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

The R-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 GsR dissociation, we constructed a membrane-tethered GsR (tetGsR), expressed it in Sf9 insect cells, and examined its ability to couple with the â2 adrenoceptor and to activate adenylyl cyclase. Compared to wild-type GsR, tetGsR coupled much more efficiently to the â2 adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTPγS binding and GTPase activity. The high coupling efficiency was abolished when GsR was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetGSR to adenylyl cyclase. These results demonstrate that regulating the mobility of GsR 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), tetGSR can facilitate the study for receptor-G protein interactions. Heterotrimeric guanine nucleotide-binding proteins (G proteins) are the signal transducers which relay information from membrane-bound receptors to effector systems. Receptor activation stimulates exchange of GDP for GTP on the R-subunit, which then dissociates from the receptor and the âγ dimer and interacts with effectors. The R-subunit remains active until the bound GTP is hydrolyzed to GDP by the intrinsic GTPase activity of the R-subunit (1-3). G protein R-subunits have a variety of posttranslational lipid modifications (4). GiR, GoR, and GzR are myristoylated by amide linkage at their N-terminal glycine residues (5, 6), and they are also palmitoylated at distinct sites (7). GsR and GqR are palmitoylated by a thioester bond at N-terminal cysteine residues (8, 9). These R-subunit modificaitons may be important for membrane attachment or interactions with âγ subunits or effector molecules (10, 11). Prenylation of the γ subunit is also known to play a role in membrane anchorage of the âγ dimer (12). Activation of the adenylyl cyclase activating G protein R subunit, GsR, by the nonhydrolyzable GTP analogues Gpp[NH]p and GTPγS, or stimulation via receptors induces palmitate turnover and the release of GsR-subunit from membrane to cytosol (11, 1317). It has been suggested that a cycle of acylation and deacylation could be a mechanism for regulating the activity and membrane association of G protein R-subunits (18, 19); however, the functional consequence of R-subunit translocation from the plasma membrane has not been determined. Other investigators have found that activation of G protein R subunits causes them to concentrate in subdomains of the plasma membrane but not to be released from the membrane