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J Biol Chem, Vol. 274, Issue 37, 26033-26043, September 10, 1999

Biological Properties of Human Prolactin Analogs Depend Not Only on Global Hormone Affinity, but Also on the Relative Affinities of Both Receptor Binding Sites

Sandrina Kinet, Sophie Bernichtein^¶, Paul A. Kelly^¶, Joseph A. Martial, and Vincent Goffin^¶

From the  Laboratory of Molecular Biology and Genetic Engineering, Allée du 6 Août, University of Liège, 4000 Sart-Tilman, Belgium and ^¶ INSERM Unit 344, Endocrinologie Moléculaire, 156, rue de Vaugirard, 75730 Paris Cedex 15, France

Zinc increases the affinity of human growth hormone (hGH) for the human prolactin receptor (hPRLR) due to the coordination of one zinc ion involving Glu-174_hGH and His-18_hGH. In contrast, binding of hPRL to the hPRLR is 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 hPRL, 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). These analogs were characterized using a stable clone expressing both the hPRLR and a PRLR-responsive reporter gene. The D183E mutation per se decreases the binding affinity and transcriptional activity of hPRL. However, this loss is partially rescued by the addition of zinc and the effect is much more 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 almost nil. Although the double mutant D183E/G129R displays lower affinity (~1 log) compared with G129R hPRL, it unexpectedly recovers partial agonistic activity in the absence of zinc. Moreover, whereas zinc increases the affinity of D183E/G129R, it paradoxically abolishes its agonistic activity. Our results demonstrate that the biological properties of hPRL analogs do not necessarily parallel their overall affinity. Rather, the relative affinities of the individual binding sites 1 and 2 may play an even more important role.

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