tailieunhanh - Báo cáo khoa học: Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of b-arrestin 1 recruitment

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-relateddisorders, includingdepressionandanxiety, detailsof CRFR1 regulation such as internalization remain unchar-acterized. In the present study, agonist-induced internal-ization of CRFR1 in HEK293 cells was visualized by confocal microcopy and quantified using the radioligand 125 I-labelled sauvagine. | Eur. J. Biochem. 271 4366-4374 2004 FEBS 2004 doi Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of p-arrestin 1 recruitment Trine N. Rasmussen1 3 Ivana Novak3 and Soren M. Nielsen2 1Department of Molecular Biology and department of Molecular Pharmacology H. Lundbeck A S Valby Denmark 3August Krogh Institute University of Copenhagen Denmark 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 microcopy and quantified using the radioligand 125I-labelled sauvagine. Recruitment of b-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 phosporylation sites in the third intracellular loop did not affect receptor internalization. However these mutations almost abolished the recruitment of b-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 b-arrestin 1 recruitment. Keywords b-arrestin 1 CRFR1 GPCR receptor internalization. .