Identification of amino-acids in the alpha-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin.

OBJECTIVE To identify amino-acids in the alpha-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) alpha-subunit combined with an equid beta-LH subunit. DESIGN Chimeric alpha-subunits possessing the first 63 amino-acids of the porcine (p) and the last 33 amino-acids of the donkey alpha-subunit (alphap-dk) and the inverse (alphadk-p) were constructed. Porcine-specific amino-acids were introduced by mutagenesis in donkey alpha-subunit at positions 70, 85, 89, 93 and 96 (alphadk5xmut), 18 (alphadkK18E) or 78 (alphadkI78A). METHODS These different alpha-subunits were co-transfected in COS-7 cells with beta-eLH, beta-dkLH and beta-eFSH. The LH and FSH bioactivities of the dimers were then assessed in two heterologous in vitro bioassays. RESULTS alphap-dk or alphadk-p exhibited FSH activity when co-expressed with beta-eLH but not with beta-dkLH. alphadkK18E or alphadkI78A gave hybrids with no FSH activity and important LH activity when expressed with beta-dkLH. alphadkI78A/betaeLH displayed an FSH/LH ratio as low as that of dkLH. However, mutation at 78 in alpha-dk had no effect on FSH bioactivity when co-expressed with beta-eFSH. CONCLUSIONS Amino-acids present in both the first two-thirds and the last third of the alpha-subunit of equid LHs are involved in their heterologous biospecificity. Ile alpha78 exerts as strong an influence on it as the beta102-103 residues. By contrast, this residue plays no role in the FSH specificity of eFSH.

[1]  M. Jeoung,et al.  Follicle-stimulating Hormone Interacts with Exoloop 3 of the Receptor* , 2002, The Journal of Biological Chemistry.

[2]  Satoshi Tanaka,et al.  Equine Follicle-Stimulating Hormone: Molecular Cloning of β Subunit and Biological Role of the Asparagine-Linked Oligosaccharide at Asparagine56 of α Subunit1 , 2001 .

[3]  P. Roey,et al.  Three-dimensional structure of human follicle-stimulating hormone. , 2001, Molecular endocrinology.

[4]  Y. Wang,et al.  The surface of alpha-subunit loop 1 distant from the subunit interface is exposed in the hCG lutropin receptor complex. , 2000, Biochimica et biophysica acta.

[5]  Y. Combarnous,et al.  Human chorionic gonadotropin with C-elongated alpha-subunit retains full receptor binding and partial agonist activity. , 2000, European journal of endocrinology.

[6]  C. Cambillau,et al.  Crystal structure of a ternary complex between human chorionic gonadotropin (hCG) and two Fv fragments specific for the alpha and beta-subunits. , 1999, Journal of molecular biology.

[7]  T. Ji,et al.  The α-Subunit of Human Choriogonadotropin Interacts with the Exodomain of the Luteinizing Hormone/Choriogonadotropin Receptor. , 1999, Endocrinology.

[8]  J. Dias,et al.  The human follitropin alpha-subunit C terminus collaborates with a beta-subunit cystine noose and an alpha-subunit loop to assemble a receptor-binding domain competent for signal transduction. , 1998, Biochemistry.

[9]  Y. Combarnous,et al.  Evidence that the alpha-subunit influences the specificity of receptor binding of the equine gonadotrophins. , 1997, The Journal of endocrinology.

[10]  O. P. Bahl,et al.  Effect of modification of the β-hairpin and long loops simultaneously in both α- and β-subunits on the function of human choriogonadotropin: part II , 1997, Molecular and Cellular Endocrinology.

[11]  Y. Combarnous,et al.  Expression of horse and donkey LH in COS-7 cells: evidence for low FSH activity in donkey LH compared with horse LH. , 1997, The Journal of endocrinology.

[12]  R. Salesse,et al.  Mapping of HCG-receptor complexes , 1996, Molecular and Cellular Endocrinology.

[13]  S. Purohit,et al.  Effect of modification of all loop regions in the α- and β-subunits of human choriogonadotropin on its signal transduction activity , 1996, Molecular and Cellular Endocrinology.

[14]  M. Chopineau,et al.  Cloning and analysis of the cDNA for the common alpha-subunit of the donkey pituitary glycoprotein hormones. , 1996, Journal of molecular endocrinology.

[15]  T. Ji,et al.  Lys91 and His90 of the alpha-subunit are crucial for receptor binding and hormone action of follicle-stimulating hormone (FSH) and play hormone-specific roles in FSH and human chorionic gonadotropin. , 1995, Endocrinology.

[16]  D. C. Harris,et al.  Crystal structure of human chorionic gonadotropin , 1994, Nature.

[17]  W A Hendrickson,et al.  Structure of human chorionic gonadotropin at 2.6 A resolution from MAD analysis of the selenomethionyl protein. , 1994, Structure.

[18]  R. Campbell,et al.  Co-evolution of ligand-receptor pairs , 1994, Nature.

[19]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[20]  T. Ji,et al.  Receptor activation of and signal generation by the lutropin/choriogonadotropin receptor. Cooperation of Asp397 of the receptor and alpha Lys91 of the hormone. , 1993, The Journal of biological chemistry.

[21]  W. Murdoch,et al.  COOH-terminal amino acids of the alpha subunit play common and different roles in human choriogonadotropin and follitropin. , 1993, The Journal of biological chemistry.

[22]  Y. Combarnous,et al.  Molecular basis of the specificity of binding of glycoprotein hormones to their receptors. , 1992, Endocrine reviews.

[23]  D. Puett,et al.  The carboxy-terminal region of the glycoprotein hormone alpha-subunit: contributions to receptor binding and signaling in human chorionic gonadotropin. , 1992, Molecular endocrinology.

[24]  C. Clay,et al.  A single gene encodes the beta-subunits of equine luteinizing hormone and chorionic gonadotropin. , 1992, Molecular endocrinology.

[25]  J. Dias,et al.  Identification of assembled epitopes on the α-subunit of human follicle stimulating hormone , 1992, Molecular and Cellular Endocrinology.

[26]  T. Ji,et al.  Conversion of lysine 91 to methionine or glutamic acid in human choriogonadotropin alpha results in the loss of cAMP inducibility. , 1991, The Journal of biological chemistry.

[27]  Y. Combarnous,et al.  Rapid in vitro desensitization of the testosterone response in rat Ley dig cells by sub‐active concentrations of porcine luteinizing hormone , 1985, FEBS letters.

[28]  J. Roser,et al.  Chemical, biological and immunological properties of pituitary gonadotropins from the donkey (Equus asinus): comparison with the horse (Equus caballus). , 1984, Biology of reproduction.

[29]  R. Canfield,et al.  Use of monoclonal antibodies to subunits of human chorionic gonadotropin to examine the orientation of the hormone in its complex with receptor. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[30]  B. Aggarwal,et al.  Purification and characterization of donkey chorionic gonadotrophin. , 1980, The Journal of endocrinology.

[31]  R. Moor,et al.  Influence of foetal genotype on the follicle-stimulating hormone:luteinizing hormone ratio of pregnant mare serum gonadotrophin. , 1977, The Journal of endocrinology.

[32]  C. Galet,et al.  (cid:1) -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 , 2001 .

[33]  J. Pierce,et al.  Glycoprotein hormones: structure and function. , 1981, Annual review of biochemistry.