Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01275 (glucagon)
 26,492 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Active glucagon receptor was solubilized with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (Chaps) from rat liver plasma membranes but rapidly (less than 8 h) lost activity. Either inclusion of 1X Hanks' balanced salt solution in the 3 mM Chaps solubilization buffer or its addition after solubilization increased the percentage of total binding attributable to specific glucagon binding from approximately 10 to greater than 80%; of great importance, it increased the stability from near zero binding at 8 h to 50% binding at 48 h (4 degrees C). Of the Hanks' solution components, either NaCl (137 mM) or CaCl2 (1.26 mM) was effective in increasing specific binding to approximately 70 and 60% respectively: Mg salts were ineffective. Soluble receptor binding activity was assayed by dextran-coated charcoal adsorption of free hormone. The assay is rapid, simple, and reproducible. It is suitable for monitoring receptor activity during purification and molecular characterization. Competition binding studies gave an IC50 value of 10-20 nM (slope factor approximately 1), with or without GTP. Dissociation assays revealed GTP sensitivity when receptors were solubilized either as glucagon-receptor complexes or free receptor. Active glucagon-receptor complexes could be eluted from wheat germ lectin-agarose: neither concanavalin A-agarose nor soybean agglutinin-agarose bind receptor. A glucagon degrading activity which co-solubilized with the receptor but did not require detergent for extraction was distinguishable from the soluble receptor not only by solubility but also by its heat stability (30 degrees C), its inhibition by bacitracin, its affinity for glucagon, its retention of activity for at least 1 week at 4 degrees C, and its size.
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PMID:Stabilization of soluble active rat liver glucagon receptor. 254 42

In order to clarify the potential roles of glucagon for energy induction during the perinatal period, the developmental changes of serum glucagon concentration, the ontogenesis of hepatic glucagon receptor and glucagon-sensitive adenylate cyclase system were estimated in comparison with insulin in rats. In fetal rats on day 18 to 20 gestation, serum glucose levels and hepatic glycogen contents gradually increased as pregnancy progressed. The levels of serum glucagon and its binding to liver microsomal membranes were significantly lower than in adults, and glucagon-sensitive c-AMP production was also markedly suppressed. On day 21 of gestation, hepatic glycogen contents markedly increased to the maximal level, serum glucagon level increased, and glucagon receptors in the fetal liver were already estimated at the same level as in the adult. However, glucagon-sensitive c-AMP production was still suppressed the same as in the fetuses of earlier gestational ages. On the other hand, serum insulin levels in fetuses were higher than those in adults, and the abundant receptor could also be observed in liver microsomal membranes. After delivery, serum glucose rapidly decreased with a marked decline of hepatic glycogen contents until 5 hours in the neonatal period. Serum glucagon and its hepatic receptors were significantly increased with a gradual development of the glucagon-sensitive adenylate cyclase system. Conversely, serum insulin levels were suppressed without any remarkable change in its receptor. From these results, it is suggested that glucagon plays an important role in the neonatal adaptation mechanism, especially in the production of endogenous glucose in place of the transplacental supply from the mother, such effects of glucagon are already initiated by the induction of its receptor in target tissues in the fetus on late gestation.
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PMID:[Studies on the fetal and neonatal secretion of glucagon and the development of glucagon receptors]. 255 4

The stimulation of adenylate cyclase by glucagon and isoproterenol in cell lysates of hepatocytes isolated from fetal and adult female rats were measured after pretreatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Glucagon stimulation of adenylate cyclase activation was found to decrease in the hepatocyte lysate of adult female rats. Application of guanyl-5'-yl-imidodiphosphate (GppNHp) and forskolin showed this effect to be localized on the receptor level. However, glucagon stimulation of glucose liberation from phorbol ester-treated hepatocytes from adult female rats was not influenced. Maximum effects of glucose liberation were observed at glucagon concentrations which did not stimulate adenylate cyclase. The results are in agreement with the proposed existence of a low and high affinity glucagon receptor coupled to two different transducing systems. It is concluded that TPA uncouples in the liver of adult female rats--most likely by phosphorylation--the low affinity receptor from the adenylate cyclase system, whereas the high affinity receptor and phospholipase C/inositol 1,4,5-trisphosphate (IP3)/diacylglycerol (DAG) signalling systems do not seem to be affected. Such TPA effects could not be found in the liver of fetal rats.
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PMID:Age-dependent effects of phorbol ester on adenylate cyclase stimulation by glucagon in liver of female rats. 275 35

The present study was designed to determine if Golgi fractions from fetal rat liver contain glucagon receptors and to characterize the properties of such receptors. Purification patterns of liver plasma membranes and Golgi fractions from fetal and adult rats were similar, as verified by morphological and biochemical approaches. Glucagon binding was greater in plasma membranes of adult than fetal rats, while in Golgi fractions glucagon binding was similar in both groups. The modifications in in glucagon binding reflect changes in glucagon receptors. Glucagon association and glucagon receptor inactivation by liver membranes were similar in the two groups of animals, while glucagon degradation was lower in fetal than in adult rats.
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PMID:Characterization of glucagon receptors in Golgi fractions of fetal rat liver. 282 Aug 4

To investigate whether guanine nucleotides regulate interconversion of the two-state hepatic glucagon receptor we have utilized kinetic assays of glucagon binding to partially purified rat liver plasma membranes. Dissociation of glucagon at 30 degrees C exhibited biexponential character in either the absence or presence of GTP, indicating that the system previously seen in intact hepatocytes is independent of intracellular modulators. In each case the receptors underwent a time-dependent conversion from a low affinity to a high affinity state. However, GTP decreased the fraction of receptors in the high affinity state. The rank order for stabilizing the low affinity state was Gpp(NH)p greater than GTP greater than GDP much greater than GMP = no nucleotides. Data from competition binding assays with increasing concentrations of GTP allow calculation of equilibrium constants which are 3.32 nM for glucagon and receptor in the absence of GTP, 18.6 nM for glucagon and receptor in the presence of GTP, 1.55 microM for the association of receptor and GTP presumably linked to an N protein, and 8.86 microM for the association of the glucagon-receptor complex and GTP again presumably linked to an N protein, Glucagon binding to receptor is noncooperative in both the absence and presence of GTP, distinguishing this system from the beta-adrenergic system. With GTP, binding to the low affinity state is favored because of the relative affinities reported. Therefore, GTP regulates the activation by slowing the conversion of the receptor from a low affinity to high affinity form.
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PMID:Guanine nucleotide regulation of the interconversion of the two-state hepatic glucagon receptor system of rat. 283 9

We have studied, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, glucagon-receptor complexes that arise from the incubation of canine hepatic plasma membranes with [[125I]iodo-Tyr10]glucagon. While a 54,000-dalton membrane protein was tentatively identified as the glucagon receptor by chemical cross-linking, an additional component having an apparent molecular weight of 30,000 was routinely identified as also resulting from glucagon-receptor interactions. The latter material, however, was not observed when gels were fixed prior to autoradiography and was not affected by the addition of cross-linking agents to membrane incubations. Subsequent analysis actually identified the material as a fragment of radiolabeled glucagon that contains at least residues 1-13, has no ability of its own to associate with plasma membranes, and remains tightly membrane bound once it has been formed by receptor-mediated processes. Formation of the fragment was inhibited by phenylmethylsulfonyl fluoride, glucagon, and GTP, but not by N-ethylmaleimide or by a variety of glucagon-related peptides. Overall, our results identify a proteolytic modification of glucagon this is linked to the binding of ligand to high affinity GTP-dependent receptors and the existence of a physically distinct state of receptor in which the binding site is tightly filled by a ligand fragment.
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PMID:Receptor-linked proteolysis of membrane-bound glucagon yields a membrane-associated hormone fragment. 283 24

Chick hepatocytes in primary culture have been used to study the homologous and heterologous pathways of glucagon-induced desensitization of adenylyl cyclase. Scatchard analysis and guanine nucleotide effects on dissociation kinetics indicate that the initial phase of homologous desensitization, an increase in low affinity glucagon receptors due to the rapid uncoupling of the receptor from Gs, is essentially complete within 5 min. These receptors recouple within 20 min upon removal of glucagon. Upon prolonged (2 h or more) exposure of hepatocytes to glucagon, disappearance of low affinity receptors from cell surface membranes constitutes the second phase of homologous desensitization. Recovery of these lost and presumably internalized receptors requires more than 12 h following the removal of glucagon but is not dependent on new protein synthesis. The heterologous phase of desensitization is slower, requiring 20-30 min of glucagon treatment to reach completion. Stimulation of adenylyl cyclase by hormonal and nonhormonal effectors is similarly reduced, indicating a common defect in this desensitized state. Agonist occupancy of other hormone receptors coupled to adenylyl cyclase in hepatocytes, such as beta-adrenergic, prostaglandin E1, and vasoactive intestinal peptide, results in heterologous desensitization. Heterologous desensitization is rapidly reversed (within 30 min) upon partial removal of glucagon, under conditions allowing the maintenance of the homologously desensitized state. Neither onset of nor recovery from heterologous desensitization requires protein synthesis. These data indicate that homologous and heterologous desensitization occurs by independent mechanisms. Homologous desensitization involves uncoupling of the glucagon receptor from Gs, followed by removal of these uncoupled receptors from the cell surface. Heterologous desensitization represents a second level of cellular control of hormonal responsiveness to be turned on when the cell is subjected to prolonged hormonal stimulation and withdrawn when hormone levels are lowered.
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PMID:Glucagon-induced desensitization of adenylyl cyclase in primary cultures of chick hepatocytes. Evidence for multiple pathways. 284 35

Previous studies have identified the association of the hepatic glucagon receptor with a membrane-bound protease which cleaves [[125I]iodo-Tyr10]glucagon in a hormone- and GTP-sensitive manner (Sheetz, M. J., and Tager, H. S. (1988) J. Biol. Chem. 263, 8509-8514). The current investigations were undertaken to characterize the receptor-linked protease. Treatment of canine hepatic membranes with buffers containing Lubrol and NaCl, followed by gel filtration of soluble material on Sepharose CL-6B and subsequent fractionation of proteins by use of (NH4)2SO4, resulted in a 50-fold purification of the enzyme. Kinetic analysis of the solubilized protease under conditions of linear initial rate (by use of glucagon as substrate and high performance liquid chromatography to separate and quantitate the products) revealed its minimal dependency on pH between values of 7 and 9, its relative preference for glucagon as substrate, and its sensitivity to the presence of salt. Initial rates and the ratio Vmax/Km typically increased 5- to 7-fold and 10-fold, respectively, in the presence of 1 M NaCl. Identification by amino acid analysis of peptide fragments resulting from the incubation of glucagon with the partially purified enzyme and analysis of related time courses for hormone processing demonstrated that the glucagon Tyr13-Leu14 peptide bond is the primary site for proteolytic cleavage by the receptor-linked protease. The implications of these and related findings are discussed.
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PMID:Characterization of a glucagon receptor-linked protease from canine hepatic plasma membranes. Partial purification, kinetic analysis, and determination of sites for hormone processing. 284 17

125I-labeled glucagon was directly crosslinked to its receptor in isolated liver plasma membranes and on the surface of intact hepatocytes, by using a UV irradiation procedure. This investigation resulted in the identification of a glucagon-receptor complex of apparent Mr 62,000. The specificity of labeling was shown by the interference of unlabeled hormone at physiological concentration with incorporation of radioactive glucagon into the 62,000 Mr species. The receptor behaved as a typical integral membrane protein: it was not released by extraction with lithium diiodosalicylate or at basic pH but was solubilized by digitonin treatment. Reduction of the receptor polypeptide with dithiothreitol resulted in a decrease in its electrophoretic mobility, suggesting the presence of intramolecular disulfide bonds. Soluble glucagon-receptor complexes adsorbed to Con A-Sepharose and could be eluted with methyl alpha-D-mannoside, indicating that the receptor molecule is a glycoprotein. Treatment of glucagon-labeled liver plasma membrane with endoglycosidase F resulted in the appearance of four intermediate species, indicating that glucagon receptor contains at least four N-linked oligosaccharide chains.
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PMID:Direct cross-linking of 125I-labeled glucagon to its membrane receptor by UV irradiation. 298 52

The glucagon receptor and the adenylyl cyclase system of human liver membranes were studied in six non-obese and six obese subjects who had elevated insulin and plasma glucagon levels. Analysis of specific glucagon binding by the method of Scatchard demonstrated a linear (monocomponent) plot with a dissociation constant of 2-3 nM, and the binding at low hormone concentrations was sensitive to guanosine triphosphate (GTP). The molecular weight of the glucagon receptor was 63,000 D as determined by an affinity labeling procedure and sodium dodecyl sulfate gel electrophoresis. Affinity labeling of this structure was specific for glucagon and inhibited by GTP. Glucagon stimulated the production of cyclic adenosine monophosphate (cAMP) by human membranes with half-maximal activation elicited by 6 nM hormone. The human cyclase system required GTP to facilitate an optimal glucagon response. NaF (10 mM) also activated the cyclase system and produced the same magnitude of response as maximum glucagon activation. A comparison of the liver adenylyl cyclase system of non-obese and obese subjects was made using glucagon (5 nM and 1 microM) and NaF (10 mM). No significant differences in cAMP production were noted between the two groups, regardless of the agent used to activate the enzyme. These findings agree with the glucagon binding studies that showed similar amounts of binding activity in the membranes from the two groups. Also, there was no influence of either age or sex of the subjects on the adenylyl cyclase response. In conclusion, human liver membranes contain a glucagon receptor and an adenylyl cyclase system that correspond closely to the well-studied system in animal liver. This system in human obesity is not altered by the approximately twofold elevation in plasma glucagon that occurs in this metabolic disorder.
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PMID:Glucagon receptor of human liver. Studies of its molecular weight and binding properties, and its ability to activate hepatic adenylyl cyclase of non-obese and obese subjects. 298 13


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