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alpha-Subunit dissociated from glycoprotein hormones has been previously shown to stimulate rat pituitary lactotroph differentiation and proliferation. However, whether the free form of the alpha-subunit (free alpha) can also play such a role is not known. To test whether free alpha may act on prolactin (PRL) release from ovine foetal pituitaries, this molecule was purified and two major isoforms, alphaA and alphaB were isolated. Free alphaA was found to be more acidic and more hydrophobic than both free alphaB and ovine LH alpha-subunit (oLHalpha). Free alphaA and oLHalpha exhibited a molecular mass of 14 kDa as determined by mass spectrometry, whereas free alphaB displayed a molecular mass of only 13.5 kDa because of its truncated N-terminus. All three alpha molecules bear mature-type N-linked saccharide chains including Nacetyl galactosamine residues but none of them contains O-linked oligosaccharide. The free alphaA isoform, more than the oLHalpha, was able to stimulate PRL release from ovine foetal pituitary explants in culture, whereas the free alphaB isoform displayed no activity. Moreover, the free alphaA and alphaB isoforms were able to recombine with the ovine LH beta-subunit (oLHbeta). The free alphaB/oLHbeta, and the oLHalpha/oLHbeta dimer were 4-fold more active than the free alphaA/oLHbeta dimer in a specific LH radioreceptor assay and in the stimulation of testosterone release from rat Leydig cells. The present study demonstrates that the two free alpha isoforms of ovine glycoprotein hormones exhibit distinct efficiencies in stimulating PRL release from ovine foetal pituitaries. Moreover, despite their identical ability to recombine with the oLHbeta, the free alpha isoform, which is the most efficient on PRL release, is the least efficient in conferring LH activity on the alpha/beta dimer.
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The C-terminal region of the beta subunit of the human chorionic gonadotrophin (hCG) is implied in heterodimer stability (beta26-110 disulphide bridge), in vitro LH bioactivity (region beta102-110) and in in vivo LH bioactivity (beta CTP). Like the hCG beta, the equine eLH and eCG beta subunits, also possess a C-terminal extension (CTP). But, in contrast to hCG, eLH and eCG bind to both LH and FSH receptors in species other than the horse. This allows investigation of the roles of the beta subunit C-terminal region of a eLH/CG recombinant molecule on both LH and FSH activities. To do so, the CTP was deleted and/or the beta26-110 disulphide bond was mutated and the resulting mutated beta subunits were transiently co-expressed with common alpha subunit in COS7 cells. These regions were also deleted in a betaalphaeLH/CG single chain also expressed in COS7 cells. The hormones produced were characterized by different ELISAs and in vitro LH and FSH bioassays. Mutation of the 26-110 disulphide bond and deletion of the betaCTP led to a decrease in eLH/CG heterodimer production. Double mutation promoted an additive effect on production of the heterodimer and of the corresponding tethered eLH/CG. The elimination of the beta26-110 disulphide bond in the betaalpha single chain had no effect on its production. However, neither the 26-110 disulphide bond nor the CTP mutations affected dimer stability and bioactivities of the secreted heterodimers and/or single chain molecules. Therefore, in contrast to hCG, the 26-110 S-S bond of the recombinant eLH/CG beta subunit does not seem to be essential for eLH/CG dimer stability upon secretion and expressing LH and FSH bioactivities.
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Horse LH/CG (eLH/CG) and donkey LH/CG (dkLH/CG) are strictly LH-specific in their respective homologous species. However, both bind to the FSH receptors from non-equid species, whereas the zebra hormone (zbLH/CG) does not. The FSH/LH ratio of eLH/CG and of the alphadkbetae hybrid is about tenfold higher than that of dkLH/CG and of the alphaebetadk hybrid, showing that the betae subunit contains the structural features responsible for the high FSH activity of eLH/CG. Only six amino acid positions (51, 94, 95, 102, 103 and 106) are unique to the betae subunit when compared with the betadk and betazb subunits. The Gly-Pro and Val-Phe sequences in positions 102-103 of betadk and betae respectively were swapped by site-directed mutations and the mutated beta-subunits cDNAs were cotransfected in COS cells with either alphae or alphadk subunit cDNA. Other mutations were also introduced in 102-103 dkLH/CG beta-subunit: Ala-Ala, Gly-Ala or Ala-Pro. These mutations with Ala-Ala, Gly-Ala or Ala-Pro in the 102-103 betadkLH/CG subunit did not change the FSH/LH ratio of dkLH/CG but the Gly(102)-Pro(103)-->Val(102)-Phe(103) mutation promoted a marked increase in the FSH/LH activity ratio. This was observed with the two heterodimers containing alphae or alphadk. Conversely, the Val(102)-Phe(103) mutation in betae led to a dramatic drop in FSH/LH activity ratio of eLH/CG, to a level similar to that of dkLH/CG. Since all FSHs possess a Gly residue at position 104, we introduced the Gly(102)-Pro(103)-Arg(104)-->Val(102)-Phe(103)-Gly(104) mutation in betadk with the expectation that the increase in FSH activity observed with the Gly(102)-Pro(103)-->Val(102)-Phe(103) mutation could be potentiated. In fact, the additional Arg(104)-->Gly(104) mutation was found to abolish the increase in FSH activity observed with Gly(102)-Pro(103)-->Val(102)-Phe(103). Mutations Gly(102)-Pro(103)-->Val(102)-Arg(103) or Gly(102)-Pro(103)-Lys(104)--> Val(102)-Arg(103)-Gly(104) were also introduced in human CGbeta (hCGbeta) to compare the impact of these amino acid changes in the well-studied gonadotrophin hCG. The betahCG mutants obtained, co-expressed either with the human or the horse alpha-subunit, did not display any FSH activity. In conclusion, the 102-104 sequence in eLH/CG beta-subunits appears to be of utmost importance for their binding to FSH receptors. However, these results obtained with equid beta-subunits are not transposable to other gonadotrophins as similar mutations in hCGbeta did not lead to any increase in FSH activity.