ABSTRACT
Cells use heterotrimeric G proteins to differentially modulate the speed and throughput of various signaling pathways by coupling one or more class of G proteins to different receptors and effectors. Receptor is quantified by radioligand binding assay using [3H]-quinuclidinyl benzylate methyl chloride. Receptor-bound radioligand is separated from free radioligand and then measured by scintillation counting. Nonspecific binding is determined by addition of a high concentration of atropine. Receptor and G protein are co-reconstituted into lipid vesicles by gel filtering a mixture of phospholipid-detergent micelles and purified proteins. Receptor is eluted with atropine, and chromatographed through a hydroxyapatite column which is subsequently eluted with potassium phosphate in step-gradient manner. Addition of effector to the reconstituted system increases the experimental complexity somewhat, but provides crucial, and otherwise unattainable, mechanistic information about receptor-G protein-effector signaling. The assay measures a major biochemical event that occurs after receptor-promoted guanosine diphosphate / guanosine triphosphate exchange on the G protein a subunit.
