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Identification of a Contact Site for Residue 19 of Parathyroid Hormone (PTH) and PTH-Related Protein Analogs in Transmembrane Domain Two of the Type 1 PTH Receptor

Endocrine Unit (R.C.G., N.S., J.T., T.J.G.) and Pediatric Endocrine Unit (R.C.G.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Thomas J. Gardella, Endocrine Unit, 50 Blossom Street, WEL 501, Boston, Massachusetts 02114. E-mail: gardella{at}helix.mgh.harvard.edu.

Recent functional studies have suggested that position 19 in PTH interacts with the portion of the PTH-1 receptor (P1R) that contains the extracellular loops and seven transmembrance helices (TMs) (the J domain). We tested this hypothesis using the photoaffinity cross-linking approach. A PTHrP(1–36) analog and a conformationally constrained PTH(1–21) analog, each containing para-benzoyl-L-phenylalanine (Bpa) at position 19, each cross-linked efficiently to the P1R expressed in COS-7 cells, and digestive mapping analysis localized the cross-linked site to the interval (Leu232-Lys240) at the extracellular end of TM2. Point mutation analysis identified Ala234, Val235, and Lys240 as determinants of cross-linking efficiency, and the Lys240Ala mutation selectively impaired the binding of PTH(1–21) and PTH(1–19) analogs, relative to that of PTH(1–15) analogs. The findings support the hypothesis that residue 19 of the receptor-bound ligand contacts, or is close to, the P1R J domain—specifically, Lys240 at the extracellular end of TM2. The findings also support a molecular model in which the 1–21 region of PTH binds to the extracellular face of the P1R J domain as an -helix.

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