Multiple sites of contact between the carboxyl-terminal binding domain of PTHrP-(1-36) analogs and the amino-terminal extracellular domain of the PTH/PTHrP receptor identified by photoaffinity cross-linking

The carboxyl-terminal portions of parathyroid hormone (PTH)-(1-34) and PTH- related peptide (PTHrP)-(1-36) are critical for high affinity binding to th e PTH/ PTHrP receptor (P1R), but the mechanism of receptor interaction for this domain is largely unknown. To identify interaction sites between the c arboxyl-terminal region of PTHrP-(1-36) and the P1R, we prepared analogs of [I-5,W-23,Y-36]PTHrP-(1-36)-amide with individual p-benzoyl-L-phenylalanin e (Bpa) substitutions at positions 22-35. When tested with LLC-PK1 cells st ably transfected with human P1R (hP1R), the apparent binding affinity and t he EC50 of agonist-stimulated cAMP accumulation for each analog was, with t he exception of the Bpa(24)-substituted analog, similar to that of the pare nt compound. The radiolabeled Bpa(23)-, Bpa(27)-, Bpa (28)-, and Bpa(33)-su bstituted compounds affinity-labeled the hP1R sufficiently well to permit s ubsequent mapping of the cross-linked receptor region. Each of these peptid es cross-linked to the amino-terminal extracellular domain of the P1R: [I-5 ,Bpa(23),Y-36]PTHrP-(1-36)-amide crosslinked to the extreme end of this dom ain (residues 33-63); [I-5,W-23 Bpa(27),Y-36]PTHrP-(1-36)-amide cross-linke d to residues 96-102; [1(5),W-23 Bpa (28),Y-36] PTHrP-(1-36). amide cross-l inked to residues 64-95; and [15,W23, Bpa(33),Y-36]PTHrP-(1-36)-amide cross -linked to residues 151-172. These data thus predict that residues 23, 279 28, and 33 of native PTHrP are each near to different regions of the amino- terminal extracellular receptor domain of the P1R. This information helps d efine sites of proximity between several ligand residues and this large rec eptor domain, which so far has been largely excluded from models of the hor mone-receptor complex.