Identification of novel sites in the ovine growth hormone receptor involved in binding hormone and conferring species specificity.
Using site-directed mutagenesis we mutated the extracellular domain of the ovine growth hormone receptor (oGHR) to the corresponding amino acids in the rat GHR at two different sites: site A is between Thr28 and Leu34 and represents a major immunogenic epitope, while site B is between Ser121 and Asp124 and is involved in the interaction of the human GHR with growth hormone (GH). Native and mutant receptors were bacterially expressed and refolded, and then RIA and GH-binding assays were carried out on the purified recombinant proteins. Mutations at the N-terminal site A of oGHR led to greatly reduced binding to bovine GH and, in addition, to significant loss of recognition by a polyclonal antiserum to bovine GHR which recognizes site A as a major epitope. The crystal structure of human GH bound to human GHR did not resolve this extreme N-terminal region of the receptor but our data indicate that the N-terminal loop undertakes a 180 degrees turn bringing it into close proximity to the hormone-binding domain in a fashion analogous to the prolactin receptor. A fourfold decrease in affinity for binding bovine GH was also observed after mutation of site B. However, this change from the ovine sequence to the equivalent sequence in the rat GHR at site B caused a 2.4-fold increase in the affinity of binding to rat GH. Taken together, the changes in binding affinity of the site-B mutant for rat and bovine GH demonstrate that this site is involved in conferring species specificity for binding GH.
Allan
G J
GJ
Hannah Research Institute, Ayr, Scotland, UK. allang@hri.sari.ac.uk
Shand
J H
JH
Beattie
J
J
Flint
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002942:Circular Dichroism; D000939:Epitopes / Q000276:immunology; D013006:Growth Hormone / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D011986:Receptors, Somatotropin / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012044:Regression Analysis; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013045:Species Specificity
D000939:Epitopes; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D013006:Growth Hormone