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.
Pawson
Adam J
AJ
Human Reproductive Sciences Unit, Medical Research Council, Edinburgh, United Kingdom EH16 4SB.
Maudsley
Stuart R
SR
Lopes
John
J
Katz
Arieh A
AA
Sun
Yuh-Man
YM
Davidson
James S
JS
Millar
Robert P
RP
D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000235:genetics / Q000378:metabolism; D002645:Chickens / Q000235:genetics*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D034281:Dynamins / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D019308:Palmitic Acid / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011966:Receptors, LHRH / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000071557:beta-Arrestins
D019390:Arrestins; C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D007295:Inositol Phosphates; D011966:Receptors, LHRH; D000071557:beta-Arrestins; D019308:Palmitic Acid; D013912:Threonine; D007987:Gonadotropin-Releasing Hormone; D034281:Dynamins; D003545:Cysteine