GENERATION AND CHARACTERIZATION OF HUMAN KIDNEY-CELL LINES STABLY EXPRESSING RECOMBINANT HUMAN PTH PTHRP RECEPTOR - LACK OF INTERACTION WITH A C-TERMINAL HUMAN PTH PEPTIDE/

Parathyroid hormone (PTH) exerts its biological action by binding to m embrane-bound, G-protein coupled receptors expressed predominantly in bone and kidney. In this study, we describe the production and charact erization of a panel of cell lines, derived from a human embryonic kid ney cell line (HEK-293), each of which stably express different amount s of the recombinant human PTH/parathyroid hormone-related protein (PT HrP) receptor (Rc). A total of 52 distinct clones displaying different levels of PTH-responsive cAMP production were analyzed; three clones were chosen for more detailed evaluation. These clones (and the recept or-lacking parental cell line) were examined for PTH binding, PTH-stim ulated cyclic AMP accumulation and PTH/PTHrP Rc mRNA expression. Recep tor-positive clones display a spectrum of PTH-responsiveness that corr elates with receptor number/cell and level of receptor mRNA present. T he interaction of a C-terminal hPTH-(52-84) peptide with the stably ex pressed human receptor was examined in cells expressing the highest am ount of Rc (>400,000 Rc/cell). There was no direct binding of hPTH-(52 -84) or specific competition versus radiolabeled PTH-(1-34). However, competition versus radiolabeled PTH-(1-34) was observed with bPTH-(1-3 4), hPTH-(1-84) and hPTHrP-(1-34). These data suggest that hPTH-(52-84 ) does not interact with the only known form of the human PTH/PTHrP Rc . Therefore, the reported effects of PTH-(52-84) in other systems must be via an alternate (as yet unidentified) mechanism(s). The expressio n of various amounts of the human PTH/PTHrP Rc in a human target cell background should facilitate characterization of the ligand-binding pr operties and physiological signal transduction mechanism of the Rc.