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 limitation hindering identification of GPRs as GRK substrates has been the necessity to use purified and reconstituted receptors in GRK assays. Here, the human m2 and human m3 (hm3) 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 beta-adrenergic receptor kinase (beta ARK)1 phosphorylated the membrane-bound human 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 hm3 mAChRs. The membrane-bound hm3 mAChRs were phosphorylated by beta ARK1 in an agonist-dependent, G beta gamma-enhanced manner. This is the first demonstration that hm3 mAChRs can serve as substrates for GRKs. The stoichiometry of receptor phosphorylation was approximately 2 mol of phosphate/mol of receptors in the absence of G beta gamma and approximately 4 mol of phosphate/mol of receptors upon addition of G beta gamma. When 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 phosphorylated the hm3 mAChRs under similar conditions. The approach of studying GRK-mediated phosphorylation of GPRs in their membrane-bound state identified the hm3 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.
Debburman
S K
SK
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611.
Kunapuli
P
P
Benovic
J L
JL
Hosey
M M
MM
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D018721:Muscarinic Agonists / Q000494:pharmacology*; D010766:Phosphorylation; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera; D013379:Substrate Specificity; D051552:beta-Adrenergic Receptor Kinases
D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins