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Originally published In Press as doi:10.1074/jbc.M002093200 on May 1, 2000

J. Biol. Chem., Vol. 275, Issue 28, 21631-21638, July 14, 2000
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Selective Stabilization of the High Affinity Binding Conformation of Glucagon Receptor by the Long Splice Variant of Galpha s*

Cecilia G. UnsonDagger §, Cui-Rong WuDagger , Thomas P. SakmarDagger , and R. B. MerrifieldDagger

From the Dagger  Rockefeller University and the  Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, Rockefeller University, New York, New York 10021

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 GTPgamma S, 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 Gs (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 desHis1[Nle9,Ala11,Ala16]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.

* This work was supported by National Institutes of Health Research Grant R01-DK24039.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence may be addressed: Box 294, Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8239; Fax: 212-327-8245; E-mail: unsonc@rockvax.rockefeller.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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