Biological properties of human prolactin analogs depend not only on globalhormone affinity, but also on the relative affinities of both receptor binding sites

Zinc increases the affinity of human growth hormone (hGH) for the human pro lactin receptor (hPRLR) due to the coordination of one zinc ion involving G lU-174(hGH) and His-18(hGH),,. In contrast, binding of h:PRL to the hPRLR i s zinc-independent. We engineered in binding site 1 of hPRL a hGH-like zinc coordination site, by mutating Asp-183(hPRL) (homologous to Glu-174(hGH)) into Glu (D183E mutation). This mutation was also introduced into G129R hPR L, a binding site 2 mutant (Goffin, V., Kinet, S., Ferrag, F., Binart, N., Martial, J. A., and Kelly, P, A. (1996) J. Biol. Chem. 271, 16573-16579), T hese analogs were characterized using a stable clone expressing both the hP RLR and a PRLR-responsive reporter gene. The D183E mutation per se decrease s the binding affinity and transcriptional activity of hPRL. However, this loss is partially rescued by the addition of zinc and the effect is much mo re marked on bioactivity than on binding affinity. These data indicate that the D183E mutation confers zinc sensitivity to hPRL biological properties. Due to an impaired site 2, the agonistic activity of G129R analog is almos t nil. Although the double mutant D183E/G129R displays lower affinity (simi lar to 1 log) compared with G129R hPRL, it unexpectedly recovers partial ag onistic activity in the absence of zinc. Moreover, whereas zinc increases t he affinity of D183E/G129R, it paradoxically abolishes its agonistic activi ty. Our results demonstrate that the biological properties of hPRL analogs do not necessarily parallel their overall affinity. Rather, the relative af finities of the individual binding sites 1 and 2 may play an even more impo rtant role.