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Biomedical subjects

L Birnbaumer

Publications and source records attributed to L Birnbaumer.

At least 253 records · Page 14Linked to original sources
PubMed1980

Regulation of hormone-receptor coupling to adenylyl cyclase. Effects of GTP and GDP.

GDP and GTP regulation of receptor-mediated stimulation of adenylyl cyclases in membranes of S49 murine lymphoma cells (S49), NS-20 murine neuroblastoma cells (NS-20), rabbit corpora lutea (CL), and turkey erythrocytes were studied under assay conditions which minimized conversion of added GTP to GDP and of added GDP to GTP. Hormonal stimulation in all systems required guanine nucleotide addition. In the presence of GTP, adenylyl cyclase activity in S49, NS-20, and CL was stimulated respectively by isoproterenol and prostaglandin E1 (PGE1), by PGE1 and the adenosine analog, phenylisopropyladenosine, and by PGE1 and isoproterenol, with the first of the listed stimulants eliciting higher activities than the second. Activity in turkey erythrocyte membranes was stimulated by isoproterenol. GDP was partially effective in promoting hormonal stimulation, being able to sustain stimulation by isoproterenol and PGE1 in S49 cell membranes and by PGE1 in CL membranes. In NS-20 membranes, both GDP and guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) were inhibitory on basal activity, yet promoted limited but significant stimulation by PGE1. In turkey erythrocytes, stimulation by isoproterenol could not be elicited with GDP or GDP beta S. Thus, although less effective than GTP in promoting hormonal stimulation of several adenylyl cyclase systems, GDP was clearly not inactive. Concentration effect curves for active hormone in the presence of GDP had higher apparent Ka values than in the presence of GTP. In spite of differences between the effects of GTP and GDP on hormonal stimulation of adenylyl cyclase activities, GTP and GDP affected equally well isoproterenol binding, regardless of whether or not its receptor could be shown to stimulate adenylyl cyclase in the presence of GDP. Determination of transphosphorylation of GDP to GTP showed that at saturating concentrations, the proportion of GDP converted to GTP is negligible and unaffected by hormonal stimulation. Concentrations giving 50% inhibition were determined for GTP- and GDP-mediated inhibition of guanyl-5'-yl imidodiphosphate stimulation in the absence and presence of stimulatory hormones. In all four systems studied, GTP and GDP interacted with about equal potency and hormonal stimulation was not accompanied by a selective decrease in affinity for GDP. One way to explain all of the results obtained is to view hormonally sensitive adenylyl cyclase systems as two-state enzymes whose activities are regulated by GTP and GDP through an allosteric site related to the catalytic moiety, and receptors as entities that are inactive and hence unable to couple unless occupied by hormones and activated by any guanine nucleotide through a distinct receptor-related process.

Adenylyl Cyclases↗
PubMed1980

Transient and steady state kinetics of the interaction of guanyl nucleotides with the adenylyl cyclase system from rat liver plasma membranes. Interpretation in terms of a simple two-state model.

Transient and steady state kinetics of the interaction of liver plasma membrane adenylyl cyclase with GTP and the GTP analogues guanyl-5'-yl imidodiphosphate (GMP-P(NH)P), guanyl-5'-yl diphosphonate (GMP-P(CH2)P), and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) was studied before and after treatment of membranes with preactivated cholera toxin. (a) In control experiments, GTP stimulated the enzyme partially and without a noticeable lag, while GTP analogues stimulated to varying degrees (GTP gamma S approximately GMP-P(NH)P greater than GMP-P(CH2)P greater than or equal to GTP) and with lag periods that varied with the nucleotide (GMP-P(NH)P approximately GMP-P(CH2)P greater than GTP gamma S). (b) In toxin-treated membranes, transient kinetics and degree of activation by GTP analogues were unaltered, while activation by GTP remained rapid and became as effective as the most effective of the analogues. (c) Toxin treatment had no effect on the concentration-effect curves for GTP analogues but resulted in a 5- to 9-fold lowering of the apparent K alpha with which GTP stimulates the system. These results indicate that the degree of stimulation of the liver adenylyl cyclase by GTP and its analogues is independent of the susceptibility of the beta-gamma bond of the guanosine triphosphate to be hydrolyzed and that lags in progress curves are not due to slow dissociation of "resident" GDP molecules. The time transients of the interaction of the rat liver adenylyl cyclase with combinations of GTP and GMP-P(NH)P were studied and found to be of the competitive type regardless of the time and sequence of addition of the two nucleotides. The results are consistent with GMP-P(NH)P interacting with and dissociating from the system very slowly and not leading to the formation of an irreversibly activated state of the enzyme. The data obtained on regulation of the basic (no hormone added) liver adenylyl cyclase by guanyl nucleotides are interpreted in terms of a two-state enzyme model (Birnbaumer, L., Bearer, C.F., and Iyengar, R. (1980) J. Biol. Chem. 255, 3552-3557) and the results are discussed in the light of current views of regulation of adenylyl cyclases and involvement of a GTPase. Our experiments are consistent with, but not proof for, presence of a cholera toxin-inhibited GTPase in intimate association with the liver adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1980

Naturally soluble component(s) that confer(s) guanine nucleotide and fluoride sensitivity to adenylate cyclase.

Supernatant fractions (300,000 x g, 60 min) from homogenates of rat liver, heart, and skeletal muscle, dog liver, and rabbit liver prepared without detergent in the homogenization medium (referred to as S300) are shown to contain an activity that restores Mg2+-dependent fluoride- and guanine nucleotide-stimulated cyclizing activity to the adenylate cyclase system [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] in cyc- S49 murine lymphoma cell membranes. Approximately 25% of the total cyc- reconstituting activity in the above tissues is present in S300. Reconstituting activity is proportional to S300, is sensitive to trypsin, is protected against heat inactivation by guanine nucleotide, and has a sedimentation coefficient of 5.3 in both H2O and 2H2O linear sucrose density gradients. Treatment with cholera toxin and NAD+ results in reconstitution of cyc- adenylate cyclase with enhanced activity in the presence of GTP. Reconstituion with S300 is stable, as seen in cyc- membranes after washing. All of these properties of S300 are similar to those of membrane-derived cyc- reconstituting activity. It is concluded that cell cytoplasm contains a naturally soluble protein or mixture of proteins having guanine nucleotide regulatory component activity of adenylate cyclase.

Adenylyl Cyclases↗
PubMed1980

LH/hCG responsive adenylyl cyclase in rat testis and testes from testicular feminized male (Tfm) rats and mice; desensitization by homologous hormone.

Testes of testicular feminized (tfm) rats and mice, as well as of normal male rats contain an LH/hCG responsive adenylyl cyclase. Basal, as well as hCG stimulated activities were higher in tfm rats and mice than in normal rats. The presence of an LH/hCG responsive adenylyl cyclase in the testis of tfm rats and mice shows that the greatly elevated LH levels present in males having this syndrome, giving 80-90% reduction in LH/hCG receptors, do not cause an uncoupling of the remaining receptors from the adenylyl cyclase. It also shows that androgens are not essential for coupling of the LH/hCG receptors to the adenylyl cyclase. Injection of 200 IU of hCG into adult normal rats and tfm rats caused, after 48 h, a complete loss of LH/hCG stimulated adenylyl cyclase whereas the FSH responsive adenylyl cyclase in both animal preparations was maintained. Desensitization of the LH responsive adenylyl cyclase by hCG in normal rats, confirms previous studies showing lack of hCG stimulated cyclic-AMP secretion after a comparable dose of hCG in vivo. Similarly, hCG (50 IU) caused a transient loss of LH/hCG responsive adenylyl cyclase in tfm mice, with a complete disappearance of response after 24 h. At 48 and 72 h after injection of hCG the response gradually returned to normal. The fact that hCG caused a complete desensitization of the LH/hCG responsive adenylyl cyclase in both tfm rats and mice, proves that androgen receptor mediated events are not involved in hCG desensitization of the adenylyl cyclase in Leydig cells.

Adenylyl Cyclases↗
PubMed1979

Coupling of glucagon receptor to adenylyl cyclase. Requirement of a receptor-related guanyl nucleotide binding site for coupling of receptor to the enzyme.

Effects of glucagon and guanyl nucleotides on the rat liver plasma membrane adenylyl cyclase were studied. It was established that: 1) glucagon stimulates the fully guanyl-5'-yl imidodiphosphate (GMP-P(NH)P)-activated enzyme between 20 and 70%, provided a guanyl nucleotide is present in the assay; 2) glucagon has no effect on adenylyl cyclase activity in membranes activated fully by GMP-P(NH)P and then washed free of nucleotides. It is concluded that occupancy of the guanyl nucleotide binding site that activates the catalytic moiety of the system is not sufficient to promote hormone-receptor coupling to adenylyl cyclase and that occupancy of a second site by guanyl nucleotides is essential to effect stimulation of adenylyl cyclase by the glucagon-receptor complex. The data presented raise the question whether the guanyl nucleotide site that promotes coupling is distinct from the guanyl nucleotide site that modulates binding of glucagon to receptor and whether the occupancy of the guanyl nucleotide site associated with the catalytic moiety is necessary for coupling.

Adenylyl Cyclases↗
PubMed1979

LH-induced desensitization of the adenylyl cyclase system in ovarian follicles.

Studies on the gonadotrophin-responsive adenylyl cyclase (AC) system of rabbit and porcine ovarian follicles reveal that hCG or LH-induced desensitization of the AC system can be divided into two phases: an initial, LH-specific phase and a second phase which is not specific for LH. The first phase occurs within the first hour after LH-hCG-receptor interaction, is agonist specific, and is not mediated by protein synthetic events or by cAMP. In view of our previous demonstration of the critical dependence of the LH-induced desensitizing process in cell-free membrane preparations of porcine follicles upon Mg2+ and ATP, we investigated the role of a phosphorylation reaction in the first phase of the AC desensitizing process. Porcine follicular membranes rich in LH-sensitive AC activity were found to contain the molecular requirements necessary for a phosphorylation reaction: namely, cAMP-dependent and cAMP-independent protein kinases as well as phosphoprotein phosphatases. The following lines of indirect evidence indicated that reversal or resensitization of the desenzitized AC system to LH was mediated by a dephosphorylation reaction. Activators of endogenous phosphoprotein phosphatases--Mn2+ and dithiothreitol--promoted a specific resensitization of the follicular AC system to LH. Likewise, a partially purified phosphoprotein phosphatase also resensitized the desensitized, LH unresponsive AC to LH, and boiling of the phosphatase prevented its effect. LH-induced desensitization of the AC system, on the other hand, did not appear to be mediated by a cAMP-dependent protein kinase, as evidenced both by the inability of beef heart protein to promote desensitization of AC and by the inability of an inhibitor of cAMP-dependent protein kinase to prevent LH-induced densensitization. The second phase of desensitization, which occurs after the first hour following hCG-LH-receptor interaction, is characterized by a loss of responsiveness to FSH as well as to LH and can be promoted by dibutryl cAMP (in the absence of LH). These results provide new evidence on the characteristics and molecular mechanism of LH-induced densensitization of the follicular AC system. These results indicate that the level of phosphorylation of membrane-associated components may, in part, regulate the activity of the AC system during this first phase of homologous desensitization.

Adenylyl Cyclases↗