Endocrine Abstracts (2001) 2 P82

A Threonine-Doublet within the Carboxyl-Terminal Tail of a Non-Mammalian Gonadotropin-Releasing Hormone Receptor is Critical for Rapid Agonist-Induced Internalization

AJ Pawson1,2, J Lopes2, AA Katz2, SR Maudsley1, R Sellar1, N Miller1, Y-M Sun2, RP Millar1,2 & JS Davidson2

1Human Reproductive Sciences Unit, Medical Research Council, Edinburgh, United Kingdom; 2Departments of Chemical Pathology and Medical Biochemistry, University of Cape Town Medical School, Cape Town, South Africa.

The chicken gonadotropin-releasing hormone receptor (cGnRH-R) is notable for having a cytoplasmic carboxyl-terminal tail, which is not present in the mammalian GnRH-Rs. We have previously shown that the cGnRH-R undergoes rapid agonist-induced internalization, and requires the carboxyl-terminal tail for this process. To investigate the role of the carboxyl-terminal tail of the cGnRH-R in relation to its rapid internalization, and to identify the key residues involved, a series of mutant constructs were generated, whereby either the tail was progressively truncated, or serine and threonine residues located in the tail substituted with alanine. Truncations of the carboxyl-terminal tail resulted in receptors that internalized GnRH agonist at a slower rate, and to a lower extent than the wild type receptor. Our data demonstrate the presence of signals for agonist-induced internalization in two regions, from Ser337 to Ser346, and Asp356 to the carboxyl-terminus. We show that a threonine-doublet located near the carboxyl-terminus plays a critical role in mediating rapid internalization. Furthermore, we provide evidence to suggest that the cGnRH-R expressed in COS-7 cells preferentially undergoes rapid agonist-induced internalization in caveolae, in a dynamin-dependent manner. Additionally, cGnRH-Rs are mobilized to the beta-arrestin- and clathrin-coated vesicle-mediated endocytic pathway upon beta-arrestin overexpression.

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