PROBING THE BIMOLECULAR INTERACTIONS OF PARATHYROID-HORMONE AND THE HUMAN PARATHYROID-HORMONE PARATHYROID HORMONE-RELATED PROTEIN-RECEPTOR .2. CLONING, CHARACTERIZATION, AND PHOTOAFFINITY-LABELING OF THE RECOMBINANT HUMAN RECEPTOR

Parathyroid hormone (PTH) acts to regulate calcium homeostasis by inte racting with a G-protein-coupled receptor that also binds PTH-related protein (PTHrP). In this report we describe the cloning, characterizat ion, and biological activity of the cloned human (h) PTH/PTHrP recepto r (Rc) and crosslinking of a benzophenone-substituted PTH analog, epsi lon-pBz(2)),L-2-Nal(23),Tyr(34)]bPTH(1-34)-NH2 (K13), to cells endogen ously expressing the Rc and cells transiently or stably transfected wi th the human Rc. A full-length cDNA clone was isolated and fully seque nced from a human kidney cDNA library. Northern blot analysis of norma l human tissues revealed a limited tissue distribution: a single trans cript of similar to 2.3 kb was detected in kidney, lung, placenta, and liver. In human embryonic kidney cells (HEK-293, clone C-21) stably t ransfected with hPTH/PTHrP Rc, a single 85-90 kDa Rc-hormone complex w as formed after photolysis in the presence of K13. This covalent cross -linking reaction was specifically inhibited by excess quantities of b iologically active 1-34 analogs of bovine (b) PTH or hPTHrP but not by C-terminal and midregion PTH peptides. Photoincorporation of I-125-la beled K13 into the Rc occurred with high efficiency (60-70%), approxim ately an order of magnitude greater than that achieved with convention al aryl azide cross-linking reagents. These results support the feasib ility of our approach for specifically cross-linking a tagged PTH anal og to the Re, as a first step in the effort to identify directly the a mino acid residues that constitute the Rc binding site.