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.
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 Lys240-->Ala 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 alpha-helix.
Gensure
Robert C
RC
Endocrine Unit, Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 02114, USA.
Shimizu
Naoto
N
Tsang
Janet
J
Gardella
Thomas J
TJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010281:Parathyroid Hormone / Q000737:chemistry*; D044162:Parathyroid Hormone-Related Protein / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
C473449:PTH1R protein, human; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone