[Norleucine30]tip(7-39) is a non-selective antagonist of human parathyroid hormone receptors (PTH1 and PTH2)

E.A. Harper, W. Xun, D. Patel, C. Austin, M. Shaxted, M. Raynor, R.A.D. Hull, S, B., Kalindjian. James Black Foundation, Dulwich, London. U.K.

Tuberoinfundibular peptide(7-39) (TIP(7-39)) acts as a potent and selective competitive antagonist of the human PTH1-receptor (PTH1 pA2~6.8, pKI~8.21; PTH2 pKI~7.02; Hoare et al., 2000; 2002) and as such, it has been used to characterise PTH1-receptor activity. However, due to the propensity for methionine30 oxidation in TIP(7-39), which could affect peptide potency (Frelinger et al,1984), we have substituted this amino acid for norleucine (Nle). Here, we have used PTH1- and PTH2 -receptor cAMP assays to investigate if [Nle30]TIP(7-39) (NleTIP) acts as a selective simple competitive antagonist of the human PTH1-receptor.

HEK293 and CHO-K1 cells expressing human PTH1- (9B3) or PTH2-receptors (96D), respectively, were lysed (10min, 10mM Tris-HCl; pH7.4, 4ºC), membranes collected by centrifugation (20,000g, 20min, 4ºC) and resuspended in 20mM Hepes-NaOH (pH 7.2) containing 150μM IBMX, 10mM phosphocreatine, 200μM ATP, 10U ml-1 creatine phosphokinase, MgCl2 (15mM 96D; 5mM 9B3) and GTP (1µM 96D; 10µM 9B3). To determine the intrinsic efficacy of NleTIP and PTH1 selective ( PTHrp(1-34)), PTH2 selective (TIP(1-39)), and non-selective PTH-receptor (PTH(1-34)) agonists, they (50µl; 2pM–20µM) were incubated with membranes (50µl; 96D=106, 9B3=3x105cells ml-1) in cAMP Flashplates. To establish whether NleTIP was a selective PTH1-receptor antagonist, it was pre-incubated with membranes (30min, 21ºC; 9B3= 0.07, 0.3, 0.7 and 3µM; 96D= 0.2, 0.7, 2 and 7µM) before addition to Flashplates containing PTH(1-34) (9B3) or TIP(1-39) (96D). Assays were terminated after 210 (96D) or 180min (9B3) at 21ºC by addition of detection buffer (100µl).

Agonist potency orders (table 1, mean ± s.e.mean ; 9B3, PTH(1-34) >PTHrp(1-34) >TIP(1-39); 96D, TIP(1-39) > PTH(1-34) > PTHrp(1-34)) indicated that cAMP accumulation (pmol ml-1) was mediated by PTH1-receptors in 9B3 cells and byPTH2-receptors in 96D cells. NleTIP did not induce cAMP accumulation in 9B3 or 96D membranes (table 1) and produced concentration-dependent, parallel dextral-shifts of PTH(1-34) and TIP(1-39) concentration effect (E/[A]) curves without change in maximal asymptote ( a ), in three and five experiments, respectively. The data was best fit to the Schild equation with slope constrained to unity (p<0.05 F-test) and pKB estimates of 8.07 ± 0.32 (PTH1) and 7.55 ± 0.28 (PTH2) were obtained.

In the recombinant receptor systems we have investigated, data suggests that NleTIP is a non-selective simple competitive antagonist at human PTH1 and PTH2 receptors. Therefore, in human tissues/ cells, this ligand cannot be used to define the PTH receptor subtype mediating the effects of a non-specific PTH receptor agonist (eg PTH(1-34)).

Hoare, S.R.J. et al. (2000) J. Pharmacol. Expt. Ther., 295, 761-770.
Hoare, S.R.J. et al. (2002) Peptides, 23, 989-998.
Frelinger, A.L. et al. (1984) J. Biol. Chem., 259, 5507-5513.