MECHANISMS OF THE DOWNREGULATION OF PROLACTIN RECEPTOR AND THEIR ROLE IN CELL PROLIFERATION

Cells react to diverse stimuli by expressing specific receptors that recognize these stimuli and initiate specific signaling pathways that enable a cell to change with the environment. Downregulation of these signaling receptors represents the most direct method for limiting the magnitude and duration of downstream signal transduction. For cell surface transmembrane receptors, ligand-stimulated endocytosis is a major mechanism by which the ability of a cell to react to a ligand is restricted. In order to investigate the downregulation of the prolactin receptor (PRLr), we investigated the mechanism and key determinants in the endocytosis and downregulation of PRLr. In Chapter 2, we show that the endocytosis of PRLr is a ligand-induced process which requires the catalytic activity of the constitutively bound Janus kinase Jak2. In Chapter 3, we show that PRLr is internalized by a clathrin-dependent mechanism which requires phosphorylation of the conserved phosphodegron motif (DS349GRGS) at Ser 349 and an active SCFβ-TrCP E3 ligase complex. Optimal PRLr endocytosis is shown to be achieved via K63-linked polyubiquitination of the receptor. In Chapter 4, we show that PRL signaling promotes cell growth in 2-D and 3-D culture systems where PRLr levels are increased/stabilized. In Chapter 5, we identify pyruvate kinase M2 (PKM2), a glycolytic enzyme whose role in tumorigenesis has been described, to be a novel interactor of PRLr. We show that prolactin (PRL) signaling works to inhibit PKM2 activity by the propagation of tyrosine-phosphorylated proteins. This inhibition of PKM2 prevents progression through glycolysis and allows PKM2 to take a pro-tumorigenic role. We show that the interaction between PRL signaling and PKM2 is required for optimal prolactin-dependent cell growth. In this thesis (model shown in Model 1), we show that there is a defined mechanism of PRLr downregulation which works to limit PRL signaling. If this mode of receptor downregulation is not properly executed, it can result in aberrant signaling whereby prolactin-mediated inhibition of pyruvate kinase M2 mediates the pro-tumorigenic effect of prolactin