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.
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
M
M
Chopineau
N
N
Martinat
C
C
Galet
F
F
Guillou
Y
Y
Combarnous
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