title: Mutational analysis of the receptor-activating region of human parathyroid hormone.
abstract: The first 4 residues of parathyroid hormone (PTH) are highly conserved in evolution and are important for biological activity. We randomly mutated codons 1-4 of human PTH (hPTH) with degenerate oligonucleotides and, after expression in COS cells, screened the mutants for receptor binding and cAMP-stimulating activity using ROS 17/2.8 cells. This survey identified Glu4 and Val2 as important determinants of receptor binding and activation, respectively. Positions 1 and 3 were more tolerant of substitutions indicating that these sites are less vital to hormone function. Activities of synthetic hPTH(1-34) analogs further demonstrated the importance of positions 2 and 4. The binding affinity of [Ala4,Tyr34] hPTH(1-34)NH2 was 100-fold reduced relative to [Tyr34]hPTH(1-34)NH2 (Kd values = 653 +/- 270 and 4 +/- 1 nM, respectively), and [Arg2, Tyr34]hPTH(1-34)NH2 was a weak partial agonist which bound well to the ROS cell receptor (Kd = 31 +/- 10 nM). The Arg2 analog was nearly as potent as PTH(3-34) as an in vitro PTH antagonist in osteoblast derived cells. However, unlike PTH(3-34), [Arg2]PTH was a full agonist in opossum kidney (OK) cells. These observations suggest that the activation domains of the OK and ROS cell PTH receptors are different. Thus, amino-terminal PTH analogs may be useful as probes for distinguishing properties of PTH receptors.
authors.affiliation: Endocrine Unit, Massachusetts General Hospital, Boston 02114.
authors.forename: T J
authors.initials: TJ
authors.lastname: Gardella
authors.forename: D
authors.initials: D
authors.lastname: Axelrod
authors.forename: D
authors.initials: D
authors.lastname: Rubin
authors.forename: H T
authors.initials: HT
authors.lastname: Keutmann
authors.forename: J T
authors.initials: JT
authors.lastname: Potts
authors.forename: H M
authors.initials: HM
authors.lastname: Kronenberg
authors.forename: S R
authors.initials: SR
authors.lastname: Nussbaum
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D003062:Codon / Q000235:genetics; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010281:Parathyroid Hormone / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D010957:Plasmids; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018016:Receptors, Parathyroid Hormone; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D003062:Codon; D010281:Parathyroid Hormone; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; D000242:Cyclic AMP