Expression of PTH1R constructs in LLC-PK1 cells: Protein nuclear targeting is mediated by the PTH1R NLS

This study demonstrates that the PTH1R NLS can target a fusion protein to the nucleus, and that this is blocked by sequences downstream of the NLS. GFP fused to the NLS showed a significant increase in nuclear targeting compared to GFP alone or GFP fused to a peptide of the same length. In previous studies, we demonstrated that the type I PTH/PTHrP receptor (PTH1R) localizes to the nucleus of cells within rat liver, kidney, uterus, ovary and gut. Similarly, nuclear localization of the PTH1R was observed in the cultured osteoblast-like cells MC3T3-E1, UMR106, ROS 17/2.8 and SaOS-2. We have identified a putative bipartite nuclear localization signal (NLS), from residues 471–488 in the protein sequence of the PTH1R. In this study, several PTH1R constructs were made in the Enhanced Green Fluorescent Protein (EGFP) expression vector (Clontech), transiently transfected into LLC-PK1 Clone 46 cells, and the resultant fusion protein expression followed by fluorescence microscopy. This particular clone of LLC-PK1 shows no biochemical response in vitro to parathyroid hormone. Constructs included the entire PTH1R sequence (PTH1R-GFP), the putative NLS fused to the C-terminus of GFP (GFP-NLS) or the NLS through to the C-terminus of the PTH1R fused to GFP (GFP-NLSCT). Deconvolution fluorescence microscopy of cells transfected with PTH1R-GFP showed abundant fluorescent signal throughout the cells with distinctly fluorescing plasma membranes. These cells also exhibited an increase in cAMP production in response to (0–10− 8 M) hPTH(1–34), with an increase in cAMP from 11 fmol/μg of protein to 101 fmol/μg. In contrast, cells transfected with the GFP-NLS construct showed significant nuclear sequestration of fluorescence as compared to GFP alone, GFP-NLSCT, or a short amino acid sequence fused to GFP (GFP-FFVAIYCFCNGEVQAEI). These results indicate that the NLS at residues 471–488 of the mature rat PTH1R is functional and plays a role in targeting the PTH1R the nucleus, also the addition of GFP to the C-terminus of the PTH1R still allows cAMP generation which will be useful for further studies.