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.

The aim of this study was to increase understanding of the occurrence and regulation of chicken gonadotropin releasing hormone I (cGnRH I) and chicken gonadotropin releasing hormone receptor (cGnRH-R) mRNA variants in the hypothalamic-pituitary-testicular axis (HPTA). The study was carried out in the cockerel. Fully processed cGnRH I mRNA (cGnRH Ia) and a variant transcript (cGnRH Ib) with a retained intron 1 were observed in the preoptic/anterior hypothalamus (POA), the basal hypothalamus, anterior pituitary gland, and testes. Fully processed cGnRH-R mRNA (cGnRH-Ra) and a variant transcript (cGnRH-Rb) with a deletion were detected in the same tissues. In juvenile cockerels, concentrations of cGnRH Ia and b in the POA increased after castration, and this was prevented by oestrogen treatment. In the anterior pituitary gland, the concentration of cGnRH-Ra increased after castration and this was reversed by oestrogen treatment. In intact adult cockerels, oestrogen treatment depressed plasma luteinizing hormone but did not affect concentrations of cGnRH I and cGnRH-R mRNAs in the POA, basal hypothalamus, and anterior pituitary gland, suggesting that locally produced oestrogen, by aromatization, may exert maximal suppression on cGnRH I and GnRH-R mRNAs. In intact adult cockerels, the concentrations of cGnRH Ia and b in the testis, but not cGnRH-Ra and b, were depressed by oestrogen treatment. It was concluded that fully processed and variant cGnRH I and cGnRH-R mRNAs occur in all components of the HPTA. Oestrogen appears to play a role in the regulation of cGnRH Ia and b in the POA and testes, and of cGnRH-Ra in the POA and anterior pituitary gland.

The majority of vertebrates have two or three forms of gonadotropin-releasing hormone (GnRH), which appear to have arisen by successive gene duplication events. This suggests the possibility of concordant gene duplications of the GnRH receptor to produce two or more cognate receptors. Since the extracellular loop 3 (EC3) domain of mammalian GnRH receptors plays a role in distinguishing the different forms of GnRH, we have contemplated that the sequence of this domain will differ significantly in the putative cognate receptors. Degenerate oliognucleotides encoding the sequences of the transmembrane domains preceding and following EC3 were used for PCR amplification of genomic DNA from zebrafish (Brachydanio rerio), goldfish (Carassius auratus), African clawed frog (Xenopus laevis), chicken (Gallus domesticus), and lizard (Agama atra). Isolation and sequencing of specific clones revealed that they fell into three groups. Two of these were most similar to the mammalian pituitary GnRH receptor and were therefore designated Type IA and Type IB. The third form (designated Type II) was most different from the others and was identified inXenopus,lizard, and human DNA. These findings support the concept of the existence of three distinct GnRH receptors, which have evolved in conjunction with three distinct GnRH ligand classes present in many vertebrates.

We report on the cloning of the full-length complementary DNA for the chicken TRH receptor. Although the TRH receptor has been cloned from several mammalian species, this is the first report from another vertebrate class. The ligand binding pocket, which is situated in the transmembrane helices of the mouse and rat TRH receptors, is completely conserved in the chicken receptor. Pharmacological studies (receptor binding and signaling) employing several TRH analogs revealed that there are no significant differences between the chicken and mouse receptors. These findings show that there have been considerable evolutionary constraints on TRH receptor structure and function. Several truncated forms of the chicken TRH receptor that appear to retain a part of an intron and are truncated in the putative third intracellular loop were also cloned, but were nonfunctional. This study provides a useful tool for further studies on the roles of TRH in avian growth and TSH regulation.

The chicken gonadotropin-releasing hormone receptor (GnRH-R) is notable for having a cytoplasmic C-terminal tail, which is not present in the mammalian GnRH-Rs. We report here that the cytoplasmic tail mediates rapid agonist-promoted receptor internalization. The chicken GnRH-R mediated internalization of gonadotropin-releasing hormone (GnRH) agonist (125I[His5-D-Tyr6]GnRH) at a rate of 11.3%.min-1, compared with only 0.71 %.min-1 for the human GnRH-R. To determine whether the presence of the cytoplasmic tail was responsible for the more rapid internalization kinetics of the chicken GnRH-R we truncated the tail after the Ile336 residue (S337stop). Receptor-mediated internalization of GnRH agonist by the S337stop-chicken GnRH-R was much slower than in the wild-type chicken receptor, and was similar to the wild-type human GnRH-R (0.55 %.min-1). These data indicate that rapid agonist-promoted internalization of the chicken GnRH-R is mediated through elements in the cytoplasmic C-terminal tail, distal to or including Ser337 and suggests that elimination of the C-terminal tail during evolution of mammalian GnRH-Rs may be related to its effects on internalization.