CHARACTERIZATION OF CDNA-ENCODING N-TERMINAL REGION OF THE QUAIL LUTROPIN RECEPTOR

For understanding the evolutionary relationships between gonadotropins [GTHs: lutropin (LH) and follitropin (FSH)] and their receptors, we a ttempted to characterize the extracellular domain of the receptors, wh ich is thought to be a key region of hormone binding, in nonmammalian species, and to compare the information to that of the known mammalian data. For this purpose, we designed two sets of sense and antisense o ligonucleotides as polymerase chain reaction (PCR) primers, referring to the known mammalian GTH receptors, such as LH receptors of human, p ig, and rat, and FSH receptors of human and rat. All possible combinat ions of the primers showed the successful amplification of cDNA of LH receptor without contamination of FSH receptor cDNA from rat testicula r RNA samples. With these primers, reverse transcription (RT)-PCR was applied to the gonads of nonmammalian species (quail, snake, tortoise, newt, and bullfrog). Only the quail, however, showed the specific amp lification when only one set of primers was used. Thus, the PCR produc t of the quail was used as a probe for Northern blot and in situ hybri dization. By Northern blot analysis, a single size of mRNA (3 kb) was identified from quail testicular poly A(+) RNA. The distribution of mR NA visualized by in situ hybridization was limited only on Leydig cell s of quail testis. These results suggest that a part of the quail LH r eceptor cDNA was amplified by RT-PCR. The nucleotide and predicted pep tide sequences of this amplified cDNA were compared with those of mamm alian receptors. The size of characterized cDNA sequence was 519 bp, w hich is completely identical with those of mammalian LH receptors. The homology of both cDNA and predicted peptide was about 70% of those of mammalian LH receptors (intramammalian, about 80%). In spite of the r elatively low homology, the positions of cystein residues and potentia l N-linked glycosylation sites in the peptide were completely conserve d in all species compared (human, pig, rat, and quail). The conserved portions indicate their importance for the molecular conformation and specific ligand binding activity of LH receptors. (C) 1994 Academic Pr ess, Inc.