Zinc(II)-mediated enhancement of the agonist activity of histidine-substituted parathyroid hormone(1-14) analogues.
Previous studies on parathyroid hormone (PTH)(1-14) revealed that residues (1-9) played a dominant role in stimulating PTH-1 receptor-mediated increases in cAMP formation. In the present study, we examined the effects of installing a metal-binding motif in the (10-14) region of rat PTH(1-14) on the peptide's agonist activity. We found that substitution of histidine for the native asparagine at position 10 of PTH(1-14) provided a peptide that was approx. 8-fold more potent as an agonist in the presence of divalent zinc salts than it was in the absence of the metal. This enhancement in potency was dependent on the native histidine at position 14, the concentration of Zn(II) utilized, and did not occur with other divalent metal ions. The zinc-activated [His(10)]-PTH(1-14) peptide was blocked by a classical PTH-1 receptor antagonist, PTHrP(7-36), and did not activate the PTH-2 receptor. The zinc-mediated enhancing effect did not require the large N-terminal extracellular domain of the PTH-1 receptor. Although we were able to demonstrate that [His(10)]-PTH(1-14) binds Zn(II) using (1)H-NMR, our spectroscopic studies (circular dichroism and nuclear magnetic resonance) were not consistent with the notion that zinc enhanced the activity of [His(10)]-PTH(1-14) simply by inducing a helical structure in the 10-14 region. Rather, the data suggest that the enhancement in cAMP potency arises from the formation of a ternary complex between [His(10)]-PTH(1-14), a zinc atom, and the extracellular loop/transmembrane domain region of the PTH-1 receptor.
Carter
P H
PH
Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, 55 Blossom Street, Boston, MA 02114, USA.
Gardella
T J
TJ
D000818:Animals; D019556:COS Cells; D002413:Cations, Divalent; D002460:Cell Line; D002712:Chlorides / Q000494:pharmacology; D002942:Circular Dichroism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D010281:Parathyroid Hormone / Q000819:agonists* / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis; D018016:Receptors, Parathyroid Hormone / Q000819:agonists; D015032:Zinc / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D017967:Zinc Compounds / Q000494:pharmacology
D002413:Cations, Divalent; D002712:Chlorides; D008024:Ligands; D010281:Parathyroid Hormone; D010455:Peptides; D018016:Receptors, Parathyroid Hormone; D017967:Zinc Compounds; D006639:Histidine; C016837:zinc chloride; D015032:Zinc