PubMed2001
The N-terminal portion of parathyroid hormone is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated whether substitution of the sterically hindered and helix-promoting amino acid alpha-aminoisobutyric acid (Aib) in N-terminal PTH oligopeptides would improve the capacity of the peptide to activate the P1R. Analysis of the effects of individual Aib substitutions at each position in [Ala(3,12),Gln(10),Har(11),Trp(14)]PTH(1-14)NH(2) ([M]PTH(1-14)) on cAMP-stimulating potency in HKRK-B28 cells revealed that Aib at most positions diminished potency; however, Aib at positions 1 and 3 enhanced potency. Thus [Aib(1,3),M]PTH(1-14) was approximately 100-fold more potent than [M]PTH(1-14) (EC(50) = 1.1 +/- 0.1 and 100 +/- 20 nm, respectively), approximately 100,000-fold more potent than native PTH(1-14), and 2-fold more potent than PTH(1-34). The shorter peptide, [Aib(1,3),M]PTH(1-11), was also fully efficacious and 1,000-fold more potent than [M]PTH(1-11) (EC(50) 4 +/- 1 nm versus 3 +/- 1 microm). In cAMP stimulation assays performed in COS-7 cells expressing P1R-delNt, a receptor that lacks most of the N-terminal extracellular domain, [Aib(1,3),M]PTH(1-14) was 50-fold more potent than [M]PTH(1-14) (EC(50) = 0.7 +/- 0.2 versus 40 +/- 2 nm) and 1,000-fold more potent than PTH(1-34) (EC(50) = 700 nm). [Aib(1,3),M]PTH(1-14), but not PTH(1-34), inhibited the binding of (125)I-[Aib(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH(1-21)NH(2) to hP1R-delNt (IC(50) = 1,600 +/- 200 nm). The Aib(1,3) substitutions in otherwise unmodified PTH(1-34) enhanced potency and binding affinity on hP1R-delNt, but they had no effect for this peptide on hP1R-WT. Circular dichroism spectroscopy demonstrated that the Aib-1,3 substitutions increased helicity in all peptides tested, including PTH(1-34). The overall data thus suggest that the N-terminal residues of PTH are intrinsically disordered but become conformationally constrained, possibly as an alpha-helix, upon interaction with the activation domain of the PTH-1 receptor.