Prolactin significantly contributes to the growth and differentiation of breast cancer. We have demonstrated that phosphorylation of tyrosine on rat PRLr is necessary for the receptor function. However, similar studies evaluating the role of tyrosines during human PRLr signaling have not been done. To that end, this research proposal has three main goals: (1) to identify sites of PRLr tyrosine phosphorylation and their relative importance to PRLr signaling, (2) to assess the role of Jak2 and Fyn kinases in hPRLr phosphorylation (3) to examine the functional significance of individual tyrosines on the hPRLr. PCR-based mutagenesis is being employed to knock-out individual or groups of intracellular tyrosine residues on the PRLr. These constructs will be used to evaluate the role of specific tyrosine residues in activating specific pathways associated with the PRLr and will foster a better understanding of the underlying mechanisms driving PRL-mediated effects on breast cancer