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Cloning and functional expression of the equine luteinizing hormone/chorionic gonadotrophin receptor.
Abstract: Pituitary equine luteinizing hormone (eLH) and fetal chorionic gonadotrophin (eCG) have identical polypeptidic chains, but different linked carbohydrates. In equine tissues, eCG and eLH bind only to the LH/CG receptor (eLH/CG-R) and have no FSH activity. However, radio-receptor assays on equine luteal or testicular tissues have shown that eCG binds to the eLH/CG-R with only 2-4% of the binding activity of eLH. In order to study the structure-function relationship of eLH and eCG in a homologous system, we undertook the cloning and functional expression of the eLH/CG-R. Based on sequence homologies among mammalian sequences for the LH/CG-R, overlapping partial fragments of LH/CG-R cDNAs were obtained from mare luteal RNA using reverse transcription-PCR and 5'-rapid amplification of cDNA ends. Ligations of the partial cDNA fragments encoded a part of the signal peptide followed by a putative 672 amino acid eLH/CG-R mature protein. The mature eLH/CG-R displayed 88.2-92.8% overall sequence homology with the other mammalian LH/CG-Rs and contained one unique seventh N-glycosylation site in its extracellular domain. COS-7 cells were transiently transfected with a cDNA construct encoding an engineered complete signal peptide and the mature eLH/CG-R. Membrane preparations from transfected COS-7 cells bound 125I-eLH with high affinity (Kd 3.8 x 10(-10) M). On a molar basis, eCG competed with 125I-eLH on membrane preparations with only 12.4% of the eLH binding activity. In transfected COS-7, both eLH and eCG increased the extracellular cAMP concentration in a dose-dependent manner, whereas eFSH did not. Furthermore, on a molar basis, eCG stimulated cAMP production with only 13.9% of the eLH stimulating activity. We conclude that the cloned cDNA encodes a The differences functional eLH/CG-R. between eLH and eCG activities towards this receptor will be useful in studies of the influence of carbohydrates on gonadotrophin receptor binding and activation.
Publication Date: 2004-12-14 PubMed ID: 15590981DOI: 10.1677/joe.1.05888Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The study focuses on the genetic cloning and functional expression of the equine luteinizing hormone (eLH) and chorionic gonadotrophin (eCG) receptor, primarily exploring the differential activities of eLH and eCG towards this receptor in horse tissues, which might aid our understanding of the impact of carbohydrates on gonadotrophin receptor binding and activation.
Study Objective and Background
- The study aims at understanding the structure-function relationship of eLH and eCG in a homologous system by cloning and functionally expressing the eLH/CG-R.
- Previous studies demonstrated that eCG and eLH bind only to the LH/CG receptor (eLH/CG-R) in equine tissues, with no FSH activity, but eCG had significantly lower binding activity to eLH/CG-R than eLH.
Methodology and Findings
- Researchers cloned overlapping partial fragments of LH/CG-R cDNAs from mare luteal RNA using methods such as reverse transcription-PCR and 5′-rapid amplification of cDNA ends.
- These cDNA fragments encoded a part of the signal peptide and a putative 672 amino acid eLH/CG-R mature protein.
- The resulting mature eLH/CG-R showed high sequence homology with other mammalian LH/CG-Rs and displayed a unique seventh N-glycosylation site in its extracellular domain.
- COS-7 cells were transiently transfected with a cDNA construct encoding the cloned signal peptide and the mature eLH/CG-R, which bound 125I-eLH with high affinity.
- Experiments found that eCG competed with 125I-eLH for binding to this receptor with only 12.4% of the eLH binding activity.
- Both eLH and eCG were found to increase the extracellular cAMP concentration in a dose-dependent manner in transfected COS-7 cells, while eFSH did not. eCG stimulated cAMP production with only 13.9% of the eLH stimulating activity.
Conclusion
- The cloned cDNA successfully encoded a functional eLH/CG-R. The differential activities of eLH and eCG towards this receptor will be useful in research studies focusing on understanding the influence of carbohydrates on gonadotrophin receptor binding and activation.
Cite This Article
APA
Saint-Dizier M, Foulon-Gauze F, Lecompte F, Combarnous Y, Chopineau M.
(2004).
Cloning and functional expression of the equine luteinizing hormone/chorionic gonadotrophin receptor.
J Endocrinol, 183(3), 551-559.
https://doi.org/10.1677/joe.1.05888 Publication
Researcher Affiliations
- Unité de Physiologie de la Reproduction et des Comportements, UMR 6175 INRA-CNRS-Université F. Rabelais de Tours-Haras Nationaux, 37 380 Nouzilly, France. stdizier@inapg.fr
MeSH Terms
- Amino Acid Sequence
- Animals
- Base Sequence
- COS Cells
- Cattle
- Cyclic AMP / metabolism
- Dose-Response Relationship, Drug
- Female
- Gene Expression
- Horses
- Humans
- Luteinizing Hormone / pharmacology
- Molecular Sequence Data
- Rats
- Receptors, LH / genetics
- Receptors, LH / metabolism
- Reverse Transcriptase Polymerase Chain Reaction
- Sequence Alignment
- Swine
- Transfection
Citations
This article has been cited 2 times.- Byambaragchaa M, Kang HJ, Park SH, Shin MG, Won KM, Kang MH, Min KS. Functional Divergence for N-Linked Glycosylation Sites in Equine Lutropin/Choriogonadotropin Receptors. Curr Issues Mol Biol 2025 Jul 25;47(8).
- Lösle M, Lin CW, Beil-Wagner J, Aebi M, Buch T. Comparison of pregnant mare serum gonadotropin products with surprising differences in protein content. Sci Rep 2025 Feb 25;15(1):6824.
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