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Mechanisms of Ligand Binding to the Parathyroid Hormone (PTH)/PTH-Related Protein Receptor: Selectivity of a Modified PTH(1–15) Radioligand for G_S-Coupled Receptor Conformations

Endocrine Unit (T.D., A.L., M.J.M., M.B., H.J., J.T.P., T.J.D.) and Department of Pediatrics (H.J.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Thomas J. Gardella, Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: gardella{at}helix.mgh.harvard.edu.

Mechanisms of ligand binding to the PTH/PTHrP receptor (PTHR) were explored using PTH fragment analogs as radioligands in binding assays. In particular, the modified amino-terminal fragment analog, ¹²⁵I-[Aib^1,3,Nle⁸,Gln ¹⁰,homoarginine¹¹,Ala¹²,Trp¹⁴,Tyr¹⁵]rPTH(1–15)NH₂, ¹²⁵I-[Aib^1,3,M]PTH(1–15), was used as a radioligand that we hypothesized to bind solely to the juxtamembrane (J) portion of the PTHR containing the extracellular loops and transmembrane helices. We also employed ¹²⁵I-PTH(1–34) as a radioligand that binds to both the amino-terminal extracellular (N) and J domains of the PTHR. Binding was examined in membranes derived from cells expressing either wild-type or mutant PTHRs. We found that the binding of ¹²⁵I-[Aib^1,3,M]PTH(1–15) to the wild-type PTHR was strongly (90%) inhibited by guanosine 5'-O-(3-thio)triphosphate (GTPS), whereas the binding of ¹²⁵I-PTH(1–34) was only mildly (25%) inhibited by GTPS. Of these two radioligands, only ¹²⁵I-[Aib^1,3,M]PTH(1–15) bound to PTHR-delNt, which lacks most of the receptor’s N domain, and again this binding was strongly inhibited by GTPS. Binding of ¹²⁵I-[Aib^1,3,M]PTH(1–15) to the constitutively active receptor, PTHR-H223R, was only mildly (20%) inhibited by GTPS, as was the binding of ¹²⁵I-PTH(1–34). In membranes prepared from cells lacking G_S via knockout mutation of Gnas, no binding of ¹²⁵I-[Aib^1,3,M]PTH(1–15) was observed, but binding of ¹²⁵I-[Aib^1,3,M]PTH(1–15) was recovered by virally transducing the cells to heterologously express G_S. ¹²⁵I-PTH(1–34) bound to the membranes with or without G_S. The overall findings confirm the hypothesis that ¹²⁵I-[Aib^1,3,M]PTH(1–15) binds solely to the J domain of the PTHR. They further show that this binding is strongly dependent on coupling of the receptor to G_S-containing heterotrimeric G proteins, whereas the binding of ¹²⁵I-PTH(1–34) can occur in the absence of such coupling. Thus, ¹²⁵I-[Aib^1,3,M]PTH(1–15) appears to function as a selective probe of G_S-coupled, active-state PTHR conformations.

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