ALTERATIONS IN RECEPTOR ACTIVATION AND DIVALENT-CATION ACTIVATION OF AGONIST BINDING BY DELETION OF INTRACELLULAR DOMAINS OF THE GLUCAGON RECEPTOR

Deletion of residues 252-259 within the putative second intracellular loop of the human glucagon receptor results in a protein with high aff inity for glucagon but with attenuated agonist activation of adenylyl cyclase, The Delta 252-259 mutant has 4-fold higher affinity for gluca gon than does the wild type receptor, The nonhydrolyzable GTP analog, guanosine 5'-(beta,gamma-imido)triphosphate (Gpp(NH)p), inhibits bindi ng of I-125-glucagon to the wild type receptor but not to the Delta 25 2-259 mutant, Divalent cations such as MgCl2 and CaCl2 stimulate the b inding of I-125-glucagon to the wild type receptor by increasing gluca gon affinity, The rate of dissociation of I-125-glucagon is decreased 4-fold by MgCl2 and increased 6-fold by Gpp(NH)p. However, divalent ca tions do not affect the binding of I-125-glucagon to the Delta 252-259 mutant, The rate of dissociation of I-125-glucagon from the Delta 252 -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 I-125-glucagon. Dele tion of residues 252-259 appears to lock the protein in the conformati on promoted by divalent cations and prevents the protein from normal c oupling to Gs.