Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of beta-arrestin 1 recruitment.

TY - JOUR

T1 - Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of beta-arrestin 1 recruitment.

AU - Rasmussen, Trine N

AU - Novak, Ivana

AU - Nielsen, Søren M

N1 - Keywords: Animals; Arrestins; Binding Sites; Cattle; Cell Line; Clathrin-Coated Vesicles; Cyclic AMP-Dependent Protein Kinases; Endocytosis; Humans; Immunohistochemistry; Isoquinolines; Microscopy, Confocal; Mutagenesis, Site-Directed; Peptides; Phosphorylation; Protein Kinase C; Radioligand Assay; Receptors, Corticotropin-Releasing Hormone; Recombinant Proteins; Staurosporine; Sulfonamides

PY - 2004

Y1 - 2004

N2 - 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.

AB - 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.

U2 - 10.1111/j.1432-1033.2004.04371.x

DO - 10.1111/j.1432-1033.2004.04371.x

M3 - Journal article

C2 - 15560778

SN - 1742-464X

VL - 271

SP - 4366

EP - 4374

JO - FEBS Journal

JF - FEBS Journal

IS - 22

ER -