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 baeLH/CG was used to identify sequences in the b-subunit involved in the secretion and activities of the hormone. The C-terminal region of the b-subunit was progressively truncated. All resulting truncated single-chains were secreted in the media as detected by an anti-bpeptide 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 b-subunit (b109aeLH/CG) resulted in a 90% decrease in the recognized correctly folded hormone compared with the full-length baeLH/CG single-chain and no properly folded hormone was detected in the secretion medium when the last 46 amino acids of the b-subunit were deleted (b103aeLH/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 baeLH/CG (b104-109Alaa) led to a 50% decrease in the production of properly folded hormone in COS-7 as well as in aT3 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 b eLH/CG subunit is essential for the secretion of a fully folded baeLH/CG and for its FSH activity but not for its LH activity.

In order to better understand the cellular mechanisms underlying LH and FSH secretion, we have addressed the contribution of lipid rafts to the secretion of gonadotropins. We used methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering agent, on an LbetaT2 murine gonadotroph cell line and on primary cultures of ovine pituitary cells. We found that in both systems, cholesterol depletion by MbetaCD induced a fast and substantial release of LH in the absence of natural stimulation by GnRH. In ovine pituitary cells, MbetaCD-mediated LH release was shown to be independent of protein synthesis. Twenty-four hours after MbetaCD treatment, there was no loss of cell viability and full recovery of LH secretory capabilities, as determined by GnRH or MbetaCD treatment. In addition, our data suggest the existence of a pool of LH that is not released by GnRH treatment but that is released by MbetaCD treatment. Finally, in ovine pituitary cells, MbetaCD treatment induced FSH secretion. Importantly, these in vitro data are supported by in vivo studies, because MbetaCD injected into the pituitary glands of anaesthetized sheep reproducibly induced a peak of LH release.

Identification of amino-acids in the a-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin M Chopineau, N Martinat, J F Gibrat 1 , C Galet, F Lecompte, F Foulon-Gauze, C Pourchet, F Guillou and Y Combarnous AbstractObjective: To identify amino-acids in the a-subunit important for expression of heterospecific FSH activity of horse (e) LH/choriogonadotropin (CG) (eLH) and donkey (dk) LH/CG (dkLH) (FSH/LH ratio ten times higher for eLH than for dkLH); this FSH activity absolutely requires an equid (donkey or horse) a-subunit combined with an equid b-LH subunit. Design: Chimeric a-subunits possessing the first 63 amino-acids of the porcine (p) and the last 33 amino-acids of the donkey a-subunit (ap-dk) and the inverse (adk-p) were constructed. Porcinespecific amino-acids were introduced by mutagenesis in donkey a-subunit at positions 70, 85, 89, 93 and 96 (adk5xmut), 18 (adk K18E ) or 78 (adk I78A ). Methods: These different a-subunits were co-transfected in COS-7 cells with b-eLH, b-dkLH and b-eFSH. The LH and FSH bioactivities of the dimers were then assessed in two heterologous in vitro bioassays. Results: ap-dk or adk-p exhibited FSH activity when co-expressed with b-eLH but not with b-dkLH. adk K18E or adk I78A gave hybrids with no FSH activity and important LH activity when expressed with b-dkLH. adk I78A /beLH displayed an FSH/LH ratio as low as that of dkLH. However, mutation at 78 in a-dk had no effect on FSH bioactivity when co-expressed with b-eFSH. Conclusions: Amino-acids present in both the first two-thirds and the last third of the a-subunit of equid LHs are involved in their heterologous biospecificity. Ile a78 exerts as strong an influence on it as the b102-103 residues. By contrast, this residue plays no role in the FSH specificity of eFSH.

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, radioreceptor 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 125 I-eLH with high affinity (K d 3·8 10 10 M). On a molar basis, eCG competed with 125 I-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 functional eLH/CG-R. The differences between eLH and eCG activities towards this receptor will be useful in studies of the influence of carbohydrates on gonadotrophin receptor binding and activation.

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