The glucagon receptor of rat liver plasma membrane can couple to adenylate cyclase without activating it (original) (raw)
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The Journal of biological chemistry, 1981
The ability of several chemically modified forms of glucagon to activate adenylate cyclase have been compared with their ability to displace 125I-glucagon from specific membrane binding sites. The results demonstrate that both NH2-terminal and COOH-terminal portions of the peptide, as well as the central region of the glucagon molecule, are all involved in receptor binding and subsequent activation of adenylate cyclase. Receptor binding was very sensitive to chemical modification of the polar residues of glucagon. For example, conversion of the sole lysine residue of glucagon to homoarginine resulted in over a 2-fold loss in receptor-binding affinity. Loss in ability to activate adenylate cyclase was at least as great as loss in receptor binding for all of the derivatives. In the case of derivatives modified at the COOH terminus, the loss in ability to activate adenylate cyclase correlated well with loss in receptor binding. In general, however, the loss of the ability to activate a...
Biochemistry, 1986
In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [ 3-M e-H i~' , A r g '~]-, [Phe',ArgI2]-, [~-A l a~, A r g " ]-, and [~-P h e~] g l u c a g o n , to compete with '251-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, [ 3-Me-Hi~',Arg'~]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe',ArgI2] glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [~-A l a~, A r g '~] g l u c a g o n and [~-P h e~] g l u c a g o n , analogues designed to examine the possible importance of a @-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [~-Ala~,Arg'*]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [~-P h e~] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal @-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists. The partial agonism of [3-Me-His',Arg12]-, [Phe1,Arg'2]-, and [~-A l a~, A r g '~] g l u c a g o n for adenylate cyclase activity in isolated liver plasma membranes observed in this study is not modulated by changes in the guanosine triphosphate (GTP) concentration. In addition, the receptor binding dose-response curve for [Phe',Arg12]glucagon is shifted to the right in the presence of G T P to the same extent as that seen with the native hormone. Thus, the partial agonism demonstrated by these three analogues in this study is not due to a lack of modulation by G T P of the receptor binding and adenylate cyclase activities measured on liver plasma membranes. The in vivo degradation rates for glucagon and [~-P h e~] g l u c a g o n , half-lives of 5.3 and 7.5 min, respectively, were determined in this study. This slightly slower rate of degradation for [~-Phe~]glucagon is not sufficient to account for its highly potent glycogenolytic activity seen in vivo. The lack of correlation between the in vitro adenylate cyclase and the in vivo glucose release activities for these compounds is discussed.
Journal of Supramolecular Structure, 1980
The cationic local anaesthetics carbocaine and unpercaine were found to increase the fluoride-stimulated adenylate cyclase up to a maximum level; above this maximum level further increases in drug concentration inhibited the enzyme. At concentrations where this activity was stimulated, a fatty acid spin label detected an increase in bilayer fluidity, which, it is suggested, is responsible for the activation of the enzyme. A solubilized enzyme was unaffected by the drugs, a finding consistent with this proposal.These cationic drugs began to inhibit the glucagon-stimulated activity at concentrations where they activated the fluoride-stimulated activity. It is suggested that this is due to their effect on the coupling interaction between the receptor and catalytic unit.The anionic drugs, phenobarbital, pentobarbital, and salicylic acid, all inhibited the fluoride-stimulated enzyme. This may be due in part to a direct effect on the protein and in part to the interaction of the drugs with the bilayer. The drugs had small inhibitory effects on the lubrol-solubilized enzyme.The glucagon-stimulated enzyme was initially inhibited by the anionic drugs at low concentrations, then activated, and finally inhibited with increasing drug concentration. The reasons for such changes are complex, but there was no evidence from electron spin resonance studies to suggest that the elevations in activity were due to increases in bilayer fluidity.
Inhibition by glucagon of the calcium pump in liver plasma membranes
The Journal of biological chemistry, 1984
The ATP-dependent calcium transport in plasma membrane vesicles prepared from rat liver was inhibited by 0.1 to 10 microM glucagon. Inhibition of the high affinity (Ca2+-Mg2+)-ATPase was observed concomitantly. This effect was neither mimicked by cyclic AMP nor by dibutyryl cyclic AMP. A study of the structure-activity relationships of six glucagon derivatives demonstrated the specificity of glucagon action since only one or two analogs markedly altered the (Ca2+-Mg2+)-ATPase activity. The study also demonstrated the total absence of correlation between adenylate cyclase activation and (Ca2+-Mg2+)-ATPase inhibition induced by these glucagon derivatives. The decrease in the maximal velocities induced by glucagon of both calcium transport and (Ca2+-Mg2+)-ATPase activity were related to a reduction in the rate of dephosphorylation of the Ca-dependent phosphorylated intermediate of the enzyme. This phosphorylated intermediate was characterized as a 32P-labeled 110,000-dalton protein whi...
N-Ethylmaleimide uncouples the glucagon receptor from the regulatory component of adenylyl cyclase
Biochemistry, 1986
125I-Glucagon binding to rat liver plasma membranes was composed of high- and low-affinity components. N-Ethylmaleimide (NEM) and several other alkylating agents induced a dose-dependent loss of high-affinity sites. This diminished the apparent affinity of glucagon receptors for hormone without decreasing the binding capacity of membranes. Solubilized hormone-receptor complexes were fractionated as high molecular weight (Kav = 0.16) and low molecular weight (Kav = 0.46) species by gel filtration chromatography; NEM or guanosine 5'-triphosphate (GTP) diminished the fraction of high molecular weight complexes, suggesting that NEM uncouples glucagon receptor-N-protein complexes. Exposure of intact hepatocytes to the impermeable alkylating reagent p-(chloromercuri)benzenesulfonic acid failed to diminish the affinity of glucagon receptors on subsequently isolated plasma membranes, indicating that the thiol that affects receptor affinity is on the cytoplasmic side of the membrane. Hormone binding to plasma membranes was altered by NEM even after receptors were uncoupled from N proteins by GTP. These data suggest that a sensitive thiol group that affects hormone binding resides in the glucagon receptor, which may be a transmembrane protein. Alkylated membranes were fused with wild-type or cyc- S49 lymphoma cells to determine how alkylation affects the various components of the glucagon-adenylyl cyclase system. Stimulation of adenylyl cyclase with fluoride, guanylyl 5'-imidodiphosphate, glucagon, or isoproterenol was observed after fusion of cyc- S49 cells [which lack the stimulatory, guanine nucleotide binding, regulatory protein of adenylyl cyclase (Ns)] with liver membranes alkylated with 1.5 mM NEM.(ABSTRACT TRUNCATED AT 250 WORDS)
Glucagon and epinephrine stimulation of adenyl cyclase in isolated rat liver plasma membranes
Biochemical and Biophysical Research Communications, 1969
Adenyl cyclase has been shown to be specifically localized in the plasma membrane of rat liver. The activity of this enzyme is stimulated by glucagon and epinephrine in isolated plasma membrane systems. The stimulation by epinephrine has a lag period of about 10 minutes. Epinephrine shows selective binding to isolated plasma membranes. The results indicate that epinephrine binds to a receptor protein rather than interacting directly with the enzyme adenyl cyclase. Calcium ions also stimulate adenyl cyclase in isolated plasma membranes but the effect shows appreciable variability. The biological significance of cyclic AMP (adenosine-3',5'-monophosphate)
Peptides, 1986
Comparative efficacy of seven synthetic glucagon analogs, modified in position I, 2 and~or 12. on liver and heart adenylate cyclasefrom rat. PEPTIDES 7: Suppl. 1, [109][110][111][112] 1986.--Crude fresh membranes from rat liver and membranes from rat heart obtained according to Snyder and Drummond were tested for adenylate cyclase activation by glucagon (Gn) and seven glucagon analogs including (Ala2)-, (Ar#2)-, (Des-His 1, Arg12), (Phe l, Argl2)-, (N-Ac-His ~, ArgO2)-, (1-Me-His ~, ArgO2)-, and (3-Me-His l, Arg12)-glucagon. (Des-His ~, Arg~Z)-glucagon acted as a competitive antagonist in heart membranes and as a partial agonist in liver membranes. Results obtained with analogs where His ~ was modified suggest that the size of the imidazole ring and the charge of its nitrogen 1, but not the charge of the free amino group of histidine, played a major role in biological activity. When comparing functional glucagon receptors in liver and heart membranes, it appears that the first receptors were more sensitive to the hormone and more efficiently coupled to adenylate cyclase.
Biochimica et Biophysica Acta (BBA) - General Subjects, 1974
The polypeptide hormone glucagon can react with the nucleophiles; glycinamide, taurine or ethylenediamine in the presence of 1-ethyl-3-(3-dimethylaminopropylcarbodiimide). The number of carboxyl groups which are modified depend on the concentration of guanidine hydrochloride in the reaction media. These results demonstrate an additional property which glucagon possesses in common with larger globular proteins and suggests that the hormone has a specific, folded structure in dilute aqueous solution. In the absence of guanidine hydrochloride only one taurine residue is incorporated into the terminal carboxyl group of the peptide. In 7 M guanidine hydrochloride all four of the carboxyl groups react with glycinamide or taurine while only two and a half residues of ethylenediamine are incorporated. All of these derivatives and glucagon have identical circular dichroism spectra in dilute aqueous solution. The taurine modified derivative has greatly enhanced solubility compared with glucagon but still associates to structures of higher helical content. Both of the taurine derivatives of glucagon have the ability to stimulate the adenyl cyclase of rat liver membranes but at concentrations several fold higher than is needed for the native hormone. It is suggested that each carboxyl group contributes to the binding of the hormone to the specific membrane receptor sites. * This work was presented in part at the Biochemistry/Biophysics 1974 Meeting. Minneapolis.
Biochemical Journal, 1986
The effects of guanosine 5′-[beta-thio]diphosphate (GDP[S]) on the kinetics of activation of rat liver membrane adenylate cyclase by guanosine 5′-[beta, gamma-imido]triphosphate (p[NH]ppG) were examined. GDP[S] caused immediate inhibition of the activation by p[NH]ppG at all time points tested. Substantial inhibition by GDP[S] was observed even after the time required for the enzyme to reach its steady-state activity, but the extent of inhibition became progressively smaller as the preincubation time with p[NH]ppG increased. The rate at which adenylate cyclase became quasi-irreversibly activated was a strictly first-order process. In the presence of glucagon, the formation of the irreversibly activated state was much slower. A combination of GDP[S] and glucagon could partially reverse the quasi-irreversible activation by p[NH]ppG. Glucagon decreased the lag time required for p[NH]ppG to activate adenylate cyclase and increased the extent of activation by p[NH]ppG. This stimulatory e...
Biochemistry, 1982
The concentration-dependent stimulation of adenylate cyclase by the photoaffinity reagent 2-[(2-nitro-4azidophenyl)sulfenyl]-Trp25-glucagon (glucagon-NAPS) and also its binding characteristics were compared with those of the native hormone. The derivative was found to be slightly more potent in stimulating adenylate cyclase than glucagon, in the presence of guanosine 5'-triphosphate (GTP). 1251-Labeled glucagon-NAPS or 1251-labeled glucagon bound rapidly to receptors and was competitively displaced by unlabeled glucagon or glucagon-NAPS. Glucagon-NAPS displaced bound radiolabeled hormone at a lower concentration than did glucagon in the absence of GTP. Scatchard analysis of the binding data obtained from displacement of bound From the Department of Biochemistry,