Restricting the Mobility of Gsα: Impact on Receptor and Effector Coupling

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Abstract
The α-subunit of the stimulatory G protein, Gs, 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 Gsα dissociation, we constructed a membrane-tethered Gsα (tetGsα), expressed it in Sf9 insect cells, and examined its ability to couple with the β2 adrenoceptor and to activate adenylyl cyclase. Compared to wild-type Gsα, tetGsα coupled much more efficiently to the β2 adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTPγS binding and GTPase activity. The high coupling efficiency was abolished when Gsα was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetGSα to adenylyl cyclase. These results demonstrate that regulating the mobility of Gsα 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, GTPγS binding), tetGSα can facilitate the study for receptor−G protein interactions.