title: Alterations in receptor activation and divalent cation activation of agonist binding by deletion of intracellular domains of the glucagon receptor.
abstract: Deletion of residues 252-259 within the putative second intracellular loop of the human glucagon receptor results in a protein with high affinity for glucagon but with attenuated agonist activation of adenylyl cyclase. The Delta252-259 mutant has 4-fold higher affinity for glucagon than does the wild type receptor. The nonhydrolyzable GTP analog, guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p), inhibits binding of 125I-glucagon to the wild type receptor but not to the Delta252-259 mutant. Divalent cations such as MgCl2 and CaCl2 stimulate the binding of 125I-glucagon to the wild type receptor by increasing glucagon affinity. The rate of dissociation of 125I-glucagon is decreased 4-fold by MgCl2 and increased 6-fold by Gpp(NH)p. However, divalent cations do not affect the binding of 125I-glucagon to the Delta252-259 mutant. The rate of dissociation of 125I-glucagon from the Delta252-259 mutant protein is equivalent to the rate of dissociation from the wild type receptor in the presence of MgCl2. These data suggest that at least three conformations of the glucagon receptor can exist in the membrane based on their differing affinities for 125I-glucagon. Deletion of residues 252-259 appears to lock the protein in the conformation promoted by divalent cations and prevents the protein from normal coupling to Gs.
authors.affiliation: Department of Molecular Pharmacology and Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
authors.forename: G G
authors.initials: GG
authors.lastname: Chicchi
authors.forename: M P
authors.initials: MP
authors.lastname: Graziano
authors.forename: G
authors.initials: G
authors.lastname: Koch
authors.forename: P
authors.initials: P
authors.lastname: Hey
authors.forename: K
authors.initials: K
authors.lastname: Sullivan
authors.forename: P P
authors.initials: PP
authors.lastname: Vicario
authors.forename: M A
authors.initials: MA
authors.lastname: Cascieri
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002413:Cations, Divalent; D002462:Cell Membrane / Q000378:metabolism; D005934:Glucagon / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011869:Radioligand Assay; D018027:Receptors, Glucagon / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D002413:Cations, Divalent; D018027:Receptors, Glucagon; D011994:Recombinant Proteins; D005934:Glucagon