AbstractsBiology & Animal Science

The human prolactin receptor (hPRLr) is a member of the class 1 cytokine receptor family, which also includes growth hormone receptor (GHr) and erythropoietin receptor (EPOr). The classic mechanism for class 1 cytokine receptor activation describes that ligand-induced receptor dimerization triggers downstream signaling and is supported by biochemical and biophysical evidence. However, the recent discoveries of ligand-independent dimerization of class 1 cytokine receptors in plasma membrane, including hPRLr, hGHr, and mouse EPOr, have challenged this classic mechanism. Several mechanistic models have been proposed for the activation of GHr and EPOr, including the scissor model, the piston model, and the rotation model. In contrast, it is unknown if hPRLr shares a similar mechanism. The specific amino acid residues that mediate ligand-independent hPRLr dimerization have not been determined, although the transmembrane (TM) domain has been suggested to be important. The role of ligand-independent hPRLr dimers in hPRLr activation is unclear. Furthermore, the presence of ligand-independent hPRLr dimers does not rule out the classic mechanism, because hPRLr may exist in equilibrium between monomers and preformed dimers in plasma membrane. This dissertation has focused upon the structural basis and the functional impact of ligand-independent dimerization of hPRLr. We aimed to systematically evaluate in hPRLr the proposed models for class 1 cytokine receptor activation. An extensive series of alanine or glycine insertions were introduced at the junctions between the TM domain and either the extracellular or intracellular domain to manipulate the relative orientations of different hPRLr domains. The basal and ligand-stimulated activities of these hPRLr insertion variants were examined in transiently transfected 293T cells. Our data demonstrate that altering the spatial relationships of hPRLr domains does not induce constitutive activity or impair ligand-induced activation. Such results do not support the rotation or piston model for hPRLr. We also identified a population of covalently linked ligand-independent hPRLr dimers that are redox-sensitive and investigated the involvement of intermolecular disulfides in ligand-independent hPRLr dimerization. Twelve cysteines in various domains of hPRLr were replaced with serines, and the dimerization status of these hPRLr mutants was examined under reducing and non-reducing conditions. Iodoacetamide, an alkylation reagent for cysteine, was employed to distinguish in vivo disulfides from ex vivo disulfides. Our data indicate that multiple cysteines from different domains of hPRLr, including but not limited to C184, C225, and C242, participate in forming intermolecular disulfides in ligand-independent hPRLr dimerization. We next examined the role of these disulfide-linked hPRLr dimers in hPRLr activation. Abolishing the formation of ligand-independent disulfide-linked hPRLr dimers by removing twelve C-terminal cysteines did not impair ligand-induced activation or…