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Type 1 Parathyroid Hormone Receptor (PTH1R) Nuclear Trafficking: Regulation of PTH1R Nuclear-Cytoplasmic Shuttling by Importin-/ß and Chromosomal Region Maintenance 1/Exportin 1

Departments of Physiology and Pharmacology, Medicine, and Biochemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B8

Address all correspondence and requests for reprints to: Patricia H. Watson, Ph.D., Room E4-155, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. E-mail: pwatson{at}lhrionhealth.ca.

The type 1 PTH/PTH-related peptide receptor (PTH1R) is a class B G protein-coupled receptor that demonstrates immunoreactivity in the nucleus as well as cytoplasm of target cells. Our previous studies on the PTH1R have shown that it associates with the importin family of transport regulatory proteins. To investigate the role of the importins in PTH1R nuclear import, we used small interfering (si)RNA technology to knock down the expression of importin-ß in the mouse osteoblast-like cell line, MC3T3-E1. Immunofluorescence microscopy as well as ligand blotting for PTH1R in nuclear fractions of importin-ß siRNA-treated cells demonstrated a decrease in nuclear localization of the PTH1R in comparison with control cells. Under normal culture conditions, PTH1R is present in both the nucleus and cytoplasm of cells. Serum starvation favors nuclear localization of PTH1R, whereas returning cells to serum or treatment with PTH-related peptide induced its cytoplasmic localization. To address the nuclear export of PTH1R, interactions between PTH1R and chromosomal region maintenance 1 (CRM1) were investigated. PTH1R and CRM1 coimmunoprecipitated from MC3T3-E1 cells, suggesting that CRM1 and PTH1R form a complex in vivo. After treatment with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, PTH1R accumulated in the nucleus. Taken together, our studies show that PTH1R shuttles from the nucleus to the cytoplasm under normal physiological conditions and that this nuclear-cytoplasmic transport is dependent upon importin-/ß and CRM1.