Restricting mobility of Gsα relative to the β2-adrenoceptor enhances adenylate cyclase activity by reducing Gsα GTPase activity

The beta(2)-adrenoceptor (beta(2)AR) activates the G-protein G(s)alpha to stimulate adenylate cyclase (AC). Fusion of the beta(2)AR C-terminus to the N-terminus of G(s)alpha (producing beta(2)ARG(s)alpha) markedly increases the efficiency of receptor/G-protein coupling compared with the non-fused state. This increase in coupling efficiency can be attributed to the physical proximity of receptor and G-protein. To determine the optimal length for the tether between receptor and G-protein we constructed fusion proteins from which 26 [beta(2)AR(Delta 26)G(s)alpha] or 70 [beta(2)AR(Delta 70)G(s)alpha] residues of the beta(2)AR C-terminus had been deleted and compared the properties of these fusion proteins with the previously described beta(2)ARG(s)alpha. Compared with beta(2)ARG(s)alpha, basal and agonist-stimulated GTP hydrolysis was markedly decreased in beta(2)AR(Delta 70)G(s)alpha, whereas the effect of the deletion on binding of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was relatively small. Surprisingly, deletions did not alter the efficiency of coupling of the beta(2)AR to G(s)alpha as assessed by GTP[S]-sensitive high-affinity agonist binding. Moreover, basal and ligand-regulated AC activities in membranes expressing beta(2)AR(Delta 70)G(s)alpha and beta(2)AR(Delta 26)G(s)alpha were higher than in membranes expressing beta(2)ARG(s)alpha. These findings suggest that restricting the mobility of G(s)alpha relative to the beta(2)AR results in a decrease in G-protein inactivation by CTP hydrolysis and thereby enhanced activation of AC.