Restricting the mobility of Gsα:: Impact on receptor and effector coupling

The or-subunit of the stimulatory G protein, G(s), has been shown to dissociate from the plasma membrane into the cytosol following activation by G protein-coupled receptors (GPCR) in some experimental systems. This dissociation may involve depalmitoylation of an amino-terminal cysteine residue. However, the functional significance of this dissociation is not known. To investigate the functional consequence of G(s)alpha dissociation, we constructed a membrane-tethered G(s)alpha (tetG(s)alpha), expressed it in Sf9 insect cells, and examined its ability to couple with the beta(2) adrenoceptor and to activate adenylyl cyclase. Compared to wild-type G(s)alpha, tetG(s)alpha coupled much more efficiently to the beta(2) adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTP gamma S binding and GTPase activity. The high coupling efficiency was abolished when G(s)alpha was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetG(s)alpha to adenylyl cyclase. These results demonstrate that regulating the mobility G(s)alpha relative to the plasma membrane, through fatty acylation or perhaps interactions with cytoskeletal proteins, could have a significant impact on receptor-G protein coupling. Furthermore, by enabling the use of more direct measures of receptor-G protein coupling (GTPase activity, GTP gamma S binding), tetG(s)alpha can facilitate the study for receptor-G protein interactions.