PROJECT SUMMARY KRAS is the oncogene most frequently mutated in human cancer. KRAS functions as a molecular switch that regulates signaling pathways only when associated with cellular membranes. KRAS associates with membranes as a consequence of farnesylation that operates in conjunction with a polybasic C-terminus. Efforts to defeat KRAS by blocking farnesylation failed because of alternative enzymes capable of prenylating KRAS. We therefore took an unbiased approach to identify previously unrecognized genes that participate in the membrane association of KRAS. We devised a dual luciferase assay that reports loss of KRAS affinity for membranes and used this assay in a genome-wide siRNA screen. Among the 13 genes identified we were surprised to find a G protein coupled receptor (GPCR) designated GPR31, which is a high affinity receptor for 12-(S)- HETE and has been shown to stimulate MAPK signaling. GPR31 is an understudied GPCR that is included in the Illuminating the Druggable Genome project. We were also surprised to find by co-immunoprecipitation a physical interaction between GPR31 and KRAS, suggesting that GPR31 might act as a secretory pathway chaperone for KRAS as it traffics from endomembrane to the plasma membrane (PM). In preliminary studies we have found that GRP31 and KRAS colocalized on endomembrane and PM and that silencing of GPR31 with siRNA inhibits KRAS dependent cell proliferation and macropinocytosis. We now propose to determine if 12-(S)-HETE signaling through GPR31 regulates KRAS trafficking and signaling with three Specific Aims. Aim 1. Ligation of GPR31 with 12-(S)-HETE and Interaction of GPR31 with KRAS. We will measure the interaction between GPR31 and KRAS by co-immunoprecipitation and FRET ± 12-(S)-HETE or control eicosinoids. Aim 2. Ligation of GPR31 with 12-(S)-HETE and Trafficking of KRAS. We will study KRAS trafficking from endomembrane to PM using live cell imaging ± 12-(S)-HETE. Aim 3. Ligation of GPR31 with 12-(S)-HETE and KRAS activation. We will study KRAS signaling ± 12-(S)-HETE. The prosecution of these aims will determine if GPR31 signaling regulates KRAS and thereby prioritize GPR31 for anti- cancer drug discovery. !