Figure 1.
Dissociation of PTH and PTHrP Ligands from the PTH Receptor and Effects of GTPγS
The radioligands [125I][Nle8,21,Tyr34]rPTH(1–34)NH2 (A), [125I][Tyr36]PTHrP(1–36)NH2 (B), and [125I][Ile5,Tyr36]PTHrP(1–36)NH2 (C) were prebound to the human PTHR in membranes prepared from HKRK-B7 cells for 90 min; then dissociation was initiated (t = 0) by the addition of the homologous unlabeled analog (5 × 10−7 m), added either alone (•) or with GTPγS (5 × 10−5 m, ○). At each time point, aliquots were withdrawn and immediately processed by rapid vacuum filtration to separate bound from free radioactivity. Nonspecific binding was determined in tubes containing the homologous unlabeled ligand (5 × 10−7 m) during both the preincubation and dissociation phases. The specifically bound radioactivity (SB) at each time point is expressed as a percentage of the specific binding observed at t = 0. Shown are aggregate data from four (A), five (B), or three (C) experiments. For each tracer radioligand, the respective values (means ± sem) of total radioactivity (counts per minute) added, total radioactivity bound at t = 0, and nonspecifically bound radioactivity (averaged over the time course of each experiment) were 26,754 ± 2,652, 12,964 ± 2,476, and 522 ± 42 for [125I]PTH(1–34) (n = 4); 31,597 ± 1,679, 5,959 ± 492, and 262 ± 17 for [125I]PTHrP(1–36) (n = 5); and 51,335 ± 10,516, 22,904 ± 5,365, and 1,332 ± 337 for [125I]Ile5-PTHrP(1–36) (n =3), respectively. Curves were fit to the data using either a two-phase (A and B) or a single-phase (C) exponential decay equation.