title: G Protein-Coupled Receptor Kinase 2 (GRK2) and 5 (GRK5) Exhibit Selective Phosphorylation of the Neurotensin Receptor in Vitro.
abstract: G protein-coupled receptor kinases (GRKs) play an important role in the desensitization of G protein-mediated signaling of G protein-coupled receptors (GPCRs). The level of interest in mapping their phosphorylation sites has increased because recent studies suggest that the differential pattern of receptor phosphorylation has distinct biological consequences. In vitro phosphorylation experiments using well-controlled systems are useful for deciphering the complexity of these physiological reactions and understanding the targeted event. Here, we report on the phosphorylation of the class A GPCR neurotensin receptor 1 (NTSR1) by GRKs under defined experimental conditions afforded by nanodisc technology. Phosphorylation of NTSR1 by GRK2 was agonist-dependent, whereas phosphorylation by GRK5 occurred in an activation-independent manner. In addition, the negatively charged lipids in the immediate vicinity of NTSR1 directly affect phosphorylation by GRKs. Identification of phosphorylation sites in agonist-activated NTSR1 revealed that GRK2 and GRK5 target different residues located on the intracellular receptor elements. GRK2 phosphorylates only the C-terminal Ser residues, whereas GRK5 phosphorylates Ser and Thr residues located in intracellular loop 3 and the C-terminus. Interestingly, phosphorylation assays using a series of NTSR1 mutants show that GRK2 does not require acidic residues upstream of the phospho-acceptors for site-specific phosphorylation, in contrast to the β2-adrenergic and μ-opioid receptors. Differential phosphorylation of GPCRs by GRKs is thought to encode a particular signaling outcome, and our in vitro study revealed NTSR1 differential phosphorylation by GRK2 and GRK5.
authors.affiliation: †Membrane Protein Structure Function Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Department of Health and Human Services, Rockville, Maryland 20852, United States.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Inagaki
authors.affiliation: ‡Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892, United States.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ghirlando
authors.affiliation: §Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Vishnivetskiy
authors.affiliation: ∥Departments of Pharmacology and Biological Sciences, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: Kristoff T
authors.initials: KT
authors.lastname: Homan
authors.affiliation: †Membrane Protein Structure Function Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Department of Health and Human Services, Rockville, Maryland 20852, United States.
authors.forename: Jim F
authors.initials: JF
authors.lastname: White
authors.affiliation: ∥Departments of Pharmacology and Biological Sciences, University of Michigan, Ann Arbor, Michigan 48109, United States.
authors.forename: John J G
authors.initials: JJ
authors.lastname: Tesmer
authors.affiliation: §Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, United States.
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Gurevich
authors.affiliation: †Membrane Protein Structure Function Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Department of Health and Human Services, Rockville, Maryland 20852, United States.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Grisshammer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000378:metabolism*; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D051381:Rats; D018028:Receptors, Neurotensin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D018028:Receptors, Neurotensin; C114036:neurotensin type 1 receptor; D054769:G-Protein-Coupled Receptor Kinase 2; D054774:G-Protein-Coupled Receptor Kinase 5