title: Phenotypic regulation of the sphingosine 1-phosphate receptor miles apart by G protein-coupled receptor kinase 2.
abstract: The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and β-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m(93) (mil(m93)), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that β-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling.
authors.affiliation: Institute for Biochemistry and Molecular Biology, Ulm University , 89081 Ulm, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Burczyk
authors.forename: Martin D
authors.initials: MD
authors.lastname: Burkhalter
authors.forename: Tamara
authors.initials: T
authors.lastname: Blätte
authors.forename: Sabrina
authors.initials: S
authors.lastname: Matysik
authors.forename: Marc G
authors.initials: MG
authors.lastname: Caron
authors.forename: Lawrence S
authors.initials: LS
authors.lastname: Barak
authors.forename: Melanie
authors.initials: M
authors.lastname: Philipp
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006321:Heart / Q000196:embryology; D006330:Heart Defects, Congenital / Q000378:metabolism / Q000473:pathology; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D049349:Receptors, Lysosphingolipid / Q000378:metabolism*; D000090742:Serine-Threonine Kinase 3; D015398:Signal Transduction; D000081025:Sphingosine-1-Phosphate Receptors; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000378:metabolism*; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000099317:Hippo Kinases
chemical_list: D019390:Arrestins; D054769:G-Protein-Coupled Receptor Kinase 2; D019204:GTP-Binding Proteins; D017346:Protein Serine-Threonine Kinases; D049349:Receptors, Lysosphingolipid; D000090742:Serine-Threonine Kinase 3; D000081025:Sphingosine-1-Phosphate Receptors; D029961:Zebrafish Proteins; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D020742:rhoA GTP-Binding Protein; C000605254:ARRB2 protein, human; C000605258:Arrb2 protein, mouse; C000596779:sphingosine-1-phosphate receptor 2, zebrafish; C541414:stk3 protein, zebrafish; D000099317:Hippo Kinases