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Biomedical subjects

E J Neer

Publications and source records attributed to E J Neer.

At least 73 records · Page 4Linked to original sources
PubMed1988

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.

Amino Acid Sequence↗
PubMed1988

Roles of G protein subunits in transmembrane signalling.

A family of proteins called G proteins couples cell surface receptors to a variety of enzymes and ion channels. Since many cells contain several very similar G proteins, an important question is how signals remain specific as they cross the cell membrane.

Animals↗
PubMed1988

Identification of cDNA encoding an additional alpha subunit of a human GTP-binding protein: expression of three alpha i subtypes in human tissues and cell lines.

The guanine nucleotide-binding proteins (G proteins), which mediate hormonal regulation of many membrane functions, are composed of alpha, beta, and gamma subunits. We have cloned and characterized cDNA from a human T-cell library encoding a form of alpha i that is different from the human alpha i subtypes previously reported [Didsbury, J. R., Ho, Y.-S. & Snyderman, R. (1987) FEBS Lett. 211, 160-164 and Bray, P., Carter, A., Guo, V., Puckett, C., Kamholz, J., Spiegel, A. & Nirenberg, M. (1987) Proc. Natl. Acad. Sci. USA 84, 5115-5119]. alpha i is the alpha subunit of a class of G proteins that inhibits adenylate cyclase and regulates other enzymes and ion channels. This cDNA encodes a polypeptide of 354 amino acids and is assigned to encode the alpha i-3 subtype of G proteins on the basis of its similarity to other alpha i-like cDNAs and the presence of a predicted site for ADP ribosylation by pertussis toxin. We have determined the expression of mRNA for this and two other subtypes of human alpha i (alpha i-1 and alpha i-2) in a variety of human fetal tissues and in human cell lines. All three alpha i subtypes were present in the tissues tested. However, analysis of individual cell types reveals specificity of alpha i-1 expression. mRNA for alpha i-1 is absent in T cells, B cells, and monocytes but is present in other cell lines. The finding of differential expression of alpha i-1 genes may permit characterization of distinct physiological roles for this alpha i subunit. mRNA for alpha i-2 and alpha i-3 was found in all the primary and transformed cell lines tested. Thus, some cells contain all three alpha i subtypes. This observation raises the question of how cells prevent cross talk among receptors that are coupled to effectors through such similar alpha proteins.

Amino Acid Sequence↗
PubMed1988

Specificity of action of guanine nucleotide-binding regulatory protein subunits on the cardiac muscarinic K+ channel.

The cardiac muscarinic receptor stimulates a potassium-selective ionic current (IK.ACh) through activation of a guanine nucleotide-binding regulatory protein. Purified alpha and beta gamma subunits of the guanine nucleotide-binding regulatory protein have each been reported to open the K+ channel. We have reported that nanomolar concentrations of purified brain beta gamma subunits activated IK.ACh in chicken embryonic atrial patches. In contrast, J. Codina, A. Yatani, D. Grenet, A.M. Brown, and L. Birnbaumer [(1987) Science 236, 442-445] subsequently reported that picomolar concentrations of activated erythrocyte alpha subunits (i.e., the 40-kDa alpha subunit that the authors call alpha K) opened K+ channels in guinea pig atrial patches. In this paper, we further explore the specificity of various beta gamma and alpha subunits in embryonic chicken and neonatal rat atrial patches. Beta gamma subunits from either human placenta (beta 35 gamma) or bovine brain (beta 35,36 gamma) activated IK.ACh whereas transducin beta gamma (beta 36 gamma) did not. The beta gamma activation was consistent in rat and chicken patches [118 of 123 patches (97%)]. Beta gamma subunits opened K+ channels at concentrations greater than or equal to 200 pM and maximally activated the channel at 10 nM. Beta gamma or guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]) channel activation could be reversed by alpha 41-GDP. The purified brain beta gamma preparation was contaminated with less than 0.01% unactivated alpha. The detergent (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHAPS), used to suspend the hydrophobic beta gamma, did not activate IK.ACh alone, with buffer, with heat-inactivated beta gamma, or with transducin beta gamma. Unactivated alpha subunits did not open K+ channels. Activated, alpha subunits purified from human erythrocytes (alpha 40-GTP[gamma-S]) or bovine brain (alpha 39-GTP[gamma-S]) at concentrations of 10 pM or higher (up to 1 nM) opened K+ channels less frequently in chicken atrial patches [5 of 27 patches (19%) and 9 of 35 patches (26%), respectively] than in rat atrial patches [5 of 11 patches (45%) and 11 of 19 patches (58%), respectively]. Negative results were not due to patch vesicle formation. Other experiments indicated that alpha and beta gamma activated the same population of channels. Activation of the channel by both beta gamma and alpha subunits implies a more complicated scheme for guanine nucleotide-binding regulatory protein action than previously proposed.

Animals↗
PubMed1988

Atrial natriuretic factor gene expression in ventricles of rats with spontaneous biventricular hypertrophy.

A subset of Wistar-Kyoto (WKY) rats that spontaneously develops biventricular hypertrophy (BVH) in response to increased cardiac output was evaluated for ventricular expression of the atrial natriuretic factor (ANF) gene. Normal WKY rats had low levels of left ventricular ANF mRNA and minimally detectable ANF transcripts in the right ventricle. In contrast, BVH rats showed a sixfold greater ANF mRNA concentration in the left ventricle than age-matched WKY controls. BVH right ventricular ANF mRNA levels equaled those found in BVH left ventricles and were dramatically greater than WKY right ventricular controls. Unlike experimental models of hypertrophy, both left and right ventricles significantly increase ANF gene transcripts in the natural development of BVH. The left and right ventricles can concordantly respond to hypertrophy and increase ANF gene transcription.

Animals↗
PubMed1988

The gene for the alpha i1 subunit of human guanine nucleotide binding protein maps near the cystic fibrosis locus.

The gene for the alpha i1 subunit of human guanine nucleotide binding (G) protein was mapped by in situ hybridization to chromosome 7 at band q21. The regional chromosomal location of the human alpha i1 gene was confirmed using human/mouse somatic-cell hybrid lines containing portions of human chromosome 7. Because the alpha i1 gene mapped near the cystic fibrosis locus and because an abnormal G protein might be expected to contribute to the pathophysiology of this disease, the alpha i1 gene was mapped with respect to the cystic fibrosis locus as defined by the Met oncogene and anonymous DNA marker pJ3.11. The location of the alpha i1 gene proved to be distinct from that of the cystic fibrosis locus.

Chromosome Banding↗
PubMed1987

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.

Adenosine Diphosphate Ribose↗
PubMed1987

The stimulatory guanine-nucleotide regulatory unit of adenylate cyclase from bovine cerebral cortex. ADP-ribosylation and purification.

Hormonal stimulation of adenylate cyclase from bovine cerebral cortex is mediated by a guanine-nucleotide regulatory protein (Gs). This protein contains at least three polypeptides: a guanine nucleotide-binding alpha s component and a beta X gamma component, which modulates the function of alpha s. The alpha s component from many tissues can be ADP-ribosylated with cholera toxin, but has been unusually difficult to modify in brain. We have improved incorporation of ADP-ribose by including isonicotinic acid hydrazide to inhibit the potent NAD glycohydrolase activity of brain. ADP-ribosylation is further improved by addition of detergent to render the substrates accessible and 20 mM-EDTA to chelate metal ions. Although Mg2+ is absolutely required for activation of adenylate cyclase by the GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), it is not obligatory for p[NH]ppG-stimulated ADP-ribosylation by cholera toxin. Under these conditions, the ADP-ribosylation of brain membranes is not enhanced by a cytosolic protein. We find that there are two major sizes of brain alpha s, which we have named 'alpha sL', with an apparent Mr of 42,000-45,000, and 'alpha sH' with an apparent Mr of 46,000-51,000 depending on the gel-electrophoretic system used. The alpha sL and alpha sH components can incorporate different amounts of ADP-ribose depending on the reaction conditions, so that one or the other may appear to predominate. Thus we show that incomplete ADP-ribosylation by cholera toxin is not a good indication of the relative amounts of alpha s units. Functionally, however, both forms of alpha s appear to be similar. Both forms associate with the catalytic unit of adenylate cyclase, but neither of them does so preferentially. There is an excess of each of them over the amount associated with catalytic unit. We have now substantially purified Gs from brain by a modification of the method of Sternweis et al. [(1981) J. Biol. Chem. 256, 11517-11526] as well as by a new, simplified, procedure. On SDS/polyacrylamide-gel electrophoresis, the purified brain Gs contains both the 45 and 51 kDa alpha s polypeptides revealed by ADP-ribosylation and a beta X gamma component. Activation of purified alpha s by guanine nucleotides or fluoride can be reversed by addition of purified beta X gamma component. The activated form of purified brain Gs has an Mr of 49,000 as determined by hydrodynamic measurements, which is consistent with the idea that the active form of brain Gs is the dissociated one.

Adenosine Diphosphate↗
PubMed1987

Genes for two homologous G-protein alpha subunits map to different human chromosomes.

Signal transduction across biological membranes is modulated by a family of related GTP-binding proteins termed G proteins. These G proteins have a heterotrimeric structure composed of alpha, beta, and gamma subunits. The alpha subunits of the G proteins bind GTP and appear to determine the biochemical specificity of the protein. We have recently cloned and characterized cDNA encoding two G-protein alpha subunits, alpha i and alpha h. The former is a substrate for ADP-ribosylation by pertussis toxin. The protein corresponding to alpha h has not yet been identified. These cDNAs encode proteins, which demonstrate 90% sequence identity to one another and also show marked similarity to other G proteins. The present studies were designed to determine whether the genes for these related proteins are clustered on a single human chromosome. Genomic DNA isolated from a panel of mouse-human hybrid cell lines was analyzed by hybridization to cDNAs for alpha i and alpha h. Based on the distribution patterns of alpha i and alpha h in cell hybrids, the gene for alpha i was assigned to human chromosome 7, and the gene for alpha h assigned to chromosome 12. These data suggest that the G-protein gene family may be distributed over at least two human chromosomes.

Animals↗
PubMed1986

Development of muscarinic cholinergic inhibition of adenylate cyclase in embryonic chick heart. Its relationship to changes in the inhibitory guanine nucleotide regulatory protein.

Parasympathetic and sympathetic innervation of the embryonic chick heart proceed non-coordinately. beta-Adrenergic agonists mediate an increase in beating rate in embryonic chick heart prior to ingrowth of the vagus nerve (Culver, N. G., and Fishman, D. A. (1977) Am. J. Physiol. 232, R116-R123) while muscarinic agonists exert relatively little effect on beating rate in hearts 2-4 days in ovo (Papanno, A. J. (1979) Pharmacol. Rev. 29, 3-33). Studies of developmental changes in the ability of muscarinic agonists to inhibit adenylate cyclase activity and their relationship to the development of a physiologic response of the embryonic chick heart to muscarinic stimulation have been inconclusive. In the current studies the ability of isoproterenol to stimulate adenylate cyclase activity did not change during development. Maximum stimulation above basal was 760 pmol of cAMP/10 min/mg of proterin with an IC50 of 1.5 X 10(-6) M for isoproterenol in homogenates of hearts 2 1/2, 3 1/2, and 10 days in ovo and 3 days posthatching. However, inhibition of isoproterenol-stimulated adenylate cyclase activity by carbamylcholine increased from 7.6% with a IC50 for carbamylcholine of 16 +/- 5.0 microM at day 2 1/2 in ovo to 29% with an IC50 of 0.4 +/- 0.1 microM at day 10 in ovo and to 43% with a IC50 of 0.6 +/- 0.1 microM at 3 days posthatching. Since previous data had demonstrated the presence of muscarinic receptors as early as 2 1/2 days in ovo (Galper, J. B., Klein, W., and Catterall, W. A. (1977) J. Biol. Chem. 252, 8692-8699), studies of developmental changes in guanine nucleotide-coupling proteins were carried out to determine whether early in development muscarinic receptors were uncoupled from a physiologic response. Studies of pertussis toxin-catalyzed ADP-ribosylation of homogenates of embryonic chick heart with [32P]NAD demonstrated the presence of two ADP-ribosylated proteins at 39,000 and 41,000 kDa, respectively. Both ADP-ribosylation and immunoblotting of homogenates with an antibody to the 39-kDa guanine nucleotide-binding protein in bovine brain demonstrated that the 39-kDa alpha protein increased 1.8-fold between days 2 1/2 and 3 1/2 in ovo and another 1.8-fold from day 3 1/2 to 10 in ovo in parallel with the increase in the extent of muscarinic inhibition of adenylate cyclase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1986

Active site specificity of the adenylate cyclase catalytic unit from bovine caudate nucleus.

ATP analogues were used to study the active site specificity of the catalytic unit (C) of solubilized and partially purified bovine brain caudate nucleus adenylate cyclase. Phenylenediamine ATP (PD-ATP), 8-azido ATP (8-N3ATP), chromium(III) 3'-beta-alanylarylazido ATP (CrATPa), and 2',3'-dialdehyde ATP (oATP) are competitive inhibitors of C in the presence of the substrate MnATP and the activator forskolin. (Km for MnATP is 50 +/- 11 microM, n = 13). The Ki values determined under initial velocity conditions are: PD-ATP, Ki = 695 +/- 60 microM, n = 5; 8-N3ATP, Ki = 155 +/- 23 microM, n = 5; CrATPa, Ki = 7 +/- 3 microM, n = 2; oATP, Ki = 42 +/- 5 microM, n = 3. Irradiation of 100 microM 8-N3ATP by UV light (254 nm) causes the first-order loss of reagent either in the presence or absence of C. Concomitant irreversible inhibition of C in the presence of 8-N3ATP was more complex and asymptotically approached 50% within 4-6 min. Loss of C activity in controls was 10-20%. The fraction of C covalently modified by 8-N3ATP, alpha, was calculated for each time point of irradiation for an increasing initial concentration ([A]o) of 8-N3ATP. Extrapolated to infinite time of photolysis, the value of alpha reached a final level, termed alpha t whose magnitude depended on [A]o. From these data we calculated an apparent KD of 4.5 microM for 8-N3ATP. ATP protected against the irreversible inhibition due to 8-N3ATP. These data are most consistent with a mechanism of photoaffinity labeling involving equilibrium binding and covalent insertion of 8-N3ATP into the active site. These results indicate that the active site binds analogues of ATP which are considerably modified in the adenine, ribose, and gamma-phosphate portions and that the affinity of C for these analogues is within an order of magnitude of the Km for ATP.

Adenosine Triphosphate↗
PubMed1986

Conformations of the alpha 39, alpha 41, and beta.gamma components of brain guanine nucleotide-binding proteins. Analysis by limited proteolysis.

We have recently purified two proteins, alpha 39 and alpha 41, from bovine cerebral cortex which are substrates for ADP-ribosylation by pertussis toxin (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229). Both proteins bind guanine nucleotides and interact with beta.gamma units. We have used limited proteolysis by trypsin to probe the structure and the conformational states of these proteins. The guanosine 5'-O-(thiotriphosphate) (GTP gamma S)-liganded alpha 41 protein is cleaved into stable 39- and 24/25-kDa products which appear at the same rate. In addition, an 18-kDa peptide is seen. These products are also formed from GDP- or GTP-liganded alpha 41 but are less stable. Cleavage of alpha 39 is different. With GTP gamma S stable 37-kDa product predominates while with GTP or GDP the 37-kDa fragment appears transiently, followed by 24/25-kDa fragments which are stable in the presence of guanine nucleotides but rapidly cleaved without ligand. A 17-kDa peptide is also formed with GTP or GDP. The beta.gamma unit is cleaved by trypsin to stable peptides, a 26/27-kDa doublet and a 14-kDa peptide. Addition of beta.gamma slows tryptic cleavage of alpha 41 but not alpha 39. ADP-ribosylation of alpha 39 and alpha 41 by pertussis toxin affects their conformation in distinct ways which are clearly brought out by the GTP-liganded state. In contrast to unmodified alpha 41, ADP-ribosylated and GTP-liganded alpha 41 is proteolyzed very slowly and without formation of a 39-kDa intermediate. GTP gamma S seems to override the effect of ADP-ribosylation so that cleavage is more rapid and goes via the 39-kDa product. ADP-ribosylation affects alpha 39 more subtly. The GTP-liganded protein is first cleaved to the 37-kDa product and then degraded without forming the 24/25-kDa fragment. These results suggest that ADP-ribosylation might affect the conformation and function of these related proteins differently. The site of [32P]ADP-ribosylation is on the 18-kDa product of alpha 41 and on the 17-kDa product of alpha 39. We have raised polyclonal antibodies against alpha 39 and beta in rabbits and used the antibodies to examine antigenic sites on alpha 39 and beta. The antigenic determinants of alpha 39 are located over most of the native tryptic peptides. Tryptic cleavage of alpha 41 leads to rapid loss of cross-reactivity with anti-alpha 39 antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1986

Subunit interactions of native and ADP-ribosylated alpha 39 and alpha 41, two guanine nucleotide-binding proteins from bovine cerebral cortex.

Bovine cerebral cortex contains two major substrates for ADP-ribosylation by pertussis toxin: a 39-kDa protein, alpha 39, and a 41-kDa protein, alpha 41 (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229). Both of these proteins bind guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with a similar affinity (Kd = 30 +/- 10 nM for alpha 39, Kd = 32 +/- 14 nM for alpha 41). Both proteins associate with a beta X gamma subunit made up of a 36-kDa beta component and a 6-kDa gamma component. We have previously shown that the beta X gamma unit is required for pertussis toxin-catalyzed ADP-ribosylation (Neer et al. (1984)). By measuring the amount of beta X gamma required for maximal incorporation of ADP-ribose, we now find that the EC50 for beta X gamma in this reaction is 3 +/- 1 times lower for alpha 41 than for alpha 39. ADP-ribosylation by pertussis toxin does not prevent dissociation of alpha 41 X beta X gamma or alpha 39 X beta X gamma by GTP gamma S. GTP gamma S decreases the sedimentation coefficient of ADP-ribosylated alpha 41 from 4.2 S to 3.0 S and the sedimentation coefficient of ADP-ribosylated alpha 39 from 4.3 S to 2.9 S. The conclusion that GTP gamma S dissociates both ADP-ribosylated heterotrimers was confirmed by the observation that GTP gamma S blocks precipitation of ADP-ribosylated alpha 39 or alpha 41 by anti-beta antibody. Neither alpha 41 X beta X gamma nor alpha 39 X beta X gamma is dissociated by GTP whether or not the proteins are ADP-ribosylated. The observation that alpha 41 more readily associates with beta X gamma than does alpha 39 may explain our earlier observation that alpha 41 is more readily ADP-ribosylated than alpha 39. In most intact membranes, only a 41-kDa ADP-ribosylated protein is seen. However, alpha 39 is also present in most tissues since we can detect it with anti-alpha 39 antibody. The functional consequences of pertussis toxin treatment may depend on whether one or both proteins are ADP-ribosylated. This in turn may depend on the ratio of alpha 41 and alpha 39 to beta X gamma in a given tissue.

Adenosine Diphosphate Ribose↗
PubMed1986

Go, a guanine nucleotide-binding protein: immunohistochemical localization in rat brain resembles distribution of second messenger systems.

We have localized a guanine nucleotide-binding protein, Go, in rat brain by immunohistochemistry with a selective polyclonal antiserum to the alpha 39 subunit of Go. Specific staining is widely distributed, abundant in neuropil, absent from neuronal cell bodies, and displays regional heterogeneity. Staining is enriched in cerebral cortex, particularly the molecular layer, neuropil of the hippocampal formation, striatum, substantia nigra pars reticulata, molecular layer of the cerebellum, substantia gelatinosa of the spinal cord, and posterior pituitary. High density staining in the substantia nigra reflects a Go-containing striatonigral pathway since striatal lesions reduce ipsilateral immunostaining in the pars reticulata. Confirming immunostaining, quantitative [32P]ADP-ribosylation of nigral membranes with pertussis toxin indicates a 66% +/- 11% (mean +/- SEM) reduction of Go ipsilateral to striatal lesions. Go may be associated with Purkinje cells in the cerebellum since membranes from mutant mice (Nervous), which postnatally lose Purkinje cells, are markedly depleted in pertussis toxin substrate. The localizations of Go correspond in many areas with those of protein kinase C, a component of the phosphatidylinositol cycle, suggesting a major role for Go in the brain related to regulation of the phosphatidylinositol cycle.

Adenosine Diphosphate Ribose↗
PubMed1986

Molecular cloning and characterization of cDNA encoding the GTP-binding protein alpha i and identification of a related protein, alpha h.

We have cloned and characterized cDNA encoding alpha i, the GTP-binding subunit of Gi, a protein that mediates hormonal inhibition of adenylate cyclase and hormonal regulation of other membrane functions. We have also identified cDNA encoding a putative protein, which we have named alpha h, that is highly homologous to alpha i but different from other known GTP-binding proteins. Both cDNAs were isolated from a bovine pituitary library. The cDNA encoding alpha i was identified by finding that the amino acid sequence determined for two tryptic peptides from alpha i agreed exactly with amino acid sequences deduced from the cDNA. We also determined the amino acid sequence of peptides derived from alpha o, a related 39-kDa protein purified from bovine brain. These sequences are approximately 75% identical to the sequence determined for alpha i. Southern blot analysis of bovine genomic DNA, using as probes radiolabeled cDNAs for alpha i, alpha h, and the alpha subunit of a related protein, transducin, showed that each probe recognized different genomic DNA fragments. Our results suggest a further level of complexity in the organization of the G-protein gene family, with multiple G proteins of very similar structural properties likely to be identified as products of distinct genes.

Adenosine Diphosphate Ribose↗
PubMed1986

Guanine nucleotide-binding proteins involved in transmembrane signaling.

Transmission of signals from a hormone-receptor complex to a number of intracellular effectors is mediated by a family of guanine nucleotide-binding proteins (G proteins). Each G protein consists of an alpha, beta, and gamma subunit and is most clearly distinguished from other G proteins by the biological and biochemical attributes of its GTP-binding alpha subunit. The alpha proteins can be divided into general classes by their susceptibility to modification by the bacterial toxins, cholera toxin, and pertussis toxin. In brain, there are three substrates for ADP-ribosylation by pertussis toxin with molecular weights of 41,000 (alpha 41), 40,000 (alpha 40) and 39,000 (alpha 39). The alpha 39 is the most abundant. We purified the alpha 41 and alpha 39 proteins from bovine brain and have used immunologic and biochemical tools to compare their structure and function. We have also isolated and characterized a cDNA clone from a bovine pituitary library that encodes alpha 41. The deduced amino acid sequence of the alpha 41 clone demonstrates marked homology to several other GTP-binding proteins. Southern blot analysis of the cloned alpha 41 cDNA suggests the presence of two genes encoding this G protein. In addition, we identified a cDNA clone for a novel, putative G protein, alpha h, which demonstrates marked sequence homology with other G proteins but which represents a clearly distinct gene product. Taken together, these data suggest a new level of complexity in the organization of the G protein supergene family, with multiple G proteins of similar overall structural and mechanistic properties likely to be identified as products of distinct genes.

Adenylyl Cyclases↗
PubMed1985

Characterization by two-dimensional peptide mapping of the gamma subunits of Ns and Ni, the regulatory proteins of adenylyl cyclase, and of transducin, the guanine nucleotide-binding protein of rod outer segments of the eye.

Ns and Ni, the regulatory proteins affecting adenylyl cyclase, and transducin, the guanine nucleotide-binding protein from rod outer segments of the eye, are structurally and functionally related proteins. Of these, the alpha subunits are between 39 and 42 kDa in mass, beta subunits are all of 35 kDa in mass, and gamma subunits are much smaller, of approximately 5-8 kDa in mass. We compared, by two-dimensional peptide mapping of iodinated peptides, the beta and gamma subunits of human erythrocyte Ns, human erythrocyte Ni, the beta gamma complex derived from purification of bovine brain N proteins, and frog and bovine eye transducins. We found that gamma subunits in human erythrocyte Ns and Ni and in bovine brain beta gamma complex are indistinguishable by this approach. In contrast, gamma subunits associated with frog and bovine transducin differed markedly between each other and from N protein-associated gamma. beta subunits, on the other hand, yielded essentially indistinguishable peptide maps regardless of whether derived from N proteins or from transducin and regardless also of species of origin: human versus bovine versus frog. These results suggest that the gamma subunit may impart functional heterogeneity of this family of proteins which is evident in the N proteins on the one hand and the transducin proteins on the other.

Animals↗