Structural characterization of the stem-stem dimerization interface between prolactin receptor chains complexed with the natural hormone.

The most promising approach to targeting the tumor-growth-promoting actions of prolactin (PRL) mediated by its autocrine/paracrine pathway has been the development of specific PRL receptor (PRLR) antagonists. However, the optimization of such antagonists requires a thorough understanding of the activation mechanism of PRLR. We have thus conducted a systematic X-ray crystallographic study in order to visualize the successive steps of PRLR activation by PRL. We report here the structure at 3.35 Å resolution of the 1:2 complex between natural PRL and two PRLR chains (PRLR1 and PRLR2), corresponding to the final activated state of PRLR. Further than our previously published structure involving an affinity-matured PRL variant, this structure allowed to visualize for the first time the loop L5 spanning PRLR2 residues Thr133-Phe140, revealing its central implication for the three intermolecular interfaces of the complex. We equally succeeded in obtaining a comprehensive picture of the PRLR-PRLR dimerization interface, also called stem-stem interface. Site-directed mutagenesis was conducted to probe the energetic importance of stem-stem contacts highlighted by the structure. Surprisingly, in spite of significant structural differences between the PRL/PRLR(2) complex and the 1:2 growth hormone/growth hormone receptor complex, our mutational data suggest that hot-spot residues that stabilize the receptor dimerization interface are equivalent in the two complexes. This study provides a new overall picture of the structural features of PRLR involved in stabilizing its complex with PRL.