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 th e rat GHR at two different sites: site A is between Thr28 and Leu34 and rep resents a major immunogenic epitope, while site B is between Ser121 and Asp 124 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 RTA and GH-binding assays were carried out on the purified recomb inant proteins. Mutations at the N-terminal site A of oGHR led to greatly r educed binding to bovine GH and, in addition, to significant loss of recogn ition 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 indica te 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 o vine 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 dem onstrate that this site is involved in conferring species specificity for b inding GH.