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.
Krumins
Andrejs M
AM
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
Gilman
Alfred G
AG
D000818:Animals; D001665:Binding Sites; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D010761:Phosphorus Radioisotopes; D021122:Protein Subunits; D020710:RGS Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000502:physiology; D013462:Sulfur Radioisotopes
D020690:GTPase-Activating Proteins; D008081:Liposomes; D010761:Phosphorus Radioisotopes; D021122:Protein Subunits; D020710:RGS Proteins; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D013462:Sulfur Radioisotopes; C098799:RGS4 protein; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D020962:Heterotrimeric GTP-Binding Proteins