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Different Effects of Gsα Splice Variants on β2-Adrenoreceptor-mediated Signaling

The β2-adrenoreceptor (β2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gsα differ by a 15-amino acid insert between the Ras-like domain and the α-helical domain. The long splice variant of Gsα (GsαL) binds GDP with lower affinity than the short splice variant (GsαS), but the impact of this difference on the interaction of Gsα with the β2AR is not known. We studied the β2AR/Gsα interaction using receptor/G-protein fusion proteins (β2ARGsαS and β2ARGsαL) expressed in Sf9 cells. Fusion of the β2AR to Gsα 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 β2AR or Gsα. The β2AR in β2ARGsαL 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 β2AR in β2ARGsαS. The apparent constitutive activity of the β2AR in β2ARGsαL may be due to the lower GDP affinity of GsαL compared with GsαS, i.e.GsαL is more often nucleotide-free than GsαS and, therefore, more frequently available to stabilize the β2AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein α-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.