Lactation is of great importance to survival of newborn animals. Prolactin plays a critical role in this process by regulating the differentiation of mammary epithelial cells into secretory epithelial cells. Much of the research on prolactin signal transduction has focused upon its role in regulating the transcription of milk protein genes. However, since milk is 25 - 30% fat and about 3% lactose, pathways that control glucose availability and lipid biosynthesis are likely to be critical for optimal milk production. Our previous studies indicate that the serine/threonine protein kinase Akt, plays a critical role in suppressing apoptosis in the involuting mammary gland. We hypothesize that prolactin regulates glucose transport and lipid biosynthesis and the Akt protein kinase is required for prolactin-mediated activation of glucose transport. In this grant we propose to map the region(s) of the prolactin receptor that are required for activation of Akt and glucose transport. We will focus on phosphorylated tyrosine residues because the binding of signaling molecules to these sites often regulates their activation, particularly SH2 domain containing molecules. We will use a chimeric prolactin receptor whose activation can be controlled by a chemical dimerizer. This chimeric receptor will be expressed in mammary epithelial cells to map the regions that regulate glucose transport and Akt activation. Transgenic mice will be generated that express the chimeric prolactin receptor in the mammary gland to test the function of specific receptor mutants. These studies will be done under conditions where the production of prolactin by the pituitary is blocked thereby preventing activation of the endogenous prolactin receptor. Mammary gland development is altered in Src-/- mice but not in Fyn-/- mice. Primary mammary epithelial cells derived from Src-/- and Fyn-/- mice will be used to determine the role of these kinases in regulating Akt activation, and glucose transport. We will also test the role of specific signaling molecules (c-Cbl, Cbl-B, IRS-1, Gab2, Gab3, SHP 1, and SHP2) in regulating the PI3-kinase/Akt pathway and glucose transport. Finally, we will determine the mechanisms by which prolactin increases glucose transport in mammary epithelial cells. Until now, the mechanisms by which glucose transport in mammary epithelial cells is regulated have remained a mystery. These studies should provide important information about other roles for prolactin in the mammary gland and identify the mechanisms by which prolactin regulates these diverse processes.
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