Studies of the N-terminal region of a parathyroid hormone-related peptide (1-36) analog: receptor subtype-selective agonists, antagonists, and photochemical cross-linking agents.
The N-terminal regions of PTH and PTH-related peptide (PTHrP) are involved in receptor-mediated signaling and subtype selectivity. To better understand the molecular basis for these processes, we first prepared a series of [I5,W23,Y36]-PTHrP(1-36)NH2 analogs having stepwise deletions of residues 1-4 and characterized them with the human (h)PTH-1 and hPTH-2 receptor subtypes stably transfected in LLC-PK1 cells. Deletions beyond residue 2 caused progressive and severe losses in cAMP-signaling efficacy without dramatically diminishing receptor-binding affinity; consistent with this, [I5,W23]-PTHrP(5-36) was a potent antagonist for both PTH receptor subtypes. We then prepared and characterized photolabile analogs of [I5,W23,Y36]-PTHrP(1-36)NH2 that were singly modified with parabenzoyl-L-phenylalanine (Bpa) along the first six residues. These full-length analogs exhibited receptor subtype-selective agonism, antagonism, and photochemical cross-linking profiles. In particular, the [Bpa2]- and [Bpa4]-substituted analogs selectively antagonized and preferentially cross-linked to the PTH-1 receptor and PTH-2 receptor, respectively. These results demonstrate that the 1-5 region of [I5,W23]-PTHrP(1-36) is critical for activating the PTH-1 and PTH-2 receptors and suggest that the individual residues in this region play distinct roles in modulating the activation states of the two receptors. The cross-linking of both agonist and antagonist ligands to these PTH receptors lays the groundwork for identifying critical signaling determinants in the ligand binding pocket of the receptor.
Carter
P H
PH
Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston 02114, USA.
Jüppner
H
H
Gardella
T J
TJ
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D003432:Cross-Linking Reagents; D006801:Humans; D007668:Kidney; D010281:Parathyroid Hormone / Q000037:antagonists & inhibitors; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D010649:Phenylalanine / Q000031:analogs & derivatives; D010777:Photochemistry; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D018016:Receptors, Parathyroid Hormone / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013552:Swine; D014162:Transfection
D003432:Cross-Linking Reagents; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D011506:Proteins; D018016:Receptors, Parathyroid Hormone; D011994:Recombinant Proteins; C079957:parathyroid hormone-related peptide (1-36); D010649:Phenylalanine; C053764:benzoylphenylalanine