Structure and function of a new class of human prolactin antagonists

Abstract

SummaryΔ41-52 hPRL (human prolactin with residues 41-52 removed) is a lead compound for a new class of hPRL antagonists. The deleted sequence contains residues that functionally couple sites 1 and 2, the two hormone surfaces that each bind receptors. Δ41-52 hPRL retains 0.03% agonist activity in FDC-1 cell bioassays, a 3,054-fold reduction in activity, and displays approximately 100-fold less agonist activity than G129R hPRL, an antagonist that reduces the binding of hPRL receptor at site 2 during the formation of the heterotrimeric hormone/receptor complex. Replacement of various numbers and types of residues into the gap created by the deletion of residues 41 through 52 created hPRLs with varying agonist activities, suggested that manipulation of the sequence connecting the C-terminal of helix 1 with the disulfide bond (cysteines 58 with 174) linking helices 1 and 4 modulates articulation of these helices and influences agonist activity. We have compared the antagonist activities of G129R and Δ41-52 hPRLs to induce apoptosis in Jurkat cells, a human lymphoid cell line displaying an autocrine/paracrine hPRL/receptor system. Δ41-52 hPRL induces apoptosis in a time and dose-dependent fashion. Under these same conditions G129R hPRL fails to induce apoptosis. We conclude Δ41-52 hPRL is a lead compound of a new class of hPRL antagonists capable at low concentrations of inducing apoptosis in human cells expressing an autocrine/paracrine hPRL/ receptor system.

Crystal Structure of an Affinity-matured Prolactin Complexed to Its Dimerized Receptor Reveals the Topology of Hormone Binding Site 2

“…The intrinsic dynamics of the hormone, favored by the presence of the two long loops, seems to be a prerequisite for its fully functional interaction with the receptor, as hypothesized by Jomain et al (20) and by the antagonistic properties of the ⌬41-52-hPRL (47). As evidenced by the superimposition of the three different hormones in complex with PRLR receptor, the binding of receptor 1 seems to impose the final structure of the hormone, at least at the level of binding site 1.…”

Structural Characterization of the Stem–Stem Dimerization Interface between Prolactin Receptor Chains Complexed with the Natural Hormone

“…This led several teams to investigate new strategies aimed at targeting PRL actions rather than PRL expression, which resulted in the development of PRL analogs acting as competitive antagonists of the PRL receptor (PRLR). [12][13][14] Unfortunately, although these antagonists are promising therapeutic candidates, their affinity for PRLR is weaker than that of wild-type (wt) PRL. To improve the affinity and efficiency of these molecules, we need to understand the precise mechanism of PRLR activation, a question on which our team has focused for several years using a crystallographic approach.…”

Functional Epitopes for Site 1 of Human Prolactin

“…Analysis of this information is critical for the rational design of antagonists or for understanding the mechanism by which hPRL agonists and antagonists regulate receptor activity. Site-directed mutagenesis frequently has been used to identify binding interfaces as well as interactions within prolactin that either maintain structure or functionally couple sites 1 and 2 (4,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). These studies have used a variety of functional end points to assess the effects of mutations, including binding of 125 I-labeled hormone to cells or membranes containing prolactin receptors or cellular biological assays that typically use growth as their end point.…”