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)

[125I-Tyr10]Monoiodoglucagon [( 125I]MIG) was cross-linked to liver membrane glucagon receptors with hydroxysuccinimidyl-p-azidobenzoate, and the products were analyzed by sodium dodecyl sulfate-gel electrophoresis. Autoradiograms of the gel obtained after a 24-h exposure showed one major band at Mr = 63,000 that was sensitive to GTP and excess unlabeled glucagon. Exposure for 7 days showed labeling of an additional Mr = 33,000 species that was also sensitive to excess unlabeled glucagon. The Mr = 33,000 peptide can be obtained by subtilisin, trypsin, elastase, or Staphylococcus aureus V8 protease treatment of the [125I]MIG-occupied receptor in the membrane or in Lubrol-PX solution. In contrast, limited proteolysis of membranes containing vacant receptors results in labeling of a Mr = 24,000 peptide. The Mr = 24,000 peptide specifically binds [125I]MIG in a GTP-sensitive manner. The Mr = 33,000 peptide also retains GTP sensitivity since it releases bound [125I]MIG upon addition of GTP. Elastase treatment of the electroeluted Mr = 33,000 peptide yields the Mr = 24,000 and 15,000 fragments. The Mr = 15,000 peptide is the smallest fragment of the receptor as yet identified. Treatment of the Mr = 63,000 receptor with [125I]MIG cross-linked to it with endo-beta-N-acetylglucosaminidase F results in four distinct fragments with Mr values of 61,000, 56,000, 51,000, and 45,000; prolonged treatment resulted in the accumulation of the last two. Neither the Mr = 33,000 nor the Mr = 24,000 fragment appeared to be substrates for endo-beta-N-acetylglucosaminidase F. These data indicate that glucagon receptor is a glycoprotein of approximately 60,000 daltons which contains at least four N-linked glycans accounting for 18,000 daltons of its mass. Both its glucagon binding function and its capacity to interact with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only approximately 21,000 daltons that does not contain any N-linked glycans. Hormone occupancy of the receptor results in a conformational change so as to expose a region that is susceptible to proteolysis by proteases of varying specificities to yield a peptide of approximately 30,000 daltons that also does not contain N-linked glycans.
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PMID:Structural analysis of the hepatic glucagon receptor. Identification of a guanine nucleotide-sensitive hormone-binding region. 632 24

The preparation, purification and characterization of N epsilon-4- azidophenylamidinoglucagon are described. This photoreactive peptide was found to be 50% as potent as native glucagon in competing with 125I-labeled glucagon for binding to glucagon receptors on rat liver plasma membranes. Similarly, the analog was 50% as potent as native glucagon in its ability to stimulate adenylate cyclase. The photoreactive glucagon analog was radioiodinated to high specific activity with iodine-125 and was used to label rat liver plasma membrane proteins. Analysis of labeled membrane proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed covalent incorporation predominantly into a protein of relative molecular mass, Mr, of 50 000-60 000. Occasionally a protein of Mr 170 000-180 000 was also labeled. Irradiation of membranes in the presence of unlabeled glucagon or GTP selectively inhibited the labeling of the 50 000-60 000-Mr protein(s). As a result of these studies we suggest that the sodium-dodecyl-sulfate-dissociated glucagon receptor is a 50 000-60 000-Mr protein.
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PMID:Photoaffinity labeling of the glucagon receptor with a new glucagon analog. 632 11

When compared to normal liver membranes, purified plasma membranes of regenerating liver and Morris hepatomas contain low but variable capacities to bind glucagon. This property is inversely related to the capacity of the isolated hepatocytes to bind to heterologous biomatrix glycoproteins. Since these parameters are characteristic of the proliferative state of the cells, it was important to further study the glucagon receptor protein and stimulation of adenylate cyclase activity. Our results show that 125I-iodinated plasma membranes obtained from normal liver contain three molecular species (117000, 98000, 86000 molecular weight) that can be eluted specifically with glucagon from a sepharose-glucagon affinity column. These proteins contain the putative glucagon receptor since binding of 125I- iodoglucagon is increased 150-fold as compared to unfractionated membranes. Plasma membranes obtained from Morris hepatoma (7800) and liver of chemically hepatectomized rats do not bind glucagon and lack these proteins. After inactivation with N-ethylmaleimide of the adenylate cyclase activity of the normal plasma membranes, they were fused with membrane of the hepatoma. The hybrid membranes showed 60% recovery of glucagon-stimulated cyclase activity. These results suggest that the plasma membranes of the proliferating liver cells do not contain receptor protein but have intact regulatory and catalytic subunits of the adenylate cyclase system.
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PMID:Lack of glucagon receptors in Morris hepatoma 7800. 632 89

During treatment of primary cultured hepatocytes with either glucagon or isoproterenol for several hours, the stimulations of cAMP formation by these hormones decreased time dependently. Glucagon treatment also reduced the response to isoproterenol, but isoproterenol treatment did not decrease the response to glucagon. Treatment with isoproterenol caused more rapid desensitization than treatment with glucagon. Assays of glucagon and beta-adrenergic receptors showed that the receptor number of only the hormone with which the cells were treated decreased and that dissociation constants of the receptors did not change. Moreover, in glucagon-desensitized cells, the effect of GTP on competition of the bindings of antagonist and agonist for the beta-adrenergic receptor did not change. After treatment with isoproterenol, stimulation of adenylate cyclase activity by the agonist was decreased without any decrease in the stimulations of activity by other effectors. In contrast, glucagon treatment greatly decreased the stimulations of activity by glucagon, isoproterenol, and guanyl-5'-yl imidodiphosphate and slightly decreased that by fluoride. However, after glucagon treatment, the cells showed normal responses to cholera toxin of activation of adenylate cyclase and ADP-ribosylation of guanine nucleotide binding regulatory protein (Ns). The maximal response of glucagon-treated cells to forskolin was about two-thirds that of untreated cells and this treatment also impaired the shift toward a low Kact value for forskolin observed in the presence of either glucagon or isoproterenol. These results indicate that isoproterenol caused homologous desensitization consisting of only a "down regulation" of the beta-adrenergic receptor, whereas glucagon caused heterologous desensitization, mainly by alteration of the Ns component, as well as "down regulation" of glucagon receptor. This altered Ns seems to be coupled normally to the beta-adrenergic receptor, but to have impaired coupling to the catalytic component of adenylate cyclase.
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PMID:Mechanism of heterologous desensitization of the adenylate cyclase system by glucagon in primary cultures of adult rat hepatocytes. 633 78

To gain insight into the mechanisms responsible for the impaired glycogenolytic response to glucagon and the diminished ketogenic capacity of newborn guinea pig, we studied the ontogeny of insulin and glucagon receptors, and the responsiveness of the adenylate cyclase complex to glucagon, PGE1, NaF, and cholera toxin in liver plasma membrane from fetal (58 d, late gestation, and 65 d, term) and adult guinea pigs. The number of insulin receptors (x 10(-10) M/L) was least in 58-d fetus (3.0 +/- 0.4; mean +/- SEM) and increased 3-fold in 65 d fetus (8.8 +/- 0.6; P less than 0.01). In adult guinea pig, both insulin receptor number (6.0 +/- 0.7) and average affinity constant (1.20 +/- 0.08 x 10(8) M-1) were significantly lower (P less than 0.01) compared with 65-d fetus. The number of glucagon receptors remained unchanged between 58-d and 65-d fetuses, but both average and high affinity association constants were significantly higher at d 65. In contrast to the lower capacity and affinity of insulin receptors in the adult compared with term fetus, the total glucagon receptor number (x 10(-10) M/L) in adults (7.2 +/- 0.8) was twice that of the 58 d (3.2 +/- 0.2) and 65 d (3.2 +/- 1.0) fetuses. The average affinity constant (x 10(8) M-1) in adult (3.8 +/- 0.2) was, however, significantly lower than the two fetal groups (58 d, 5.0 +/- 0.3; P less than 0.05 and 65 d, 8.1 +/- 1.0; P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ontogeny of insulin and glucagon receptors and the adenylate cyclase system in guinea pig liver. 633 Jun 58

Synthetic bombesin (100 ng/min) was infused under pentobarbital anesthesia in normal dogs and in insulin deprived depancreatized dogs 4 days after surgery. The release of gut glucagon-like immunoreactive materials (gut GLI) calculated as the difference between the values measured using cross-reacting antiserum and a so-called pancreatic glucagon specific antiserum was markedly stimulated by bombesin infusion. Plasma glucagon immunoreactivity (GI) measured by pancreatic glucagon specific antiserum also showed a small increase, whereas plasma glucose decreased significantly with a transient rise in insulin. The plasma glucose level did not decrease in depancreatized dogs. Gut GLI response in the regional mesenteric vein to 5% glucose administered into the loop of the ileum was strongly augmented by bombesin infusion. It is concluded that (1) bombesin infusion decreased blood glucose level in normal dogs but not in depancreatized dogs. (2) Bombesin infusion markedly augmented the release of GLI from the intestine. (3) Bombesin also stimulated the release of glucagon which was probably of gastrointestinal origin. (4) Insulin release was stimulated transiently by bombesin infusion. Thus, a competition of gut GLI with glucagon at the glucagon receptor site may be an explanation of the reduction in blood glucose.
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PMID:Decrease in blood glucose and release of gut glucagon-like immunoreactive materials by bombesin infusion in the dog. 701 1

The synthesis of the heterobifunctional cross-linking reagent 2-nitro-4-azidophenylsulfenyl chloride (NAPSCl) is described. This reagent can be used to specifically attach a photoactivatable nitrophenyl azide to tryptophan-containing polypeptides and proteins lacking sulfhydryl groups. The sulfenyl chloride group of NAPSCl reacts with the indole ring of tryptophan following second-order reaction kinetics in 50-100% acetic acid. The labeled product can be effectively photolyzed at wavelengths above 300 nm. The reaction of glucagon, a peptide hormone containing a single tryptophan residue at position 25 and no cysteine, with NAPSCl gave one major product, the photosensitive derivative glucagon-NAPS. The structure and properties of the purified derivative were established by amino acid analysis, absorption spectroscopy, and photolysis. Only the tryptophan residue of this derivative was modified. The photosensitive glucagon was shown to activate the adenylate cyclase of hepatocyte plasma membranes to the same extent as the native hormone at equimolar concentrations. Glucagon-NAPS could be radiolabeled by the lactoperoxidase-catalyzed iodination of the peptide. A glucagon-specific antibody bound both radiolabeled glucagon and glucagon-NAPS peptides. The covalent labeling of protein molecules with radiolabeled glucagon-NAPS peptide upon photolysis was demonstrated. Glucagon-NAPS can be used as an effective photoaffinity probe for labeling the glucagon receptor site in plasma membranes of target cells.
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PMID:Synthesis and characterization of a heterobifunctional photoaffinity reagent for modification of tryptophan residues and its application to the preparation of a photoreactive glucagon derivative. 742 13

The synthesis of 2-thioltryptophan-glucagon is described. Oxidation of this compound gives the dimer (Trp-S-glucagon)2. Both monomer and dimer are equi-potent on a molar basis with native glucagon as activators of adenylate cyclase in hepatic plasma membranes. However, from the ability to compete with 125I-glucagon for binding at the glucagon receptor, the dimer has one-fourth the binding affinity for the receptor as does native glucagon, 2-thiol-Trp-glucagon, and Trp-(2,4-dinitrophenylsulfenyl)-glucagon which have equal affinities for the receptor. Addition of GTP, which converts the receptor from a tight-binding to a lower affinity form and which activates adenylate cyclase in the presence of glucagon, allows (Trp-S-glucagon)2 to bind equally with native glucagon and the other thiol derivatives of the hormone. This effect of GTP on the binding of the glucagon dimer and the uses of 2-thiol-Trp-glucagon in the semisynthesis of new glucagon derivatives are discussed.
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PMID:Preparation of 2-thioltryptophan-glucagon and (tryptophan-S-glucagon)2. Differences in binding to the glucagon receptor in the hepatic adenylate cyclase system. 743 Jan 60

Glucagon receptor mutants were characterized with the aim of elucidating minimal structural requirements for proper biosynthesis, ligand binding, and adenylyl cyclase coupling. One N-terminal deletion mutant and five truncation mutants with progressively shorter C termini were expressed in transiently transfected monkey kidney (COS-1) cells. Each truncation mutant was designed so that the truncated C-terminal tail would remain on the cytoplasmic surface of the receptor. In order to characterize the cellular location of the expressed receptor mutants, a highly specific, high affinity antipeptide antibody was prepared against the extracellular, N-terminal tail of the receptor. Immunoblot analysis and immunofluorescence microscopy showed that the presence of all seven putative transmembrane segments, but not not an intact N-terminal tail, was required for cell surface expression of the receptor. Membranes from cells expressing receptor mutants lacking a large portion of the N-terminal tail or any of the seven putative transmembrane segments failed to bind glucagon. Membranes from cells expressing the C-terminal tail truncation mutants, which retained all seven transmembrane segments, bound glucagon with affinities similar to that of the native receptor and activated cellular adenylyl cyclase in response to glucagon. These results indicate that all seven helices are necessary for the proper folding and processing of the glucagon receptor. Glycosylation is not required for the receptor to reach the cell surface, and it may not be required for ligand binding. However, the N-terminal extracellular portion of the receptor is required for ligand binding. Most of the distal C-terminal tail is not necessary for ligand binding, and the absence of the tail may increase slightly the receptor binding affinity for glucagon. The C-terminal tail is also not necessary for adenylyl cyclase coupling and therefore does not play a direct role in G protein (GS) activation by the glucagon receptor.
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PMID:Characterization of deletion and truncation mutants of the rat glucagon receptor. Seven transmembrane segments are necessary for receptor transport to the plasma membrane and glucagon binding. 749 39

A human glucagon receptor has been cloned from human liver tissue. The 1578-bp cDNA clone encodes a protein of 477 amino acids with 82% identity to the rat glucagon receptor. The predicted secondary structure and homology to known proteins places this receptor within the superfamily of seven transmembrane domain G protein coupled receptors. Transfection of the human glucagon receptor into COS-7 cells confers upon them high affinity binding for [125I] glucagon. In membranes prepared from COS-7 cells transfected with the human glucagon receptor, the binding of [125I] glucagon is inhibited with the rank order of potency glucagon > oxyntomodulin > glucagon-like peptide 1 (7-36) amide >> glucagon-like peptide 2 = gastric inhibitory peptide = secretin.
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PMID:Cloning and expression of a human glucagon receptor. 750 21


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