title: Clathrin required for phosphorylation and internalization of beta2-adrenergic receptor by G protein-coupled receptor kinase 2 (GRK2).
abstract: Clathrin is a major component of clathrin-coated pits and serves as a binding scaffold for endocytic machinery through the binding of a specific sequence known as the clathrin-binding motif. This motif is also found in cellular signaling proteins other than endocytic components, including G protein-coupled receptor kinase 2 (GRK2), which phosphorylates G protein-coupled receptors and promotes uncoupling of receptor-G protein interaction. However, the functions of clathrin in the regulation of GRK2 are unknown. Here we demonstrated that overexpression of GRK2 mutated at the clathrin-binding motif with alanine (GRK2-5A) results in inhibition of phosphorylation and internalization of the beta2-adrenergic receptor (beta2AR). However, the interaction of beta2AR with GRK2-5A is the same as that of wild type GRK2 as determined by bioluminescence resonance energy transfer. Furthermore, GRK2-5A phosphorylates rhodopsin essentially to the same extent as wild type GRK2 in vitro. Depletion of the clathrin heavy chain using small interference RNA inhibits agonist-induced phosphorylation and internalization of beta2AR. Thus, clathrin works as a regulator of GRK2 in cells. These results indicate that clathrin is a novel player in cellular functions in addition to being a component of endocytosis.
authors.affiliation: Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka 812-8582, Japan.
authors.forename: Supachoke
authors.initials: S
authors.lastname: Mangmool
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Haga
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Kyeong-Man
authors.initials: KM
authors.lastname: Kim
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Nakata
authors.forename: Motohiro
authors.initials: M
authors.lastname: Nishida
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kurose
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002966:Clathrin / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D004705:Endocytosis; D054769:G-Protein-Coupled Receptor Kinase 2; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D051552:beta-Adrenergic Receptor Kinases / Q000378:metabolism*
chemical_list: D002966:Clathrin; D018343:Receptors, Adrenergic, beta-2; C496567:GRK2 protein, human; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2