title: Expression of PTH1R constructs in LLC-PK1 cells: protein nuclear targeting is mediated by the PTH1R NLS.
abstract: 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/mug of protein to 101 fmol/microg. 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.
authors.affiliation: Department of Biochemistry, University of Western Ontario, and The Lawson Health Research Institute, London, Ontario, Canada N6A 4V2.
authors.forename: Eric K
authors.initials: EK
authors.lastname: Patterson
authors.forename: Patricia H
authors.initials: PH
authors.lastname: Watson
authors.forename: Anthony B
authors.initials: AB
authors.lastname: Hodsman
authors.forename: Geoffrey N
authors.initials: GN
authors.lastname: Hendy
authors.forename: Lucie
authors.initials: L
authors.lastname: Canaff
authors.forename: F Richard
authors.initials: FR
authors.lastname: Bringhurst
authors.forename: Clare H
authors.initials: CH
authors.lastname: Poschwatta
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Fraher
mesh_terms: D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D019913:Nuclear Localization Signals; D044168:Receptor, Parathyroid Hormone, Type 1; D000242:Cyclic AMP