Agonist-dependent phosphorylation of XXXX muscarinic receptors in Spodoptera frugiperda insect cell membranes by G protein-coupled receptor kinases.

@entity1 @entity47643 @entity437 @entity47645 @entity1391 @entity47644 @entity16237
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 limitation hindering identification of GPRs as GRK substrates has been the necessity to use purified and reconstituted receptors in GRK assays. Here, the @entity1 m2 and @entity1 m3 ( @entity16237 ) muscarinic cholinergic receptors (mAChRs), which couple to attenuation of adenylyl cyclase and stimulation of phospholipase C, respectively, were expressed in Spodoptera frugiperda insect cells and an in vitro approach to studying GPR phosphorylation by GRKs in crude membranes was developed. The m2 mAChR, a known substrate of certain GRKs, was used to validate the approach. The GRK isoform @entity437 phosphorylated the membrane-bound @entity1 m2 mAChRs in an agonist-dependent manner. The results demonstrated that endogenous membrane-bound beta gamma subunits of G proteins stimulated the phosphorylation of the membrane-bound m2 mAChR. To reveal new GRK substrates, we tested the expressed @entity16237 mAChRs. The membrane-bound @entity16237 mAChRs were phosphorylated by @entity437 in an agonist-dependent, G beta gamma-enhanced manner. This is the first demonstration that @entity16237 mAChRs can serve as substrates for GRKs. The stoichiometry of receptor phosphorylation was approximately 2 mol of @entity1391 /mol of receptors in the absence of G beta gamma and approximately 4 mol of @entity1391 /mol of receptors upon addition of G beta gamma. When the specificity of various GRKs towards mAChRs was assessed, @entity47643 phosphorylated the agonist-activated @entity16237 mAChRs as efficiently as did @entity437 ; however, neither @entity47644 nor @entity47645 significantly phosphorylated the @entity16237 mAChRs under similar conditions. The approach of studying GRK-mediated phosphorylation of GPRs in their membrane-bound state identified the @entity16237 mAChRs as new substrates for GRKs. This approach should be valuable in identifying other new substrates of GRKs and should aid in studies that elucidate GRK/GPR pairing.
