DIFFERENT EFFECTS OF G(S)ALPHA SPLICE VARIANTS ON BETA(2)-ADRENORECEPTOR-MEDIATED SIGNALING - THE BETA(2)-ADRENORECEPTOR COUPLED TO THE LONG SPLICE VARIANT OF G(S)ALPHA HAS PROPERTIES OF A CONSTITUTIVELY ACTIVE RECEPTOR

The beta(2)-adrenoreceptor (beta(2)AR) couples to the G-protein G(s) t o mediate adenylyl cyclase activation. The splice variants of G(s) alp ha differ by a 15-amino acid insert between the Ras-like domain and th e alpha-helical domain. The long splice variant of G(s) alpha (G(s) al pha(L)) binds GDP with lower affinity than the short splice variant (G (s) alpha(S)), but the impact of this difference on the interaction of G,cu with the beta(2)AR is not known. We studied the beta(2)AR/G(s) a lpha interaction using receptor/G-protein fusion proteins (beta(2)ARG( s) alpha(S) and beta(2)ARG(s) alpha(L)) expressed in Sf9 cells. Fusion of the beta(2)AR to G(s) alpha promotes efficient coupling as shown b y high-affinity agonist binding and GTPase and adenylyl cyclase activa tion and ensures fixed stoichiometry between receptor and G-protein. I mportantly, fusion does not change the fundamental properties of the b eta(2)AR or G(s) alpha. The beta(2)AR in beta(2)ARG(s) alpha(L) showed hallmarks of constitutive activity (increased potency and intrinsic a ctivity of partial agonists, increased efficacy of inverse agonists, a nd increased basal GTPase activity) compared with the beta(2)AR in bet a(2)ARG(s) alpha(S). The apparent constitutive activity of the beta(2) AR in beta(2)ARG(s) alpha(L) may be due to the lower GDP affinity of G (s) alpha(L) compared with G(s) alpha(S), i.e. G(s) alpha(L) is more o ften nucleotide-free than G(s) alpha(S) and, therefore, more frequentl y available to stabilize the beta(2)AR in the active (R) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for t he functional properties of a G-protein-coupled receptor.