Assay of RGS protein activity in vitro using purified components.

Single-turnover and steady-state GTPase assays are an effective means to identify and characterize interactions between RGS and G alpha proteins in vitro. The advantage of the single turnover GTPase assay is that it permits simple and rapid assessment of RGS protein activity toward a putative G alpha-GTP substrate. Moreover, once an interaction between an RGS protein and a G alpha-GTP subunit has been identified, the single-turnover assay can be used to determine Michaelis-Menten constants and/or KI values for other competing G alpha substrates. A disadvantage of the single-turnover assay is that a negative result does not preclude the possibility of an interaction between given RGS and G alpha proteins in vivo. Inappropriate reaction conditions or the presence (or absence) of appropriate posttranslational modifications may result in small or undetectable increases in RGS protein-dependent GTPase activity. In these cases it may be tempting to examine RGS protein activity using steady-state GTPase assays in phospholipid vesicles reconstituted with receptors and heterotrimetric G proteins. The advantage to monitoring steady-state GTPase activity in reconstituted proteoliposomes is that ligand-dependent activation of the receptor facilitates GDP dissociation, such that effects of RGS proteins can be observed; multiple cycles of GTP binding and hydrolysis then amplify the GTPase signal. Additionally, the presence of the phospholipid membrane can increase the local RGS protein concentration approximately 10(4)-fold, permitting observation of interactions that are weak in solution. The primary disadvantage of the reconstituted system is the requirement for receptor purification, a technically demanding undertaking in comparison to the purification of G alpha, G beta gamma, and most RGS proteins.