title: Sequestration of muscarinic acetylcholine receptor m2 subtypes. Facilitation by G protein-coupled receptor kinase (GRK2) and attenuation by a dominant-negative mutant of GRK2.
abstract: Sequestration of m2 receptors (muscarinic acetylcholine receptor m2 subtypes), which was assessed as loss of N-[3H]methylscopolamine ([3H]NMS) binding activity from the cell surface, was examined in COS 7 and BHK-21 cells that had been transfected with expression vectors encoding the m2 receptor and, independently, vectors encoding a G protein-coupled receptor kinase (GRK2) (beta-adrenergic receptor kinase 1) or a GRK2 dominant-negative mutant (DN-GRK2). The sequestration of m2 receptors became apparent when the cells were treated with 10(-5) M or higher concentrations of carbamylcholine. In this case, approximately 40% or 20-25% of the [3H]NMS binding sites on COS 7 or BHK-21 cells, respectively, were sequestered with a half-life of 15-25 min. In cells in which GRK2 was also expressed, the sequestration became apparent in the presence of 10(-7) M carbamylcholine. Approximately 40% of the [3H]NMS binding sites on both COS 7 and BHK-21 cells were sequestered in the presence of 10(-6) M or higher concentrations of carbamylcholine. When DN-GRK2 was expressed in COS 7 cells, the proportion of [3H]NMS binding sites sequestered in the presence of 10(-5) M or higher concentrations of carbamylcholine was reduced to 20-30%. These results indicate that the phosphorylation of m2 receptors by GRK2 facilitates their sequestration. These results are in contrast with the absence of a correlation between sequestration and the phosphorylation of beta-adrenergic receptors by the GRK2 and suggests that the consequences of phosphorylation by GRK2 are different for different receptors.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Tsuga
authors.forename: K
authors.initials: K
authors.lastname: Kameyama
authors.forename: T
authors.initials: T
authors.lastname: Haga
authors.forename: H
authors.initials: H
authors.lastname: Kurose
authors.forename: T
authors.initials: T
authors.lastname: Nagao
mesh_terms: D000818:Animals; D001483:Base Sequence; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005799:Genes, Dominant; D008647:Mesocricetus; D008969:Molecular Sequence Data; D009154:Mutation; D019832:N-Methylscopolamine; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D012602:Scopolamine Derivatives / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D017931:DNA Primers; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D002217:Carbachol; D020794:Receptor Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins; D019832:N-Methylscopolamine