Photoaffinity cross-linking identifies differences in the interactions of an agonist and an antagonist with the parathyroid hormone/parathyroid hormone-related protein receptor.
Analogs of parathyroid hormone (PTH)-related protein (PTHrP), singularly substituted with a photoreactive L-p-benzoylphenylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmacologically evaluated in human embryonic kidney cells stably expressing the recombinant human PTH/PTHrP receptor. Two of these analogs, in which the photoreactive residue is either in position 1 or 2 (Bpa(1)- and Bpa(2)-PTHrP, respectively) displayed high affinity binding. Bpa(1)-PTHrP also displayed high efficacy for the stimulation of increased cAMP levels. Surprisingly, Bpa(2)-PTHrP was found to be a potent antagonist, despite the presence of the principal activation domain (sequence 1-6). Analysis of the digestion profiles of the ligand-receptor photoconjugates revealed that both the agonist and the antagonist cross-link to the S-CH(3) group of Met(425) in transmembrane domain 6 of the human PTH/PTHrP receptor. However, the antagonist Bpa(2)-PTHrP also cross-links to a proximal site within the receptor domain Pro(415)-Met(425). Unlike the antagonist Bpa(2)-PTHrP, the potent agonist Bpa(2)-PTH, also bearing the Bpa residue in position 2, cross-links only to the S-CH(3) group of Met(425) (similar to Bpa(1)-PTHrP and Bpa(1)-PTH). Taken together, these results suggest that the antagonist Bpa(2)-PTHrP is able to distinguish between two distinct conformations of the receptor. The comparison between PTHrP analogs substituted by Bpa at two consecutive positions and across PTH and PTHrP reveals insights into the PTH/PTHrP ligand-receptor bimolecular interaction at the level of a single amino acid.
Behar
V
V
Division of Bone and Mineral Metabolism, Charles A. Dana Laboratories, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Bisello
A
A
Bitan
G
G
Rosenblatt
M
M
Chorev
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives*; D019920:Photoaffinity Labels; D017354:Point Mutation; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000494:pharmacology; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction
C403165:1-Bpa-parathyroid hormone-related protein, recombinant; C403166:2-Bpa-parathyroid hormone-related protein, recombinant; D003432:Cross-Linking Reagents; C470121:PTHLH protein, human; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D011506:Proteins; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D011994:Recombinant Proteins; D010649:Phenylalanine; C053764:benzoylphenylalanine; D000242:Cyclic AMP