AGONIST-DEPENDENT PHOSPHORYLATION OF HUMAN MUSCARINIC RECEPTORS IN SPODOPTERA-FRUGIPERDA INSECT-CELL MEMBRANES BY G-PROTEIN-COUPLED RECEPTOR KINASES

Agonist-dependent phosphorylation of G protein-coupled receptors (GPRs ) by G protein-coupled receptor kinases (GRKs) is proposed to be a key event initiating homologous receptor desensitization. A technical lim itation hindering identification of GPRs as GRK substrates has been th e necessity to use purified and reconstituted receptors in GRK assays. Here, the human m2 and human m3 (hm3) muscarinic cholinergic receptor s (mAChRs), which couple to attenuation of adenylyl cyclase and stimul ation of phospholipase C, respectively, were expressed in Spodoptera f rugiperda insect cells and an in vitro approach to studying GPR phosph orylation by GRKs in crude membranes was developed. The m2 mAChR, a kn own substrate of certain GRKs, was used to validate the approach. The GRK isoform beta-adrenergic receptor kinase (beta ARK)1 phosphorylated the membrane-bound human m2 mAChRs in an agonist-dependent manner. Th e results demonstrated that endogenous membrane-bound beta gamma subun its of G proteins stimulated the phosphorylation of the membrane-bound m2 mAChR, To reveal new GRK substrates, we tested the expressed hm3 m AChRs. The membrane-bound hm3 mAChRs were phosphorylated by beta ARK1 in an agonist-dependent, G(beta gamma)-enhanced manner. This is the fi rst demonstration that hm3 mAChRs can serve as substrates for GRKs. Th e stoichiometry of receptor phosphorylation was similar to 2 mol of ph osphate/mol of receptors in the absence of G beta gamma, and similar t o 4 mol of phosphate/mol of receptors upon addition of G beta gamma. W hen the specificity of various GRKs towards mAChRs was assessed, beta ARK2 phosphorylated the agonist-activated hm3 mAChRs as efficiently as did beta ARK1; however, neither GRK5 nor GRK6 significantly phosphory lated the hm3 mAChRs under similar conditions. The approach of studyin g GRK-mediated phosphorylation of GPRs in their membrane-bound state i dentified the hm3 mAChRs as new substrates for GRKs. This approach sho uld be valuable in identifying other new substrates of GRKs and should aid in studies that elucidate GRK/GPR pairing.