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Biomedical subjects

L Birnbaumer

Publications and source records attributed to L Birnbaumer.

At least 199 records · Page 11Linked to original sources
PubMed1986

Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system.

The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O-(thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neither alpha T . GTP gamma S nor beta gamma T having any direct effect on the intrinsic activity of the catalytic moiety. Increasing the MgCl2 concentration in the assay incubations significantly decreases the inhibitions by both alpha T . GTP gamma S and beta gamma T. Similarly, when the pure hamster lung beta-adrenergic receptor is included in the lipid vesicles with Ns and C, the levels of inhibition of the GppNHp-stimulated activity by both alpha T . GTP gamma S and beta gamma T are reduced compared to those obtained in vesicles containing just Ns and C (but not stimulatory receptor). These inhibitions are reduced still further under conditions where the agonist stimulation of adenylate cyclase activity is maximal, i.e. when stimulating with isoproterenol plus GTP. In these cases the alpha T . GTP gamma S inhibitory effects are completely eliminated and the inhibitions observed with holotransducin can be fully accounted for by the beta gamma T complex. The ability of the beta-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to Ns (or alpha s) during the activation of the regulatory protein and the stimulation of adenylate cyclase. These results also suggest that under physiological conditions the beta gamma subunit complex is primarily responsible for mediating the inhibition of adenylate cyclase activity.

Adenylyl Cyclase Inhibitors↗
PubMed1986

Functional reconstitution of the alpha 2-adrenergic receptor with guanine nucleotide regulatory proteins in phospholipid vesicles.

We describe the successful reconstitution of functional interactions between an inhibitory adenylate cyclase-coupled receptor and various nucleotide-binding regulatory proteins in phospholipid vesicles. The receptor is the alpha 2-adrenergic receptor (alpha 2AR) which has been partially purified (approximately 500-5000-fold) from human platelet membranes. The nucleotide-binding regulatory proteins include purified preparations of human erythrocyte Ni and Ns, bovine retinal transducin and the recently discovered bovine brain No. Addition of the physiologic ligand, epinephrine, to vesicles containing the alpha 2AR and Ni results in stimulation of the GTPase activity in Ni. This stimulation of GTPase activity by epinephrine is prevented in the presence of the alpha-adrenergic antagonist, phentolamine, which indicates that a functional reconstitution of the alpha 2AR and Ni has been established. The maximum turnover number for the alpha 2AR-mediated epinephrine-stimulated GTPase activity in Ni is similar to the maximal turnover numbers obtained for the beta-adrenergic receptor-mediated isoproterenol-stimulated GTPase activity in Ns and the rhodopsin-mediated light-stimulated GTPase activity in transducin (0.5-1.5 mol of Pi released per min per mol of nucleotide regulatory protein). Functional similarities between the alpha 2AR and rhodopsin are observed in their interactions with the various nucleotide-binding regulatory proteins. Thus, both of these receptor proteins are capable of promoting the maximal activation of Ni and No while being much less effective in promoting the activation of Ns. However, there are differences between the alpha 2AR and rhodopsin in their interactions with transducin. Specifically, while rhodopsin will maximally activate transducin, the alpha 2AR is much less effective in promoting this activation (i.e. approximately 20% as effective as rhodopsin). Overall, these results suggest the following specificities of interaction: for rhodopsin, transducin approximately equal to Ni approximately equal to No much greater than Ns; while for alpha 2AR, Ni approximately equal to No greater than transducin greater than or equal to Ns.

Adenylyl Cyclases↗
PubMed1986

Seasonal variations in the responses of luteal and follicular luteinizing hormone-stimulated adenylyl cyclases to estradiol implants and implant removal in pseudopregnant rabbits.

Summer rabbits appeared to be less sensitive to the presence of exogenous estradiol or to the withdrawal of exogenous estradiol than were Winter rabbits. Suppression of the luteinizing hormone(LH)-stimulated adenylyl cyclase of corpora lutea by estradiol-filled silastic capsules, which resulted in serum estradiol concentrations approximately 3.8 X control (high level), appeared to be greater in Winter rabbits than in Summer rabbits. Both high level and low level (2.1 X control) estradiol implants suppressed follicular LH-stimulated adenylyl cyclase in Winter rabbits but neither size capsule had an effect in Summer rabbits. Serum LH concentrations were equally low in Winter rabbits using either high or low level estradiol implants, while only the high level implants caused a decrease in serum LH in Summer rabbits. Withdrawal of the exogenous estradiol caused a precipitous fall in serum progesterone concentrations in Winter rabbits; estradiol withdrawal in Summer rabbits was without effect.

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↗
PubMed1985

Transducin and the inhibitory nucleotide regulatory protein inhibit the stimulatory nucleotide regulatory protein mediated stimulation of adenylate cyclase in phospholipid vesicle systems.

The adenylate cyclase coupled inhibitory nucleotide regulatory protein (Ni) and the bovine retinal nucleotide regulatory protein transducin (T) appear to share some common functional properties since their GTPase activity is stimulated to similar extents by the retinal photoreceptor rhodopsin. In the present work, we sought to assess whether these functional similarities might extend to their interaction with adenylate cyclase. This necessitated the development of reconstitution systems in which guanine nucleotide regulatory protein mediated inhibition of adenylate cyclase activity could be demonstrated and characterized in a lipid milieu. In the absence of the pure human erythrocyte stimulatory nucleotide regulatory protein (Ns), the insertion into phospholipid vesicles of either pure Ni from human erythrocytes or pure bovine T with the resolved catalytic moiety of bovine caudate adenylate cyclase (C) does not establish GppNHp inhibition of either Mg2+- or forskolin-stimulated adenylate cyclase. However, the coinsertion into lipid vesicles of either Ni or T with Ns and resolved C results in an inhibition of Ns(GppNHp) stimulatable C activity. As is the case in intact membranes, the reconstituted inhibition of the Ns-stimulated C activity extends into the steady-state phase of time courses of activity. This inhibition is highly sensitive to the MgCl2 concentration. At 2 mM MgCl2, the inhibition is greater than 80% while at 50 mM MgCl2 it is only approximately 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1985

Regulation of glucagon receptor binding. Lack of effect of Mg and preferential role for GDP.

The effects of Mg2+ and guanine nucleotides on glucagon binding to its receptor were studied using [125I-Tyr10]monoiodoglucagon. Contrary to findings with beta-adrenergic receptors, high affinity binding of the stimulatory hormone was not dependent on Mg2+ and low affinity binding could be obtained on nucleotide addition regardless of presence of Mg2+. GDP, guanyl-5'-yl thiophosphate (GDP beta S), GTP, and guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) were all able to induce low affinity hormone binding. Since the Ns component of adenylyl cyclase, with which the receptor interacts, is inactive in stimulating the catalytic component C of adenylyl cyclase in the absence of Mg2+, both before and after GDP addition, it is suggested that Ns has at least two domains that change conformation independently of each other: a r domain, that interacts with the receptor and confers to it high affinity binding, and a c domain, that interacts with the catalyst C and stimulates it. It is suggested further that Ns is r+c- when stabilizing the receptor in its conformation with high affinity for hormone, and r-c- when under the influence of GDP which results in the receptor adopting the conformation that exhibits low affinity for the hormone. Comparison of potencies of the four nucleotides to induce low affinity binding showed that GDP and GDP beta S were equipotent and 10 times more potent than GTP and 100 times more potent than GMP-P(NH)P. Under the conditions used it was impossible to substantiate that the effects of GTP or GMP-P(NH)P were not due to formation of GDP from GTP or presence of GDP-like material in GMP-P(NH)P. It is suggested that, contrary to widely held opinions, GDP and GDP-like compounds, and not GTP or its analogs, are responsible for the lowering of the affinity of adenylyl cyclase stimulating receptors for their hormones or agonists. Furthermore, the experiments suggest that the c+ conformation of the c domain of Ns co-exists with the r+ and not the r- conformation of its r domain.

Adenylyl Cyclases↗
PubMed1985

Guanine nucleotide regulation of a mammalian myocardial muscarinic receptor system. Evidence for homo- and heterotropic cooperativity in ligand binding analyzed by computer-assisted curve fitting.

Highly purified dog heart sarcolemmal membranes, with a content of approximately 5 pmol of muscarinic acetylcholine receptor (mAChR)/mg of protein, were analyzed for mAChR-mediated inhibition of adenylyl cyclase and ligand binding in the absence and the presence of guanine nucleotides. Adenylyl cyclase was found to be coupled to the mAChR, being attenuated approximately 30% in a GTP-dependent manner. Direct binding studies, using 3H-labeled oxotremorine M, showed high affinity binding (apparent KD = 10 nM) that was reduced on nucleotide addition. Dose-response curves for GDP, GTP, and guanyl-5'-yl imidodiphosphate showed them to be equipotent. On the basis of pirenzepine binding, only one type of mAChR, commonly referred to as M2, was detected. Direct binding of [3H]quinuclidinyl benzilate [( 3H]QNB) uncovered 50% more binding sites than 150 nM 3H-labeled oxotremorine M; addition of guanine nucleotides uncovered the existence of positive cooperativity in the binding of [3H]QNB. Agonist displacement curves of [3H]QNB binding, without and with guanine nucleotides, extended over several orders of magnitude, which is inconsistent with single site competitive kinetics. The results and their analysis by computer-assisted curve fitting indicated that the data are well fitted by a model in which a receptor is at least bivalent and exists in two states: one with and the other without cooperativity between its sites, with guanine nucleotides decreasing both the degree of cooperativity between the sites and the proportion of the receptor that is in the cooperative form. Since the guanine nucleotide effect is mediated by the Ni coupling protein, it is suggested that direct binding detects R'Ni complexes (cooperative), R"NiG complexes (cooperative but distinct from R'Ni), and R0 complexes (non-cooperative and unaffected by Ni or NiG), where R = mAChR, Ni = the inhibitory regulatory component of adenylyl cyclase unaffected by guanine nucleotide, and NiG = Ni affected by guanine nucleotide (G).

Adenylyl Cyclases↗
PubMed1985

Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles.

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung beta-adrenergic receptor (beta AR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (Ns) and inhibitory (Ni) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of Ns with beta AR in lipid vesicles increases the extent of binding of [35S] GTP gamma S to Ns and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of Pi released/min/mol of Ns-guanine nucleotide complexes). Enhancement of this turnover rate requires the beta-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTP gamma S binding to Ns. The co-insertion of the beta AR with Ni or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTP gamma S binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with beta AR is Ns much greater than Ni greater than T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T approximately equal to Ni much greater than Ns. Nonetheless the fold stimulation of GTPase activity and [35S]GTP gamma S binding in T, induced by light-activated rhodopsin, is significantly greater than the "fold" stimulation of these activities in Ni. This reflects the greater intrinsic ability of Ni to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S] GTP gamma S) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both Ni and T are similar to those obtained for isoproterenol-stimulated activity in Ns. This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.

Adenylyl Cyclases↗
PubMed1985

Antisera against a guanine nucleotide binding protein from retina cross-react with the beta subunit of the adenylyl cyclase-associated guanine nucleotide binding proteins, Ns and Ni.

Antisera were produced in rabbits against a guanine nucleotide binding protein (N protein), transducin, purified from bovine retina. Antiserum AS/1, which recognized all three subunits (alpha, beta, and gamma) of the holoprotein, was tested for cross-reactivity with the subunits of the adenylyl cyclase [adenylate cyclase; ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]-associated stimulatory (Ns) and inhibitory (Ni) N proteins purified from human erythrocytes. As/1 showed strong reactivity against the beta subunits of both Ns and Ni but failed to cross-react with either the alpha or gamma subunits of Ns and Ni. Seven additional antisera against transducin reacted with the beta subunits but not with the alpha or gamma subunits of Ns and Ni. A single antiserum against transducin reacted with the alpha subunit of Ni but not of Ns. Immunostaining of the beta subunits of Ns and Ni was proportional to the amount of beta subunit blotted and to the antiserum concentration. Immunostaining of either human erythrocyte or bovine cerebral cortical plasma membrane proteins with AS/1 showed a single band, comigrating with the beta subunit of transducin; this band was absent in bovine erythrocyte membranes. Estimation of the amount of beta subunit by immunoblotting with AS/1 showed that the beta subunit comprises approximately equal to 2% of bovine cerebral cortical plasma membrane protein, approximately equal to 100-fold more than in human erythrocyte membranes. These findings provide immunochemical evidence for similarities in the beta subunits and differences in the alpha and gamma subunits of this family of N proteins. Antisera against transducin react specifically with the beta subunits of Ns and Ni in crude plasma membranes and, thus, can serve as specific probes for the beta subunit.

Adenylyl Cyclases↗
PubMed1985

The membrane-bound spermatozoal adenylyl cyclase system does not share coupling characteristics with somatic cell adenylyl cyclases.

Membrane-bound adenylyl cyclases from ram, dog, and human sperm are unresponsive to fluoride and guanylylimidodiphosphate [GMP-P(NH)P], two agents that stimulate the adenylyl cyclases of somatic cells by an action on the stimulatory guanine nucleotide-binding regulatory (Ns) component of adenylyl cyclase. We have investigated whether this is because the sperm cell catalytic unit is functionally uncoupled from Ns but, nevertheless, capable of interacting with it, or because the sperm cell adenylyl cyclase system is unique and regulated differently from that of somatic cells. Sperm cells were found to be deficient in Ns, as evidenced by the inability of detergent extracts from sperm cell membranes and fractions to reconstitute Ns-mediated regulation of the adenylyl cyclase of cyc- S49 cells. In addition, attempts to label Ns in sperm cell membranes by [32P]ADP ribosylation with cholera toxin revealed that, if present, Ns is less than 1% of that found in human erythrocyte membranes. This, however, was not the only reason for the unresponsiveness of sperm cell adenylyl cyclase, since fluoride stimulation of the sperm cell enzyme could not be induced by reconstituting it with Ns purified from human erythrocytes (hRBC). When intact hRBC membranes were added to sperm cell fractions in the presence of fluoride, the activities that resulted were greater than the sum of the individual activities. This apparent reconstitution of fluoride regulation of sperm cell adenylyl cyclase could be blocked by lima bean trypsin inhibitor and appears to have resulted from proteolytic activation of the hRBC adenylyl cyclase by sperm proteases. Sperm cell membranes also appear to lack a functional inhibitory regulatory protein of the adenylyl cyclase system (Ni), since they did not contain an ADP-ribosylatable substrate for pertussis toxin action. These results suggest that the sperm cell adenylyl cyclase system is unique and different from that of somatic cells. Sperm cells appear to neither contain Ns or Ni nor possess the ability of their adenylyl cyclase system to interact with Ns from an exogenous source.

Adenylyl Cyclases↗
PubMed1985

Evidence for a physiological role of gonadotropin-releasing hormone (GnRH) or GnRH-like material in the ovary.

The possibility that GnRH or a GnRH-like material of ovarian origin may play a physiological role in follicular development was explored in immature hypophysectomized rats by testing whether a potent synthetic antagonist of GnRH action [( N-acetyl-dehydro-Pro1,D-p-chloro-Phe2,D-Trp3,6]GnRH), would potentiate FSH-induced maturation of ovarian follicles to an ovulable stage. Rats were hypophysectomized on day 25 of their life and implanted with a Silastic capsule containing diethylstilbestrol. On day 30, they were started on injections of 10 micrograms NIH FSH-S12 twice daily alone (control) or in combination with 10 micrograms of either native GnRH or GnRH antagonist. On day 35, all rats received 30 IU hCG to trigger ovulation and luteinization of mature follicles. Rats were killed 25.5-28 h later and inspected for number of ova in Fallopian tubes, ovarian weight, number of corpora lutea (CL) on ovarian surface, and appearance of hematoxylin-eosin-stained ovarian slices. In control animals (n = 6), we found some ovulations (mean +/- SEM, 3.2 +/- 1.1/rat), many more CL (16.5 +/- 4.5/rat), and ovarian weights of 37.7 +/- 1.1 mg/rat. In GnRH-treated rats (n = 5), there were no CL formed, no ova were found, and ovarian weights were 16.0 +/- 1.5 mg/rat. In contrast, in GnRH antagonist-treated rats (n = 5), 16.4 +/- 1.6 ova/rat were recovered from the Fallopian tubes, and ovaries contained 20.8 +/- 2.5 CL/rat and weighed 52.7 +/- 3.2 mg/rat. All changes were statistically significant. We conclude that an antagonist of GnRH action is able to potentiate the action of FSH on ovarian follicle development and suggest that it does so by inhibiting the action of an endogenous GnRH or GnRH-like substance that may play a role as a physiological atretic signal.

Animals↗