Nuclear trafficking of the G-protein-coupled parathyroid hormone receptor.

G-protein-coupled receptors are a family of receptors that signal primarily through heterotrimeric G proteins. However, new evidence has emerged to show that the signaling capabilities of the receptors are beyond those of traditional signaling cascades. One such example is that the parathyroid hormone (PTH) type 1 receptor is found not only at the plasma membrane but also in the nucleus of cells in cell lines and tissues. This review discusses the emerging concepts of nuclear PTH signaling and relates this information to the growing field of nuclear G-protein-coupled receptors. We review recently published studies on the mechanism of PTH nuclear localization, its role in the cell nucleus, and the contrasting roles ligands play in regulating the receptors' nuclear localization. The review also discusses the importance of nuclear G-protein-coupled receptors and future directions for research in this field.

Type 1 parathyroid hormone receptor (PTH1R) nuclear trafficking: regulation of PTH1R nuclear-cytoplasmic shuttling by importin-alpha/beta and chromosomal region maintenance 1/exportin 1.

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-beta in the mouse osteoblast-like cell line, MC3T3-E1. Immunofluorescence microscopy as well as ligand blotting for PTH1R in nuclear fractions of importin-beta 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-alpha/beta and CRM1.

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

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.