Quantitative cell membrane-based radioligand binding assays for parathyroid hormone receptors

Most current assays of PTH receptor ligand binding employ whole cells as th e vehicle for receptor. Whole cell binding does not easily permit the estim ation of physically meaningful binding parameters, the detection of multipl e receptor states, or the evaluation of the effects of receptor modulators such as guanine nucleotides. We have developed quantitative methods for the measurement of equilibrium ligand binding parameters at cloned parathyroid hormone (PTH) receptors in cell membrane preparations. Centrifugation is u sed to separate bound and free [I-125]-labeled peptide radioligands, and no nfat dried milk is used as a blocking agent to reduce nonspecific binding. This method is useful for measurement of agonist and antagonist radioligand binding at the PTH-1 receptor and binding of [I-125]PTH(1-34) at the PTH-2 receptor. Less than 25% of [I-125]PTH(1-34) or [I-125]PTHrP(1-36) is degra ded during the assay incubation. We demonstrated the utility of the assay u sing measurements of ligand binding properties at the PTH-1 receptor. (I) H omologous displacement experiments provided estimates of K-d and B-max for the radioligands. (2) Displacement of radiolabeled antagonist binding ([I-1 25]PTH(3-34)) by an unlabeled agonist (RS-66271) revealed multiple affinity states of agonist-receptor interaction. (3) Comparison of RS-66271 displac ement in the presence and absence of GTP gamma S demonstrated that the high est affinity state is guanine nucleotide-sensitive, suggesting that this st ate requires stabilization by G-protein. This assay thus allows more mechan istic interpretation of binding data than PTH binding assays in current use . A more convenient rapid-filtration method was also developed for measurem ent of radioligand binding at PTH-1 and PTH-2 receptors. (C) 1999 Elsevier Science Inc. All rights reserved.