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.

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