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][Aib^1,3,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][Nle^8,21,Tyr³⁴]rPTH(1–34)NH₂ as a tracer radioligand, and adding GTPγS to the reactions. The unlabeled ligands used were [Nle^8,21,Tyr³⁴]rPTH(1–34)NH₂ (A); [Tyr³⁶]hPTHrP(1–36)NH₂ (B); [His⁵,Nle^{8, 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][Aib^1,3,M]PTH(1–15), respectively.