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Multiple Determinants for Rapid Agonist-Induced Internalization of a Nonmammalian Gonadotropin-Releasing Hormone Receptor: A Putative Palmitoylation Site and Threonine Doublet within the Carboxyl-Terminal Tail Are Critical

Human Reproductive Sciences Unit, Medical Research Council (A.J.P., S.R.M., R.P.M.), Edinburgh, United Kingdom EH16 4SB; and Divisions of Chemical Pathology and Medical Biochemistry, University of Cape Town Medical School (A.J.P., J.L., A.A.K., Y.-M.S., J.S.D., R.P.M.), Cape Town 7925, South Africa

Address all correspondence and requests for reprints to: Prof. Robert P. Millar, Human Reproductive Sciences Unit, Medical Research Council, University of Edinburgh Chancellor’s Building, 49 Little France Crescent, Edinburgh, United Kingdom EH16 4SB. E-mail: r.millar{at}hrsu.mrc.ac.uk.

The chicken GnRH receptor (cGnRH-R) differs from all mammalian GnRH-Rs in possessing a cytoplasmic carboxyl-terminal tail. We have previously demonstrated that the cGnRH-R undergoes more rapid agonist-induced internalization than the mammalian GnRH-Rs and requires the carboxyl-terminal tail for this process. To investigate the structural determinants mediating this rapid internalization, a series of mutant receptors was generated, including progressive truncations of the tail and substitution of serine and threonine residues with alanine. Truncation of the carboxyl-terminal tail to position 366 and then to position 356 resulted in a progressive attenuation of the rate and total extent of receptor internalization. However, truncation between positions 356 and 346 did not alter the kinetics of internalization further, whereas a further truncation to position 337 resulted in an additional marked reduction of internalization. We show that the membrane-proximal Cys³²⁸ and the Thr³⁶⁹Thr³⁷⁰ doublet located in the distal carboxyl terminus play a critical role in mediating rapid internalization. We demonstrate that the cGnRH-R, when expressed in both COS-7 and HEK 293 cells, preferentially undergoes rapid agonist-induced internalization in a caveolae-like, dynamin-dependent manner. These conclusions are based on our observation that pretreatments with filipin and methyl-ß-cyclodextrin, agents that disrupt lipid rafts such as caveolae, and coexpression of dominant-negative dynamin-1 (K44A) and caveolin-1 (1–81) mutants, effectively inhibited rapid agonist-induced internalization. Furthermore, cGnRH-Rs appeared to be mobilized to the ß-arrestin- and clathrin-coated, vesicle-mediated endocytic pathway upon ß-arrestin overexpression.

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