The genomic structure of the LH receptor is important to our understanding of its expression mechanisms, functional domains, relationships with other hormone receptors, and evolution. We have isolated four overlapping cosmid clones and six subgenomic clones of the rat LH receptor gene. They span a total of 95.6 kilobases (kb) and extend from 23 kb upstream of the translation start site to 13 kb down-stream of the stop codon. In addition, part of the human LH receptor gene has been isolated. The coding region of the rat hormone receptor gene spans over 60 kb and consists of 11 exons and 10 introns. Southern blots hybridized with exon 1 and exon 11 probes as well as gene dose analyses demonstrate that a single copy gene encodes the rat LH receptor. Sequence comparison suggests that the porcine and human LH receptor genes have similar, if not identical, exon-intron structures. There is no consensus cAMP-responsive element within 600 basepairs up-stream of the translation start site in spite of the cAMP responsiveness of the LH receptor gene. There are, however, unconventional cAMP-responsive elements in the region: one which is identical, several which are homologous to the activating protein-2-binding elements, CCCCAGGC, and several sequences which are similar to the G-rich cAMP-responsive element found in P450c21, a steroid 21-hydroxylase. The first 10 exons encode the N-terminal half of the molecule, while exon 11 encodes the C-terminal half of the molecule. This last exon is the same in the rat and human genes. The DNA and amino acid sequences of the first 10 exons show significant similarities and reveal repetitive sequence motifs. They have similar sizes which occur in the range of 69 and 183 bases; 8 of them are from 69-81 bases. Despite these remarkable similarities, structural predictions of exons 1-10 show a diversity of structures. The N-terminal half of the LH receptor appears to have a folded structure, with frequent turns and an extensive surface area. Part of the surface is predicted to be covered by amphiphilic helices and beta structures, types of secondary structure frequently found at the interfaces between subunits or between 2 interacting molecules. The introns dividing these exons also share many similarities.(ABSTRACT TRUNCATED AT 400 WORDS)

Trophoblastic neoplasms and choriocarcinoma cells express high levels of the hCG receptor. The hCG receptor is encoded by a single gene in chromosome 2p21-p16, spanning over -70 kb with 11 exons and 10 introns. Multiple mRNA species are produced from the gene utilizing two proximal promoters and several Sp-1 elements as well as proximal and distal suppressors. In fact, regulatory proteins which bind to one of these suppressors are expressed less in choriocarcinoma cell lines than in placenta. The LH/CG receptor is comprised of two structurally and functionally distinct domains, extracellular N-terminal exodomain and membrane embedded endodomain. These two domains can separately be expressed and processed, including folding. The exodomain alone has the high affinity hormone binding site but is not capable of generating hormonal signal. In contrast, the endodomain alone has the site for receptor activation. These two domains contact each other in holo-receptor and split receptor. This interaction, particularly through exoloops 2 and 3, constrains the high affinity hormone binding at the exodomain. Conversely, the exodomain could be involved in receptor activation. Therefore, these two domains are not entirely independent although they can be independently synthesized and processed. The existing evidence indicate that hCG and the receptor undergo multiple stages of interactions leading to receptor activation. Initial high affinity binding of hCG to the exodomain results into conformational adjustments of the hCG/exodomain complex. This leads to the secondary, low affinity contact of the hCG/exodomain complex with the endodomain. This secondary contact is responsible for generating signals. They are transduced through TM to the cytoplasmic portion (cytoloops and the C-terminal tail) of the receptor and then, transferred to cytoplasmic signaling molecules, such as G protein. Mutations in the exodomain and endodomain (N-extension, exoloops, TM, cytoloops, and cytoplasmic tail) have the potential to interfere with receptor activation at different steps, signal generation, transduction and transfer. Binding of hCG to the LH/CG receptor are known to induce two signals, one for adenylyl cyclase/ cAMP and the other for phospholipase C/inositol phosphate/diacylglycerol. The cAMP signal and IP signal diverge at the surface of the receptor. These independent signals are separately transduced through the transmembrane domains to the cytoplasmic part of the receptor, indicating the existence of the distinct transducers for each of the signals. Furthermore, it is likely that the divergent signals are separately transferred to cytoplasmic signal molecules such as G protein. In addition, each of the cAMP signal and IP signal consists of at least three separate subsignals: affinity signal, maximal production (efficacy) signal and basal level signal. In heterodimeric hCG, there are distinct parts responsible for high affinity receptor binding and receptor activation. Particularly, the C-terminal reduces of the alpha subunit play a cr...

The LH/CG receptor is uniquely expressed in the gonads of both sexes at specific stages of development. In ovaries, its expression marks particular steps of the ovulation cycle. An enigmatic aspect of expression of the LH/CG receptor is the dramatically diverse transcript sizes [from 7 to < 1 kilobase (kb)] and development-dependent expression of different sizes of mRNAs. It has been thought that mRNAs larger than 2.1 kb encode full-length receptors, whereas those smaller than 2.1 kb encode truncated receptor, because the full-length coding sequence is 2.1 kb. As a first step in elucidation of these diverse mRNAs and corresponding proteins, we have produced a series of cDNA clones and determined their DNA sequences and deduced the amino acid sequences of the resulting proteins. Our data demonstrate that variant mRNAs are produced by alternate splicing and polyadenylation, and they encode significantly shorter truncated receptor peptides. Surprisingly, many of these variant mRNAs are larger than 2.1 kb, and some are 4.2 kb. Some of them are polyadenylated in introns 3, 4, and 10. These alternate mRNAs were successfully expressed in 293 cells to produce receptor peptides 81, 116, and 294 amino acids in length compared to the wild-type receptor, which consists of 674 amino acids. Although these receptor peptides are not secreted, they are capable of binding the hormone, indicating the presence of a hormone contact site(s) in the short peptide fragments, particularly the N-terminal 81-amino acid segment. The data presented here will be helpful for understanding the functions of different sizes of mRNAs and also be valuable in studies designed to investigate whether individual cells express a specific message or multiple messages and how different classes of LH/CG receptor mRNAs are selectively expressed dependent on differentiation and development of the gonads.

The LH/CG receptor is uniquely expressed in the gonads of both sexes at specific stages of development. In ovaries, its expression marks particular steps of the ovulation cycle. An enigmatic aspect of expression of the LH/CG receptor is the dramatically diverse transcript sizes [from 7 to < 1 kilobase (kb)] and development-dependent expression of different sizes of mRNAs. It has been thought that mRNAs larger than 2.1 kb encode full-length receptors, whereas those smaller than 2.1 kb encode truncated receptor, because the full-length coding sequence is 2.1 kb. As a first step in elucidation of these diverse mRNAs and corresponding proteins, we have produced a series of cDNA clones and determined their DNA sequences and deduced the amino acid sequences of the resulting proteins. Our data demonstrate that variant mRNAs are produced by alternate splicing and polyadenylation, and they encode significantly shorter truncated receptor peptides. Surprisingly, many of these variant mRNAs are larger than 2.1 kb, and some are 4.2 kb. Some of them are polyadenylated in introns 3, 4, and 10. These alternate mRNAs were successfully expressed in 293 cells to produce receptor peptides 81, 116, and 294 amino acids in length compared to the wild-type receptor, which consists of 674 amino acids. Although these receptor peptides are not secreted, they are capable of binding the hormone, indicating the presence of a hormone contact site(s) in the short peptide fragments, particularly the N-terminal 81-amino acid segment. The data presented here will be helpful for understanding the functions of different sizes of mRNAs and also be valuable in studies designed to investigate whether individual cells express a specific message or multiple messages and how different classes of LH/CG receptor mRNAs are selectively expressed dependent on differentiation and development of the gonads.

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