CLONING AND FUNCTIONAL EXPRESSION OF THE LUTEINIZING-HORMONE RECEPTORCOMPLEMENTARY DEOXYRIBONUCLEIC-ACID FROM THE MARMOSET MONKEY TEST - ABSENCE OF SEQUENCES ENCODING EXON-10 IN OTHER SPECIES

Based on sequence homologies among the human, porcine, rat, and mouse genes for the LH receptor (LHR), overlapping partial fragments of LHR complementary DNAs (cDNAs) were multiplied from marmoset monkey testic ular RNA using reverse transcription-PCR. Ligations of the individual cDNA fragments generated a full-length monkey LHR cDNA (2031 bp) conta ining the complete amino acid-coding sequence (676 amino acids). North ern hybridization analysis of monkey testicular RNA, using a complemen tary RNA probe corresponding to the full-length cDNA, demonstrated maj or transcripts of 5.5 and 1.4 kilobases and minor ones of 4.0, 2.7, an d 1.9 kilobases. Sequence analysis of the monkey LHR cDNA revealed a s triking feature, i.e. the absence of an 81-bp nucleotide sequence corr esponding to exon 10, present in the LHR cDNAs of all other species st udied to date. The monkey LHR cDNA displayed 83-94% overall sequence h omology with the other mammalian LHR cDNAs. Reverse transcription-PCR with human exon 10-specific primers demonstrated the total absence of this sequence from the monkey LHR messenger RNA. Southern hybridizatio n of monkey genomic DNA using a human exon 10 probe demonstrated its p resence in the monkey gene and that it is totally spliced out from the primary transcript. COS cells transfected with the monkey LHR cDNA sh owed similar high affinity (K-d = 0.25 nmol/liter) of [I-125]iodo-hCG binding as those transfected with human LHR cDNA (K-d = 0.20 nmol/lite r). The cells expressing the recombinant monkey and human LHR displaye d similar responses of extracellular cAMP and inositol trisphosphate t o hCG. In conclusion, marmoset monkey LHR seems to lack the sequence c orresponding to exon 10 of the LHR gene in other mammalian species. Th e truncation does not alter LHR function, as the monkey receptor prote in bound hCG and evoked cAMP and inositol trisphosphate responses comp arable to those of the human LHR containing the exon 10-encoded struct ure. As the sequence homologous to exon 10 is missing in the other two glycoprotein receptors, i.e. those of FSH and TSH, this extra exon is apparently inserted into the LHR messenger RNA of some species during evolution from intronic sequences by a change in alternative splicing .