Type 1 parathyroid hormone receptor (PTH1R) nuclear trafficking: association of PTH1R with importin alpha1 and beta

Abstract

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 alpha(1), and importin beta 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 alpha(1), or importin beta. 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 alpha(1) and beta, the complex was purified from total cell protein of osteoblast-like cells using a PTH-linked affinity chromatography column. Using an anti-importin alpha(1) antibody, Western blots detected importin alpha(1) at 58 kDa in the purified sample. Also, using an anti-importin beta antibody, Western blots detected importin beta 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 alpha(1) and importin beta, and that nuclear PTH1R is associated with the nucleoplasm.