beta-Subunit 102-104 residues are crucial to confer FSH activity to equine LH/CG but are not sufficient to confer FSH activity to human CG.
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.
Chopineau
M
M
Station de Physiologie de la Reproduction et des Comportements (PRC), Institut National de la Recherche Agronomique, UMR 6073, 37380 Nouzilly, France. chopinea@tours.inra.fr
Martinat
N
N
Galet
C
C
Guillou
F
F
Combarnous
Y
Y
D000818:Animals; D001681:Biological Assay; D019556:COS Cells; D006063:Chorionic Gonadotropin / Q000235:genetics; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D018397:Equidae / Q000378:metabolism*; D005640:Follicle Stimulating Hormone / Q000235:genetics* / Q000378:metabolism; D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics; D006801:Humans; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism; D007986:Luteinizing Hormone / Q000235:genetics* / Q000378:metabolism; D008297:Male; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013045:Species Specificity; D013739:Testosterone / Q000096:biosynthesis; D014162:Transfection
D006063:Chorionic Gonadotropin; D017931:DNA Primers; D037201:Follicle Stimulating Hormone, beta Subunit; D015292:Glycoprotein Hormones, alpha Subunit; D011994:Recombinant Proteins; D013739:Testosterone; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone