A structure-function analysis of bovine prolactin

The 23 kDa pituitary protein hormone bovine prolactin (bPRL) belongs to a family of hormones including growth hormones, prolactins, and placental lactogens, and is involved in regulating a variety of physiological processes including lactation and immune response. The biological actions of bPRL are mediated by cell surface receptors and it is anticipated, as reported for human growth hormone (hGH) and based on sequence homology among members of this hormone family, that the hormone-receptor interaction involves receptor dimerization by a single hormone molecule. The structure of bPRL is not known, but is believed to resemble that of hGH in consisting of four a-helices arranged in a left twisted helical bundle. Variants of recombinant methionyl bovine prolactin (met-bPRL) containing single amino acid changes were generated by site-directed mutagenesis in order to carry out a structure-function analysis of the interaction of bPRL with the Nb2 PRL receptor. The changes involved the replacement, in most cases with alanine, of residues in the loop region joining putative helices 1 and 2, in putative helix 3, and in putative helix 4; regions homologous with functionally important portions of closely related proteins including human prolactin (hPRL) and hGH. The variant proteins were produced as inclusion bodies in E. coli, extracted with N-lauryl sarcosine (sarcosyl), and renatured by air oxidation at pH 10. The contribution of the residues to the biological activity of bPRL was assessed using the Nb2 lymphoma cell bioassay. In this assay, in which met-bPRL was as active as pituitary-derived bPRL, growth factor activity is measured, and the rate of cell proliferation is proportional to the concentration of lactogen present. The findings are discussed in the light of a putative threedimensional structure of bPRL, modelled using the structural coordinates of hGH. None of the exchanges of residues of the loop region resulted in drastic reductions in the mitogenic activity. The variants H59A, L63A, Q71V, Q73V, and Q74V exhibited an approximate two- to three-fold reduction in bioactivity compared to unmodified met-bPRL, suggesting that these residues may be involved in the mitogenic activity of bPRL. Of the 18 putative helix 4 residues examined only substitutions of R177 and K181 led to marked decreases in mitogenic activity, indicating that these residues are very important to the bioactivity of bPRL Replacements of L171, R176, and D183 resulted in variants with bioactivities 2.5- to 3-fold less than that of unmodified met-bPRL and only 9% activity was exhibited by the mutant D178E. However, according to the putative model of bPRL the side chains of 1159, L63, L171, and D178 point into the molecule; thus, these residues may play a structural role in maintaining the shape of the binding site rather than directly contacting the receptor. The variant G129R had no mitogenic activity, and E128A had reduced bioactivity, suggesting that bPRL contains a second receptor binding site analogous to that described for hGH. Comparison of these results with those reported for related hormones has confirmed that the bPRL residues involved in mediating the mitogenic activity of the hormone, and presumably also in interacting with the Nb2 PRL receptor, are located in regions similar to those identified for other hormones. However, many differences exist in that the specific residues involved in eliciting the biological actions are not at equivalent positions; these differences likely contribute to the specificity of hormone-receptor interactions. The results of this study thus not only provide insight into the interaction between bPRL and the Nb2 PRL receptor, but also, when considered in light of the putative three-dimensional structure of bPRL and with respect to related hormones, help to give a greater understanding of the general mechanism of receptor binding by members of the growth hormone/prolactin/placental lactogen family of hormones.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat