Altered selectivity of parathyroid hormone (PTH) and PTH-related protein (PTHrP) for distinct conformations of the PTH/PTHrP receptor

Abstract

PTH and PTHrP use the same G protein-coupled receptor, the PTH/PTHrP receptor (PTHR), to mediate their distinct biological actions. The extent to which the mechanisms by which the two ligands bind to the PTHR differ is unclear. We examined this question using several pharmacological and biophysical approaches. Kinetic dissociation and equilibrium binding assays revealed that the binding of [(125)I]PTHrP(1-36) to the PTHR was more sensitive to GTPgammaS (added to functionally uncouple PTHR-G protein complexes) than was the binding of [(125)I]PTH(1-34) ( approximately 75% maximal inhibition vs. approximately 20%). Fluorescence resonance energy transfer-based kinetic analyses revealed that PTHrP(1-36) bound to the PTHR more slowly and dissociated from it more rapidly than did PTH(1-34). The cAMP signaling response capacity of PTHrP(1-36) in cells decayed more rapidly than did that of PTH(1-34) (t(1/2) = approximately 1 vs. approximately 2 h). Divergent residue 5 in the ligand, Ile in PTH and His in PTHrP, was identified as a key determinant of the altered receptor-interaction responses exhibited by the two peptides. We conclude that whereas PTH and PTHrP bind similarly to the G protein-coupled PTHR conformation (RG), PTH has a greater capacity to bind to the G protein-uncoupled conformation (R(0)) and, hence, can produce cumulatively greater signaling responses (via R(0)-->RG isomerization) than can PTHrP. Such conformational selectivity may relate to the distinct modes by which PTH and PTHrP act biologically, endocrine vs. paracrine, and may help explain reported differences in the effects that the ligands have on calcium and bone metabolism when administered to humans.

Figures

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 At each time point, aliquots were withdrawn and immediately processed by rapid vacuum filtration to separate bound from free radioactivity.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.

Figure 2

GTPγS Sensitivity of PTH and PTHrP Ligand Binding to the Human and Rat PTHRs Radioligand binding to the PTHR in membranes prepared from HKRK-B7 (A) or ROS 17/2.8 cells (B) was assessed under approximate equilibrium conditions in the presence of varying concentrations of GTPγS. Data are expressed as a percentage of the radioactivity specifically bound (SB) in the absence of GTPγS. The radioligands used were [125I][Nle8,21,Tyr34]PTH(1–34)NH2, [125I][Tyr36]PTHrP(1–36)NH2, [125I][Ile5,Tyr36]PTHrP(1–36)NH2, and [125I][Aib1,3,Nle8,Gln10,Har11,Ala12,Trp14,Tyr15]hPTH(1–15)NH2. Nonspecific binding was determined in wells containing the homologous unlabeled PTH or PTHrP ligand (5 × 10−7 m). In A, the values of total radioactivity (counts per minute) added, total radioactivity bound, and nonspecifically bound radioactivity were 21,045 ± 1,627, 2,806 ± 296, and 1,163 ± 325 for [125I]PTH(1–34) (n =3); 27,489 ± 3,507, 3,164 ± 570, and 489 ± 85 for [125I]PTHrP(1–36) (n =5); and 34,001 ± 711, 9,601 ± 959, and 569 ± 113 for [125I]Ile5-PTHrP(1–36) (n =5), respectively. In B, the corresponding values were 21,408 ± 1,245, 3,900 ± 170, and 1,214 ± 184 for [125I]PTH(1–34) (n =6); 20,373 ± 951, 2,079 ± 211, and 467 ± 34 for [125I]PTHrP(1–36) (n =6); 23,553 ± 891, 6,570 ± 1,142, and 1,040 ± 116 for [125I]Ile5-PTHrP(1–36) (n =6); and 30,986 ± 4,567, 847 ± 137, and 237 ± 27 for [125I][Aib1,3,M]PTH(1–15) (n =6), respectively. Data are means (±sem) from the number of experiments indicated by n, each performed in duplicate. Note that we have shown previously that GTPγS inhibits the binding of the [125I][Aib1,3,M]PTH(1–15) analog to HKRK-B7 cell membranes by 88% and with an IC50 of 2.9 nm (16).

Figure 3

Binding of PTH and PTHrP Analogs to the G Protein-Coupled and G Protein-Uncoupled PTHR Conformations The binding of unlabeled PTH and PTHrP analogs to the G protein-coupled PTHR conformation (RG) and G protein-uncoupled PTHR conformation (R0) was assessed by competition methods using membranes prepared from transiently transfected COS-7 cells. Binding to RG was assessed using membranes prepared from cells cotransfected with the hPTHR and a negative-dominant Gαs subunit, and [125I][Aib1,3,Nle8,Gln10,Har11,Ala12,Trp14,Tyr15]hPTH(1–15)NH2 as a tracer radioligand. Binding to R0 was assessed using membranes prepared from cells transfected with the hPTHR alone, [125I][Nle8,21,Tyr34]rPTH(1–34)NH2 as a tracer radioligand, and adding GTPγS to the reactions. The unlabeled ligands used were [Nle8,21,Tyr34]rPTH(1–34)NH2 (A); [Tyr36]hPTHrP(1–36)NH2 (B); [His5,Nle8, 21,Tyr34]rPTH(1–34)NH2 (C); and [Ile5,Tyr36]hPTHrP(1–36)NH2 (D). Each curve shows data (mean ± sem) from three to six experiments, each performed in duplicate (also see Table 1). Fifteen experiments were performed for each radioligand, for which the mean values of total radioactivity (counts per minute) added, total radioactivity bound, and nonspecifically bound radioactivity were 30,059 ± 2,263, 3,495 ± 417, and 398 ± 37 for [125I]PTH(1–34) and 24,277 ± 2,022, 4,539 ± 848, and 236 ± 33 for [125I][Aib1,3,M]PTH(1–15), respectively.

Figure 4

FRET Analysis of Ligand Binding to the PTHR in HEK-293 Cells HEK-PTHR-CFPIC3/YFPCT cells were used to assess the kinetics of ligand binding to and dissociation from the PTHR. In each panel, the trace shows the FRET ratio (FYFP(535)/FCFP(480), normalized for channel spillover) obtained in cells superfused with buffer alone or with buffer containing a PTH peptide ligand (peptide additions indicated by black bars above each trace). The ligands used were hPTH(1–34) (A); [Aib1,3,Gln10,Har11,Ala12,Trp14]hPTH(1–14)NH2 (B); [Tyr36]hPTHrP(1–36)NH2 (C), and [Ile5,Tyr36]hPTHrP(1–36)NH2 (D). Data are from a single experiment; identical results were obtained in at least three others.

Figure 5

Duration of cAMP-Signaling Responses Induced by PTH and PTHrP Analogs in Cells Stably Expressing the Human PTHR The duration of cAMP signaling capacity of [Tyr36]hPTHrP(1–36)NH2 and [Ile5,Tyr36]hPTHrP(1–36)NH2 was assessed by time-course experiments in HKRK-B7 cells (950,000 hPTHRs per cell, A). The cells were pretreated for 10 min with either buffer alone (basal) or buffer containing ligand (100 nm), washed (t = 0), incubated in buffer for the times indicated (washout phase), treated with IBMX for a final 5 min, and then assessed for intracellular cAMP accumulation. The response to each peptide observed by incubating cells concomitantly with peptide and IBMX for 10 min and omitting the was-out phase was 185 ± 16 and 198 ± 18 pmol/well for PTHrP(1–36) and Ile5-PTHrP(1–36), respectively; the cAMP level in cells treated with IBMX in the absence of ligand was 2.0 ± 0.3 pmol/well. Data are means (±sem) of three experiments, each performed in duplicate. In these experiments, [Nle8,21,Tyr34]rPTH(1–34)NH2 was also analyzed and induced responses at each time point that were not different from those induced by [Ile5,Tyr36]hPTHrP(1–36)NH2. Analogs were also assessed in HKRK-B64 cells (90,000 hPTHRs per cell, B). The cAMP was assessed 60 min after ligand washout, and the data are expressed as a percentile of the maximal cAMP response induced by each peptide (indicated in the side panel) in cells treated concomitantly with that ligand and IBMX for 10 min and omitting the washout phase. Data are means (±sem) of four experiments, each performed in triplicate. Asterisks indicate statistical analyses of paired responses: PTHrP(1–36) vs. Ile5-PTHrP(1–36) (A), or as indicated by brackets (B): *, P < 0.05; **, P < 0.003.