title: Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of beta-arrestin 1 recruitment.
abstract: The corticotropin releasing factor receptor 1 (CRFR1) belongs to the superfamily of G-protein coupled receptors. Though CRF is involved in the aetiology of several stress-related disorders, including depression and anxiety, details of CRFR1 regulation such as internalization remain uncharacterized. In the present study, agonist-induced internalization of CRFR1 in HEK293 cells was visualized by confocal microscopy and quantified using the radioligand 125I-labelled sauvagine. Recruitment of beta-arrestin 1 in response to receptor activation was demonstrated by confocal microscopy. The extent of 125I-labelled sauvagine stimulated internalization was significantly impaired by sucrose, indicating the involvement of clathrin-coated pits. No effect on the extent of internalization was observed in the presence of the second messenger dependent kinase inhibitors H-89 and staurosporine, indicating that cAMP-dependent protein kinase and protein kinase C are not prerequisites for CRFR1 internalization. Surprisingly, deletion of all putative phosphorylation sites in the C-terminal tail, as well as a cluster of putative phosphorylation sites in the third intracellular loop, did not affect receptor internalization. However, these mutations almost abolished the recruitment of beta-arrestin 1 following receptor activation. In conclusion, we demonstrate that CRFR1 internalization is independent of phosphorylation sites in the C-terminal tail and third intracellular loop, and the degree of beta-arrestin 1 recruitment.
authors.affiliation: Department of Molecular Biology, H. Lundbeck A/S, Valby, Denmark.
authors.forename: Trine N
authors.initials: TN
authors.lastname: Rasmussen
authors.forename: Ivana
authors.initials: I
authors.lastname: Novak
authors.forename: Søren M
authors.initials: SM
authors.lastname: Nielsen
mesh_terms: D029845:Amphibian Proteins; D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004705:Endocytosis; D006801:Humans; D007150:Immunohistochemistry; D007546:Isoquinolines / Q000494:pharmacology; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D036361:Peptide Hormones; D010455:Peptides / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011869:Radioligand Assay; D018019:Receptors, Corticotropin-Releasing Hormone / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000819:agonists / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D000099052:CRF Receptor, Type 1
chemical_list: C000605253:ARRB1 protein, human; D029845:Amphibian Proteins; D019390:Arrestins; D007546:Isoquinolines; D036361:Peptide Hormones; D010455:Peptides; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; D013449:Sulfonamides; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D000099052:CRF Receptor, Type 1; C025727:sauvagine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019311:Staurosporine; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide