title: Selective stabilization of the high affinity binding conformation of glucagon receptor by the long splice variant of Galpha(s).
abstract: To analyze functional differences in the interactions of the glucagon receptor (GR) with the two predominant splice variants of Galpha(s), GR was covalently linked to the short and the long forms Galpha(s)-S and Galpha(s)-L to produce the fusion proteins GR-Galpha(s)-S and GR-Galpha(s)-L. GR-Galpha(s)-S bound glucagon with an affinity similar to that of GR, while GR-Galpha(s)-L showed a 10-fold higher affinity for glucagon. In the presence of GTPgammaS, GR-Galpha(s)-L reverted to the low affinity glucagon binding conformation. Both GR-Galpha(s)-L and GR-Galpha(s)-S were constitutively active, causing elevated basal levels of cAMP even in the absence of glucagon. A mutant GR that failed to activate G(s) (G23D1R) was fused to Galpha(s)-L. G23D1R-Galpha(s)-L bound glucagon with high affinity, but failed to elevate cAMP levels, suggesting that the mechanisms of GR-mediated Galpha(s)-L activation and Galpha(s)-L-induced high affinity glucagon binding are independent. Both GR-Galpha(s)-S and GR-Galpha(s)-L bound the antagonist desHis(1)[Nle(9),Ala(11),Ala(16)]glucagon amide with affinities similar to GR. The antagonist displayed partial agonist activity with GR-Galpha(s)-L, but not with GR-Galpha(s)-S. Therefore, the partial agonist activity of the antagonist observed in intact cells appears to be due to GRs coupled to Galpha(s)-L. We conclude that Galpha(s)-S and Galpha(s)-L interact differently with GR and that specific coupling of GR to Galpha(s)-L may account for GTP-sensitive high affinity glucagon binding.
authors.affiliation: Rockefeller University and the Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, Rockefeller University, New York, New York 10021, USA. unsonc@rockvax.rockefeller.edu
authors.forename: C G
authors.initials: CG
authors.lastname: Unson
authors.forename: C R
authors.initials: CR
authors.lastname: Wu
authors.forename: T P
authors.initials: TP
authors.lastname: Sakmar
authors.forename: R B
authors.initials: RB
authors.lastname: Merrifield
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D017398:Alternative Splicing; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics* / Q000378:metabolism*; D014644:Genetic Variation; D005934:Glucagon / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D051381:Rats; D018027:Receptors, Glucagon / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: D018027:Receptors, Glucagon; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005934:Glucagon; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases