Pituitary and placental ovine growth hormone variants differ in their receptor-binding ability and in their biological properties.
The wild-type (WT) GH2-N ovine growth hormone (oGH) and duplicated GH2-Z genes differ in their open reading frame by two nonsynonymous substitutions, predicting a two-amino-acid difference in their product (G9R/G63S). Three recombinant oGH muteins: G9R, G63S and G9R/G63S, were prepared by site-directed mutagenesis of the WT oGH gene, expressed in E. coli, refolded and purified as monomers with over 98% homogeneity. Gel-filtration experiments with WT oGH and the three muteins indicated formation of 1:2 complexes with oGH receptor extracellular domain (oGHR-ECD). Interactions of oGHR-ECD with the WT and the muteins were studied by surface plasmon resonance. Kinetics constants calculated using a two-site model predicted that G9R/G63S has the highest affinity to oGHR-ECD, WT oGH the lowest, and G9R and G63S have intermediate affinities. These relative affinities were further investigated by radioreceptor assay with EC50 values were the lowest for G9R/G63S, highest for WT oGH, and intermediate for G9R and G63S. Bioactivity of the WT oGH and oGH muteins was determined by proliferation assay with FDC-P1-3B9 cells stably transfected with rabbit GHR. Relative proliferation rates of cells in cultures treated with the WT, G63S, G9R or G9R/G63S variants were 100%, 183%, 259% and 498%, respectively. In COS-7 transfected with oGHR, LHRE-TK-luciferase and beta-galactosidase plasmids G9R/G63S showed 18% higher activity than WT oGH (P<0.001). Thus the product of the oGH duplicated copy has higher affinity for GHR and higher somatogenic activity. As the GH2-Z gene copy is expressed in the placenta, allelic differences at the oGH locus may influence feto-placental development.
Reicher
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Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
Niv-Spector
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Gertler
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Gootwine
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D000818:Animals; D020440:Gene Duplication; D013006:Growth Hormone / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics; D010907:Pituitary Hormones / Q000235:genetics / Q000378:metabolism*; D010926:Placental Hormones / Q000235:genetics / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000378:metabolism*; D011994:Recombinant Proteins; D012756:Sheep
D050505:Mutant Proteins; D010907:Pituitary Hormones; D010926:Placental Hormones; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D013006:Growth Hormone