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Identification of Regions of the -1 Receptor Ligand Binding Site Using a Novel Photoprobe

Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin (A.P., D.F., S.R., U.B.C., T.M., A.E.R.); and Pharmaceutical Research Laboratory, College of Chemistry, Isfahan University of Technology, Isfahan, Iran (A.R.H.)

Receptors, once considered a class of opioid receptors, are now regarded as a unique class of receptors that contain binding sites for a wide range of ligands, including the drug 1-N(2',6'-dimethylmorpholino)3-(4-t-butylpropylamine) (fenpropimorph), a yeast sterol isomerase inhibitor. Because fenpropimorph has high-binding affinity to the -1 receptor, we have synthesized a series of fenpropimorph-like derivatives with varying phenyl ring substituents and have characterized their binding affinities to the -1 receptor. In addition, we have synthesized a carrier-free, radioiodinated fenpropimorph-like photoaffinity label, 1-N-(2',6'-dimethyl-morpholino)-3-(4-azido-3-[¹²⁵I]iodo-phenyl)propane ([¹²⁵I]IAF), which covalently derivatized the -1 receptor (25.3 kDa) in both the rat liver and guinea pig liver membranes and the -2 receptor (18 kDa) in rat liver membranes with high specificity. Furthermore, after cleaving the specific [¹²⁵I]IAF-photolabeled -1 receptor in guinea pig and rat liver membranes and the pure guinea pig -1 receptor with EndoLys-C and cyanogen bromide, the [¹²⁵I]IAF label was identified both in a peptide containing steroid binding domain-like I (SBDLI) (amino acids 91–109) and in a peptide containing steroid binding domain-like II (SBDLII) (amino acids 176–194). Because a single population of binding sites (R² = 0.992) for [¹²⁵I]IAF interaction with the -1 receptor was identified by (+)-[³H]pentazocine competitive binding with nonradioactive [¹²⁷I]IAF, it was concluded that SBDLI (amino acids 91–109) and SBDLII (amino acids 176–194) comprises, at least in part, regions of the -1 receptor ligand binding site(s).

Address correspondence to: Dr. Arnold E. Ruoho, Department of Pharmacology, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706. E-mail: aeruoho{at}wisc.edu

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