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Type 1 Parathyroid Hormone Receptor (PTH1R) Nuclear Trafficking: Association of PTH1R with Importin ₁ and ß

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 G443, Lawson Health Research Institute, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. E-mail: pwatson{at}lhrionhealth.ca.

Previous studies have shown that the type 1 PTH receptor (PTH1R), a class B G protein-coupled receptor, appears in the nucleus of target cells. Through immunofluorescence and deconvolution microscopy, we demonstrate that PTH1R, importin ₁, and importin ß are present within the nucleus and cytoplasm of osteoblast-like cell lines with the nuclear PTH1R being restricted to the nucleoplasm. Immunofluorescence studies showed that nuclear accumulation of PTH1R was associated with specific stages of the cell cycle. Using immunoprecipitation and affinity chromatography, we show that the PTH1R forms a complex with the importin family of transport molecules. Total cell protein from osteoblast-like cells was immunoprecipitated with antibodies for PTH1R, importin ₁, or importin ß. When the immunoprecipitates were separated and subsequently exposed to biotinylated PTH (1–84) a single band was present on the gel at 66.3 kDa, corresponding to the PTH1R. To confirm the interaction between PTH1R and both importin ₁ and ß, the complex was purified from total cell protein of osteoblast-like cells using a PTH-linked affinity chromatography column. Using an anti-importin ₁ antibody, Western blots detected importin ₁ at 58 kDa in the purified sample. Also, using an anti-importin ß antibody, Western blots detected importin ß at 94 kDa. These results indicate that the importins were associated with the PTH1R at the time of the purification. In conclusion, we show that the PTH1R forms a complex with the transport regulatory proteins, importin ₁ and importin ß, and that nuclear PTH1R is associated with the nucleoplasm.

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