Molecular cloning of a novel luteinizing‐hormone/ human‐chorionic‐gonadotropin‐receptor cDNA

Abstract

The biological action of luteinizing hormone/human chorionic gonadotropin (lutropidchoriogonadotropin) in the ovary is mediated by interaction with its specific receptor. Lutropinlchoriogonadotropin-receptor hnRNA is processed into multiple mRNAs. However, nucleotide sequences for many of the transcripts, including the major form (6.7 kb), have yet to be determined. In an attempt to identify a cDNA encoding the major transcript, we have isolated a 3.5-kb cDNA clone from a rat ovary cDNA library. The 3.5-kb cDNA recognized only two (6.7 kb, 4.4 kb) of the three (6.7, 4.4, 2.6 kb) ovarian lutropidchoriogonadotropin-receptor transcripts when used as a probe. The first 732 nucleotides of the newly identified 3.5-kb cDNA showed 98% identity to the 3' untranslated region (3' UTR) of the previously cloned cDNA corresponding to the 4.4-kb transcript. Southern blot analysis indicated that the 3.5-kb cDNA and the C-terminal domain of the lutropinlchoriogonadotropin-receptor originate from the same gene. Oligonucleotide-directed cleavage of the 6.7-kb lutropin/ choriogonadotropin-receptor mRNA by RNase H revealed that the newly identified 3.5-kb cDNA is a 3' extension of the 4.4-kb transcript. We propose that the nucleotide sequence of the 6.7-kb lutropidchoriogonadotropin-receptor transcript, the major form found in rat ovary, contains a long 3' UTR, which has not been previously identified.The luteinizing hormonehuman chorionic gonadotropin (lutropidchoriogonadotropin) receptor is a glycoprotein expressed in rat ovary. The interaction of lutropidchoriogonadotropin with cell-surface receptors leads to activation of adenylate cyclase, which in turn, stimulates a number of intracellular functions, including steroidogenesis od. In rat ovary, one major 6.7-kb transcript, and two minor transcripts corresponding to 4.4 kb and 2.6 kb, and a less abundant 1.8-kb transcript have been reported [12, 14, 151. It is thought that the 3.0-kb cDNA cloned by Mac Farland et al. corresponds to the 4.4-kb transcript in rat ovary [4]. Although the major transcript in the ovary is 6.7 kb, its nucleotide sequence has not been established. One objective of this study was to determine the identity of the 6.7-kb transcript.A second objective was to determine whether the lutropidchoriogonadotropin-receptor transcript contains a previously unidentified 3' untranslated region (3' UTR), since this region has been implicated as a determinant of mRNA stability in other systems [ 171. We have previously shown that the receptor mRNA is post-transcriptionally regulated during ligand-induced down-regulation [ 141. Since specific sequences in the 3' UTR can modulate message stability 1171, we hypothesized that the major transcript may contain additional regulatory sequences, and thus a longer 3' UTR than formerly recognized. Our results show that we have cloned a lutropidchoriogonadotropin-receptor cDNA that encodes a novel 3' UTR that may serve as a focal point for determining mRNA slability. MATERIALS AND METHODS Construction of the C-terminal lutropid ...

hMAF, a Small Human Transcription Factor That Heterodimerizes Specifically with Nrf1 and Nrf2

“…Extension in the 3Ј direction is more likely considering that the 3Ј-untranslated region of our clone is extremely AT-rich and has a noncanonical poly(A) addition signal. The association of a short coding region with a relatively large mRNA, on the other hand, has already been reported for the small Maf proteins and for other genes (57)(58)(59). Alternatively the higher molecular mass bands could derive from cross-hybridization to related MAF oncogenes or be highly expressed splicing isoforms of the same hMAF gene.…”

G protein expression and second messenger formation in human granulosa cells

“…RM/l is a well characterized antibody that has been used successfully to demonstrate Gas in a variety of animal and human tissues (Morris et al, 1990;Europe-Finner et al, 1993). Strong functional evidence for an active Gas pathway is given by the finding that two unrelated agonists, hCG and PGE2, the receptors of which belong to the G protein-linked family (Hipkin et al, 1993;Bastien et al, 1994;Lu and Menon, 1994;Regan et al, 1994), provoke a large and rapid increase in cAMP production by granulosa cells. Strong functional evidence for an active Gas pathway is given by the finding that two unrelated agonists, hCG and PGE2, the receptors of which belong to the G protein-linked family (Hipkin et al, 1993;Bastien et al, 1994;Lu and Menon, 1994;Regan et al, 1994), provoke a large and rapid increase in cAMP production by granulosa cells.…”

“…The purpose of this paper was to study G protein expression in human granulosa cells, and to investigate the functional involvement in receptor activation of G proteins by measuring second messenger formation in response to agonists that oper¬ ate through G protein-linked receptors (Kimura et al, 1992;Lu and Menon, 1994). Human chorionic gonadotrophin was used to stimulate adenylyl cyclase (McFarland et al, 1989;Cooke et al, 1989), and oxytocin was used to activate PLC (Schrey et al, 1987;Phaneuf et al, 1993).…”

Regulation of Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Expression: A Perspective1

“…Four rat LH/hCG receptor transcripts of 6.7, 4.4, 2.6, and 1.8 kb in length have been identified in ovarian tissue [10]. Studies conducted in our laboratory have shown that the 6.7-kb transcript is the most abundant form found in rat ovary and is an extension of the 4.4-kb transcript in the 3Ј untranslated region [46]. In fact, all four transcripts appear to be derived from the same cDNA.…”