The seven transmembrane-spanning receptor GPR30 has been linked to specific estrogen binding and rapid estrogen action attributed to two classes of plasma membrane-associated enzymes, matrix metalloproteinases and adenylyl cyclase. Activation of these GPR30-regulated enzymatic functions results in increased intracellular cAMP and triggers surface release of proHB-EGF from human breast cancer cells. While the influence of these cellular signaling events on human breast cancer cell biology remains unclear, recent data indicates that expression of GPR30 in primary human breast cancer is associated with tumor size and the development of frank metastases. Experiments outlined here further characterize GPR30 and are aimed at establishing its biological role in breast cancer. This proposal has five specific aims. First, GPR30 will be purified to determine the structural requirements of GPR30 for estrogen binding. Second, the endocytic fate of GPR30 will be evaluated to gain insight into the mechanism(s) that attenuate GPR30 activation (receptor desensitization). Third, human ER-negative breast tumor cell models in which GPR30 has been restored or inactivated will be analyzed to determine the influence of GPR30 on tumor cell survival, growth and invasion. Fourth, the capacity of these tumor cells to form tumor xenografts, invade regional lymph nodes, and form frank metastases will be measured in immunodeficient mice for the purpose of determining the relative contribution of GPR30 to tumor progression. The fifth aim extends prior work that associates GPR30 expression in primary human breast cancer to metastasis. Here, DNA ploidy and survivorship data are correlated with GPR30 and metastases in archival breast cancer specimens. Collectively, the aims detailed in this proposal will assess the role of GPR30 as a structurally distinct form of membrane estrogen receptor whose biochemical actions, and biological influence, appear to be distinct from the known estrogen receptors, ERa and ER?.
