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

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 Cys(328) and the Thr(369)Thr(370) 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-beta-cyclodextrin, agents that disrupt lipid rafts such as caveolae, and coexpression of dominant-negative dynamin-1 (K44A) and caveolin-1 (Delta 1-81) mutants, effectively inhibited rapid agonist-induced internalization. Furthermore, cGnRH-Rs appeared to be mobilized to the beta-arrestin- and clathrin-coated, vesicle-mediated endocytic pathway upon beta-arrestin overexpression.