma subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in HL-60 granulocytes. Only one of these forms was sensitive to stimulation by beta gamma t, demonstrating that stimulation of phospholipase C by beta gamma subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein beta gamma subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins.     

Interaction between elongation factors 1beta and 1gamma from Bombyx mori silk gland

Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of four subunits: alpha (51 kDa), beta (26 kDa), gamma (49 kDa), and delta (33 kDa). The EF-1alpha subunit catalyzes the binding of aminoacyl-tRNA to the ribosome concomitant with the hydrolysis of GTP. The EF-1alpha-bound GDP is then exchanged for GTP by the EF-1betagammadelta complex. To facilitate analysis of the roles of the individual EF-1beta, gamma, and delta subunits in GDP/GTP exchange on EF-1alpha, we cloned the cDNAs for these subunits and expressed them in Escherichia coli. EF-1beta, EF-1gamma, and the carboxyl-terminal half of EF-1delta were expressed, purified, and examined for protein:protein interactions by gel filtration chromatography and by a quartz-crystal microbalance method. An 80-kDa species containing EF-1beta and gamma subunits in a 1:1 molar ratio was detected by gel filtration. A higher molecular weight species containing an excess of EF-1gamma relative to EF-1beta was also detected. The amino-terminal region of EF-1beta (amino acid residues 1-129) was sufficient for binding to EF-1gamma. The carboxyl-terminal half of EF-1delta did not appear to form a complex with EF-1gamma.     

In vitro synthesis of G protein beta gamma dimers

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

Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping

The binding domains of 28 monoclonal antibodies (mAbs) against the alpha, beta, and delta subunits of the Torpedo acetylcholine receptor were mapped on the primary sequences of these subunits. Small peptide fragments (2000-20,000 daltons) of the purified subunits were obtained by digestion with staphylococcal V8 protease and papain, separated on a discontinuous polyacrylamide gel electrophoretic system, and electroblotted onto diaminophenyl thioether paper. The blots were probed with the various monoclonal antibodies and also with antibodies against carboxy-terminal decapeptides of the alpha, beta, and delta subunits to identify the carboxy-terminal fragments. From inspection of the binding patterns of the various antibodies to the subunits fragments and the molecular weights of these fragments, and by using the carboxy termini of the subunits as reference points, it was possible to deduce the regions on the primary sequence of each subunit in which the antibodies bound and in some cases to order the binding sites within these sequences. mAb 148, which inhibits receptor function by cross-linking receptor molecules on the cytoplasmic side, was mapped to the sequence beta 368-406. The main immunogenic region of the native receptor, which is of pathological importance in the autoimmune disease myasthenia gravis, was mapped by using mAb 210 to within 80 amino acid residues (alpha 46-127). The overall antigenic structure of alpha subunits was examined. Synthetic peptides have been used to locate determinants responsible for 83% of the antibodies in antisera to denatured alpha subunits and 46% of the antibodies to denatured alpha subunits in antisera to intact receptor. Theoretical models of the transmembrane orientation of the subunit polypeptide chains were tested by determining whether mapped monoclonal antibodies bound to the extracellular or intracellular surface of receptor-rich membranes. Our results confirm previous reports that the carboxy termini of the subunits are exposed on the intracellular surface, as is part of the region between a putative channel-forming domain (M5) and a putative membrane-spanning region (M3). However, contrary to current theoretical models, the region between M5 and the putative membrane-spanning sequence M4 also appears to be on the intracellular surface, implying that M4 and M5 are not membrane-spanning domains.(ABSTRACT TRUNCATED AT 400 WORDS)     

Computer-graphics interpretations of residue exchanges between the alpha, beta and gamma subunits of human-liver alcohol dehydrogenase class I isozymes

Three-dimensional models of human alcohol dehydrogenase subunits have been constructed, based on the homologous horse enzyme, with computer graphics. All types of class I subunits (alpha, beta, and gamma) and the major allelic variants (beta 1/beta 2 and gamma 1/gamma 2) have been studied. Residue differences between the E-type subunit of the horse enzyme and any of the subunits of the human isozymes occur at 64 positions, about half of which are isozyme-specific. About two thirds of the substitutions are at the surface and all differences can be accommodated in highly conserved three-dimensional structures. The model of the gamma isozyme is most similar to the crystallographically analyzed horse liver E-type alcohol dehydrogenase, and has all the functional residues identical to those of the E subunit except for one which is slightly smaller: Val-141 in the substrate pocket. The residues involved in coenzyme binding are generally conserved between the horse enzyme and the alpha, beta, and gamma types of the human enzyme. In contrast, single exchanges of these residues are the ones involved in the major allelic differences (beta 1 versus beta 2 and gamma 1 versus gamma 2), which affects the overall rate of alcohol oxidation since NADH dissociation is the rate-determining step. Residue 47 is His in beta 2 and Arg in the beta 1, gamma 1, and gamma 2 subunits, and in horse liver alcohol dehydrogenase. Both His and Arg can make a hydrogen bond to a phosphate oxygen atom of NAD; hence the lower turnover rate of beta 1 apparently derives from a charge effect. The substitution to Gly in the alpha subunit results in one less hydrogen bond in NAD binding, and consequently in rapid dissociation. This may explain why the overall rate is an order of magnitude faster than that of beta 1. The important difference between gamma 1 and gamma 2 is an exchange at position 271 from Arg to Gln which can give a hydrogen bond from Gln in gamma 2 to the adenine of NAD. The tighter binding to gamma 2 can account for the slower overall catalytic rate in this isozyme. The kinetics and interactions of cyclohexanol and benzyl alcohol with the isozymes were judged by docking experiments using an interactive fitting program.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification and characterization of Mg2+ binding sites in isolated alpha and beta subunits of H(+)-ATPase from Bacillus PS3

The properties of the nucleotide binding sites in the isolated beta and alpha subunits of H(+)-ATPase from Bacillus PS3 (TF1) have been examined by studying the EPR properties of bound VO(2+), which is a paramagnetic probe for the native Mg2+ cation cofactor. The amino acid ligands of the VO2+ complexes with the isolated beta subunit, with the isolated alpha subunit, with different mixtures of both alpha and beta subunits, and with the catalytic alpha 3 beta 3 gamma subcomplex have been characterized by a combination of EPR, ESEEM, and HYSCORE spectroscopies. The EPR spectrum of the isolated beta subunit with bound VO2+ (1 VO2+/beta) is characterized by (51)V hyperfine coupling parameters (A( parallel) = 168 x 10(-)(4) cm(-)(1) and A( perpendicular) = 60 x 10(-)(4) cm(-)(1)) that suggest that VO2+ binds to the isolated beta subunit with at least one nitrogen ligand. Results obtained for the analogous VO2+ complex with the isolated alpha subunit are virtually identical. ESEEM and HYSCORE spectra are also reported and are similar for both complexes, indicating a very similar coordination scheme for VO2+ bound to isolated alpha and beta subunits. In the isolated beta (or alpha) subunit, the bound VO2+ cation is coordinated by one nitrogen ligand with hyperfine coupling parameters A( parallel)((14)N) = 4.44 MHz, and A( perpendicular)((14)N) = 4.3 MHz and quadrupole coupling parameters e(2)()qQ approximately 3.18 MHz and eta approximately 1. These are typical for amine-type nitrogen ligands equatorial to the VO2+ cation; amino acid residues in the TF1 beta and alpha subunits with nitrogen donors that may bind VO2+ are reviewed. VO2+ bound to a mixture of alpha and beta subunits in the presence of 200 mM Na2SO4 to promote the formation of the alpha 3 beta 3 hexamer has a second nitrogen ligand with magnetic properties similar to those of a histidine imidazole. This situation is analogous to that in the alpha 3 beta 3 gamma subcomplex and in the whole TF1 enzyme [Buy, C., Matsui, T., Andrianambinintsoa, S., Sigalat, C., Girault, G., and Zimmermann, J.-L. (1996) Biochemistry 35, 14281-14293]. These data are interpreted in terms of only partially structured nucleotide binding sites in the isolated beta and alpha subunits as compared to fully structured nucleotide binding sites in the alpha 3 beta 3 heterohexamer, the alpha 3 beta 3 gamma subcomplex, and the whole TF1 ATPase.