Selective Stabilization of the High Affinity Binding Conformation of Glucagon Receptor by the Long Splice Variant of Gαs

To analyze functional differences in the interactions of the glucagon receptor (GR) with the two predominant splice variants of G alpha(s), GR was covalently linked to the short and the long forms G alpha(s)-S and G alpha(s)-L to produce the fusion proteins GR-G alpha(s)-S and GR-G alpha-L. GR-G alpha(s)-S bound glucagon with an affinity similar to that of GR, while GR-G alpha(s)-L showed a 10-fold higher affinity for glucagon. In the presence of GTP gamma S, GR-G alpha(s)-L reverted to the low affinity glucagon binding conformation. Both GR-G alpha(s)-L and GR-G alpha(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 G alpha(s)-L. G23D1R-G alpha(s)-L bound glucagon with high affinity, but failed to elevate cAMP levels, suggesting that the mechanisms of GR-mediated G alpha(s)-L activation and G alpha(s)-L-induced high affinity glucagon binding are independent. Both GR-G alpha(s)-S and GR-G alpha(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-G alpha(s)-L, but not with GR-G alpha(s)-S, Therefore, the partial agonist activity of the antagonist observed in intact cells appears to be due to GRs coupled to G alpha(s)-L. We conclude that G alpha(s)-S and G alpha(s)-L interact differently with GR and that specific coupling of GR to G alpha(s)-L may account for GTP-sensitive high affinity glucagon binding.