PROJECT SUMMARY/ABSTRACT The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that signals in response to changes in extracellular calcium. It functions as an obligate dimer and couples to different G-proteins, including Gai or Gas, in a context- or cell type-specific manner. The CaSR is widely expressed and alters proliferation, differentiation, death and ion transport in a variety of cell types, including mammary epithelial cells (MECs), where it regulates the production of parathyroid hormone-related protein (PTHrP) and milk calcium transport. During lactation, activation of the CaSR on MECs inhibits PTHrP secretion by stimulating Gai and reducing cAMP levels. However, in breast cancer cells, activation of the CaSR increases, rather than decreases, PTHrP secretion because, in these cells, the CaSR activates Gas and increases cAMP levels. The CaSR has important functions in both lactation physiology and breast cancer pathophysiology, so it is critical to better understand the regulation of its downstream signaling events in breast epithelial cells. Emerging data demonstrate that the CaSR can heterodimerize with the 2 metabotropic, GabaB receptors, GABBR1 and GABBR2 and the central premise of this application is that heterodimerization of the CaSR with GABBR1 and/or GABBR2 alters CaSR signaling, PTHrP production and transepithelial calcium transport by mammary epithelial cells. Specifically, we hypothesize that, during lactation, reductions in GABBR1 expression and increases in GABBR2 expression promote the formation of CaSR/GABBR2 heterodimers in MECs, which, in turn, increases membrane CaSR expression and reinforces coupling of the CaSR to Gai. In order to explore this hypothesis, we propose 3 Specific Aims. Aim 1 will examine how heterodimerization with GABBR1 or GABBR2 alters CaSR expression, signaling and PTHrP production in breast epithelial cells in vitro. Aim 2 will use genetically modified mice to examine whether heterodimerization with GABBR2 regulates CaSR expression and function in the lactating breast in vivo. Aim 3 will use genetically modified mice to examine whether heterodimerization with GABBR1 regulates CaSR expression and function in the lactating breast in vivo. The studies outlined in this proposal will explore novel biology of great importance for lactation physiology that also has implications for breast cancer pathophysiology. These studies have the potential to discover novel ways to target the CaSR therapeutically.

PROJECT NARRATIVE The calcium-sensing receptor (CaSR) is a G protein-coupled receptor (GPCR) that is expressed on normal breast cells, where it regulates the production of parathyroid hormone-related protein (PTHrP) and calcium transport during lactation in order to coordinate maternal calcium and bone metabolism with calcium demand for milk production. This project will study how heterodimerization of the CaSR with the metabotropic GabaB receptors, GABBR1 and GABBR2, affects the ability of the CaSR to regulate PTHrP production and calcium transport into milk. These studies will advance the understanding of how the CaSR and the GabaB receptors affect the normal breast and have the potential to discover new ways to target the CaSR therapeutically.