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A High Affinity Gonadotropin-Releasing Hormone (GnRH) Tracer, Radioiodinated at Position 6, Facilitates Analysis of Mutant GnRH Receptors¹

Medical Research Council Research Unit for Molecular Reproductive Endocrinology, Department of Chemical Pathology (C.A.F., B.J.F., J.S.D., R.P.M.), and Endocrine Laboratory, Department of Medicine (C.A.F., R.P.M.), University of Cape Town Medical School, Observatory 7925, South Africa

Address all correspondence and requests for reprints to: Robert P. Millar, Director, Medical Research Council Reproductive Biology Unit, 37 Chalmers Street, Edinburgh EH, United Kingdom. E-mail: Bob{at}ed-rbu.mrc.ac.uk

Cloning of GnRH receptors from several animal species has made it possible to investigate receptor function using site-directed mutagenesis. However, many mutant GnRH receptors exhibit decreased ligand binding, which makes analysis of their ligand binding characteristics technically difficult. To increase the affinity of binding to the GnRH receptor, a novel tracer ligand, ¹²⁵I-[His⁵,D-Tyr⁶]GnRH, was designed and synthesized to allow radioiodination at position 6 rather than the usual position 5.

In competition binding assays, total binding of ¹²⁵I-[His⁵,D-Tyr⁶]GnRH was higher than binding of a conventional tracer ligand, ¹²⁵I-[D-Ala⁶,N-MeLeu⁷,Pro⁹NHEt]GnRH. The bindable fractions and specific activities of both peptides were similar, and the receptor binding affinities of the unlabeled peptides were indistinguishable. However, comparison of the radiolabeled peptides in saturation binding assays showed that the affinity of the peptide,¹²⁵I-[His⁵,D-Tyr⁶]GnRH, (K_d, 0.19 nM), was approximately 2-fold higher than that of the conventional tracer. The increased binding of ¹²⁵I-[His⁵,D-Tyr⁶]GnRH has allowed the development of a sensitive GnRH receptor binding assay for analysis of mutant GnRH receptors that exhibit decreased ligand binding.

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