Crystal Structure of a Prolactin Receptor Antagonist Bound to the Extracellular Domain of the Prolactin Receptor

Abstract

The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5 Å resolution by x-ray crystallography. This structure represents the first experimental structure reported for a PRL variant bound to its cognate receptor. The binding of PRL variants to the PRLR extracellular domain was furthermore characterized by the solution state techniques, hydrogen exchange mass spectrometry, and NMR spectroscopy. Compared with the binding interface derived from mutagenesis studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Prolactin (PRL)4 is a protein hormone secreted by the anterior pituitary in vertebrates and possesses physiological functions of remarkable diversity, including effects on reproduction, lactation, and growth. PRL belongs to a family of homologous proteins comprising PRL, growth hormone (GH), and placental lactogen (PL). The biological effects associated with this cytokine family are mediated by two distinct classes of cell surface receptors, the PRL receptors (PRLR) and the GH receptors (GHR). The PRL/PL/GH biology is governed by a delicate balance between receptor cross-reactivity and selectivity; PRL and PL bind selectively to PRLR, whereas GH is capable of binding both PRLR and GHR.After being proposed more than a decade ago (1), hormoneinduced receptor dimerization became generally accepted as the model for cytokine receptor activation. For the PRL family members, the model describes the signaling molecular entity as a ternary complex between one hormone molecule and a receptor homodimer assembled in a strictly sequential and hormonedependent fashion; first the hormone ligand engages via binding site 1 (BS1) in high affinity binding to one receptor chain forming a 1:1 hormone-receptor complex. This complex constitutes the template for binding a second, identical receptor molecule via binding site 2 (BS2), resulting in the active 1:2 complex. However, the model has been challenged by an increasing body of experimental evidence, initially reported for the homologous human erythropoietin receptor (2) and later for GHR (3) and PRLR (4). These studies suggest that preformed, inactive dimers exist in the absence of hormone. Thus, receptor dimerization is a necessary but not sufficient event for receptor activation and, notably, not strictly ligand-dependent. For both human erythropoietin receptor (5) and GHR (3), mechanistic models have been proposed, where receptor activation involves relative rotations and movements of receptor subunits induced by hormone binding. Allosteric reorganization of the intracellular receptor domains brings associated JAK2 kinases into close proximity, allowing their activation by cross-phosphorylation. This initial activation step triggers a cascade of molecular events leading to the functional receptor response (6).The...

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

“…Binding site 1 is a flat surface of ϳ1180 Å 2 , which is slightly larger than in the oPL⅐PRLR 2 complex (ϳ1000 Å 2 ), but smaller than in hGH⅐hGHR 2 complex (ϳ1300 Å 2 ). It is closely similar to that in the recently determined structure of the 1:1 hPRL⅐hPRLR complex (25), although 7 of 28 residues involved in the binding interface differ between human and rat PRLR (supplemental Fig. S4).…”

“…4) aligned with strand A, which occupies a space between the two small strands BЈ and GЈ pushing them apart but without forming a ␤-sheet. This particular N terminus conformation is also observed in the 1:1 hPRL⅐hPRLR complex (3D45) (25).…”

“…As for hPRL, its structure in solution was obtained by NMR, confirming its four antiparallel ␣-helix bundle fold (24). More recently, we reported the first set of x-ray coordinates for a PRL-core hormone and determined the structure of the pure antagonist Del1-9-G129R-hPRL (20), and the structure of this compound bound to the hPRLR-ECD (1:1 complex) was then determined (25).…”

“…PRL , it drags along the "lower part" of the hormone (regions [15][16][17][18][19][20][21][22][23][24][25][26][27][28] PRL and 125-136 PRL ). Region 15-28 PRL reaches its final position only when the second PRLR-ECD interacts at binding site 2.…”

Prolactin and oestrogen synergistically regulate gene expression and proliferation of breast cancer cells

“…Human recombinant PRL was expressed in Escherichia coli and purified as described previously (Svensson et al 2008); E 2 was purchased from SigmaAldrich. ICI 182 780 was obtained from Tocris Bioscience (Bristol, UK).…”