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The dual LH and FSH activity of the equine LH (eLH)/equine chorionic gonadotropin (eCG) in heterologous species makes eLH/CG a good model to study structure/function relationships of gonadotropins. In order to bypass the problem of intracellular association of the heterodimer, a recombinant single-chain βαeLH/CG was used to identify sequences in the β-subunit involved in the secretion and activities of the hormone. The C-terminal region of the β-subunit was progressively truncated. All resulting truncated single-chains were secreted in the media as detected by an anti-βpeptide antibody in reducing conditions. However, using a conformation sensitive ELISA we show that the truncated single-chains were differently recognized: deletion of the last 40 amino acids of the β-subunit (β109αeLH/CG) resulted in a 90% decrease in the recognized correctly folded hormone compared with the full-length βαeLH/CG single-chain and no properly folded hormone was detected in the secretion medium when the last 46 amino acids of the β-subunit were deleted (β103αeLH/CG). We thus focused on the six amino acids sequence 104–109, which belongs to the seat-belt region. Mutation of the 104–109 sequence in alanines in the full-length βαeLH/CG (β104–109Alaα) led to a 50% decrease in the production of properly folded hormone in COS-7 as well as in αT3 pituitary cells. Moreover, the FSH activity of this mutant was decreased by 70% whereas its LH activity remained intact. These data lead us to conclude that the 104–109 region of the β eLH/CG subunit is essential for the secretion of a fully folded βαeLH/CG and for its FSH activity but not for its LH activity.

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 sytem, 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 (K _d 3.8 × 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.