Concerted stimulation and deactivation of pertussis toxin-sensitive G proteins by chimeric G protein-coupled receptor-regulator of G protein signaling 4 fusion proteins: analysis of the contribution of palmitoylated cysteine residues to the GAP activity of RGS4

Agonists stimulated high-affinity GTPase activity in membranes of HEK293 cells following coexpression of the alpha(2A) -adrenoceptor and a pertussis toxin-resistant mutant of G(o1) alpha. Enzyme kinetic analysis of V (max) and K (m) failed to detect regulation of the effect of agonist by a GTPase activating protein. This did occur, however, when cells were also transfected to express RGS4. Both elements of a fusion protein in which the N-terminus of RGS4 was linked to the C-terminal tail of the alpha(2A) -adrenoceptor were functional, as it was able to provide concerted stimulation and deactivation of the G protein. By contrast, the alpha(2A) -adrenoceptor-RGS4 fusion protein stimulated but did not enhance deactivation of a form of G(o1) alpha that is resistant to the effects of regulator of G protein signaling (RGS) proteins. Employing this model system, mutation of Asn128 but not Asn88 eliminated detectable GTPase activating protein activity of RGS4 against G(o1) alpha. Mutation of all three cysteine residues that are sites of post-translational acylation in RGS4 also eliminated GTPase activating protein activity but this was not achieved by less concerted mutation of these sites. These studies demonstrate that a fusion protein between a G protein-coupled receptor and an RGS protein is fully functional in providing both enhanced guanine nucleotide exchange and GTP hydrolysis of a coexpressed G protein. They also provide a direct means to assess, in mammalian cells, the effects of mutation of the RGS protein on function in circumstances in which the spatial relationship and orientation of the RGS to its target G protein is defined and maintained.