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J Biol Chem, Vol. 273, Issue 9, 5109-5116, February 27, 1998

Different Effects of G_s Splice Variants on ₂-Adrenoreceptor-mediated Signaling
THE ₂-ADRENORECEPTOR COUPLED TO THE LONG SPLICE VARIANT OF G_s HAS PROPERTIES OF A CONSTITUTIVELY ACTIVE RECEPTOR

Roland Seifert, Katharina Wenzel-Seifert, Tae Weon Lee, Ulrik Gether, Elaine Sanders-Bush, and Brian K. Kobilka^**

From the  Howard Hughes Medical Institute, ^** Division of Cardiovascular Medicine, Stanford University Medical School, Stanford, California 94305-5428

The ₂-adrenoreceptor (₂AR) couples to the G-protein G_s to mediate adenylyl cyclase activation. The splice variants of G_s differ by a 15-amino acid insert between the Ras-like domain and the -helical domain. The long splice variant of G_s (G_sL) binds GDP with lower affinity than the short splice variant (G_sS), but the impact of this difference on the interaction of G_s with the ₂AR is not known. We studied the ₂AR/G_s interaction using receptor/G-protein fusion proteins (₂ARG_sS and ₂ARG_sL) expressed in Sf9 cells. Fusion of the ₂AR to G_s promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the ₂AR or G_s. The ₂AR in ₂ARG_sL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the ₂AR in ₂ARG_sS. The apparent constitutive activity of the ₂AR in ₂ARG_sL may be due to the lower GDP affinity of G_sL compared with G_sS, i.e. G_sL is more often nucleotide-free than G_sS and, therefore, more frequently available to stabilize the ₂AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein -subunits can have important consequences for the functional properties of a G-protein-coupled receptor.

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