The WSXWS Motif in Cytokine Receptors Is a Molecular Switch Involved in Receptor Activation: Insight from Structures of the Prolactin Receptor

Abstract

The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane proximal domain of the human PRLR and find that the tryptophans of the motif adopt a T-stack conformation in the unbound state. By contrast, in the hormone bound state, a Trp/Arg-ladder is formed. The conformational change is hormone-dependent and influences the receptor-receptor dimerization site 3. In the constitutively active, breast cancer-related receptor mutant PRLR(I146L), we observed a stabilization of the dimeric state and a change in the dynamics of the motif. Here we demonstrate a structural link between the WSXWS motif, hormone binding, and receptor dimerization and propose it as a general mechanism for class 1 receptor activation.

A Residue Quartet in the Extracellular Domain of the Prolactin Receptor Selectively Controls Mitogen-activated Protein Kinase Signaling

“…Plasmids-The pQE-70-rPRLR-ECD and pET11a-hPRLR-D2 prokaryotic expression plasmid, pcDNA3(ϩ)-hPRLR WT and -rPRLR WT eukaryotic expression vectors (long PRLR isoforms), and the lactogenic hormone-response element-luciferase (firefly) reporter plasmid were described previously (15)(16)(17). Interface substitutions were introduced using the QuikChange II mutagenesis kit (Stratagene), and coding sequences were verified on both strands.…”

“…Hormones and Receptors-Recombinant hPRL and rPRL, hPRLR/rPRLR-ECDs (WT and variants), hPRLR-D2 (WT and variants, unlabeled and stable isotope-labeled), and the PRLR antagonist Del1-9-G129R-hPRL were produced in Escherichia coli and purified as described (15,17,18). Pituitary bPRL was obtained from Dr. Parlow (NIDDK, National Institutes of Health).…”

“…The samples were prepared at concentrations (for 15 15 N-hPRLR-D2 K168A samples with nonlinear sampling to 20%. NMR spectra were referenced to 2-dimethyl-2-silapentane-5-sulfonic acid at 0.00 ppm ( 1 H) and transformed using weighted Fourier transformation and two-dimensional spectra transformed using NMRPipe (22).…”

“…Such coupling, known as exciton coupling, occurs in the D2 domain and stems from residues Trp-191 and Trp-194 of the WS motif. Thus, the CD spectrum of rPRLR-ECD has a positive ellipticity at 230 nm, which obscures the signature from the dominant ␤-strand structure (15). Interestingly, both rPRLR-ECD K168A and rPRLR-ECD F170A gave rise to a smaller contribution from aromatic exciton couplings, suggesting the following: (i) the substitutions interfered with the organization of the WS motif; (ii) a contribution from additional aromatic exciton couplings of an opposite sign at the same wavelength appeared, or (iii) couplings of a similar sign disappeared.…”

“…Cell-based studies have shown that at least parts of the GHR and PRLR population expressed in eukaryotic cells are pre-dimerized in the absence of ligand, suggesting that signaling activation involves ligand-induced conformational changes within the receptor homodimer rather than alteration of receptor stoichiometry (12)(13)(14). For the PRLR, we recently used nuclear magnetic resonance (NMR) spectroscopy to show that in the unbound state the two tryptophan residues of the WS motif adopt a T-stack conformation that stabilizes the receptor off-state, whereas in the ligandbound state these residues form a Trp-Arg ladder characteristic of the receptor on-state (15). Chemical shift perturbation studies following PRL binding to the D2 domain further point to structural rearrangements involving various residues, including specific sites within the receptor-receptor dimerization interface, also called site 3 (Fig.…”