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Desensitization and Internalization of Human and XenopusGonadotropin-Releasing Hormone Receptors Expressed in T4 Pituitary Cells Using Recombinant Adenovirus¹

University Research Centre for Neuroendocrinology (J.N.H., M.T.M., H.M.E., T.H., J.B.U., C.A.M.), University of Bristol, Bristol, United Kingdom; Department of Cell Physiology and Pharmacology (G.B.W.), University of Leicester, Leicester LE1 9HN, United Kingdom; Medical Research Council Human Reproductive Science Unit (R.P.M.), Centre for Reproductive Biology, Edinburgh EH3 9ET, Scotland, United Kingdom; and Medical Research Council Research Unit for Molecular Reproductive Endocrinology (B.E.T., R.P.M., J.S.D.), Department of Medical Biochemistry, University of Cape Town, Observatory 7925, South Africa

Address all correspondence and requests for reprints to: Dr. Craig A. McArdle, University of Bristol, Department of Medicine, Bristol Royal Infirmary, Marlborough Street, Bristol BS2 8HW, United Kingdom. E-mail: craig.mcardle{at}bris.ac.uk

Nonmammalian vertebrates express at least two forms of GnRH and distinct forms of GnRH receptor (GnRH-R) have coevolved with their ligands. Mammalian and nonmammalian GnRH-R have key structural differences (notably the lack of C-terminal tails in mammalian GnRH-R) and comparative studies are beginning to reveal their functional relevance. However, cellular context and receptor density influence G protein-coupled receptor function and may be important variables in such work using heterologous expression systems. Here we report a comparative study using T4 cells (gonadotrope progenitors that lack endogenous GnRH-R) transfected with a mammalian (human) or nonmammalian (Xenopus laevis type I) GnRH-R. Because conventional transfection strategies proved inefficient, recombinant adenovirus expressing these receptors were constructed, enabling controlled and efficient GnRH-R expression. When expressed in T4 cells at physiological density, these GnRH-Rs retain the pharmacology of their endogenous counterparts (as judged by ligand specificity in radioligand binding and inositol phosphate accumulation assays) but do not activate adenylyl cyclase and are not constitutively active. Moreover, the Xenopus GnRH-R rapidly desensitizes and internalizes in these cells, whereas the human GnRH-R does not, and the internalization rates are not dependent upon receptor number. These data extend studies in COS, HEK, and GH₃ cells showing that other GnRH-R with C-terminal tails desensitize and internalize rapidly, whereas tail-less mammalian GnRH-R do not. Retention of these distinctions at physiological receptor density in gonadotrope lineage cells, supports the argument that the evolution of nondesensitizing mammalian GnRH-Rs is functionally relevant and related to the development of mammalian reproductive strategies.

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