JCB_201609096 2329..2338

KRAS is the oncogene most frequently mutated in cancer. KRAS encodes a small GTPase that regulates signaling pathways for cell growth but functions in this way only when associated with the plasma membrane (PM). Accordingly, much effort has been devoted to understanding how KRAS associates with the PM in the hope of interfering with this process for therapeutic benefit (Cox et al., 2015). KRAS4B, the primary splice variant of the KRAS locus, is a peripheral membrane protein that gains affinity for the PM through posttranslational modification of its C-terminal CAAX motif (CVIM sequence) with a farnesyl lipid, followed by endoproteolytic removal of the VIM residues and methyl esterification of the farnesylcysteine. These sequential modifications are catalyzed by farnesyltransferase (FTase), RAS-converting enzyme 1 (RCE1), and isoprenylcysteine carboxylmethyltransferase (ICMT; Wright and Philips, 2006). Modification of the CVIM sequence is necessary but not sufficient for trafficking to the PM; also required is a polybasic sequence in the hypervariable region (HVR) immediately upstream of CVIM (Hancock et al., 1990; Choy et al., 1999). The polybasic sequence allows for an electrostatic interaction with the negatively charged headgroups of the inner leaflet of the PM (Hancock et al., 1990). The interaction can be modulated by phosphorylation of serine 181 within the polybasic region (Bivona et al., 2006). The characterization of RCE1 (Schmidt et al., 1998) and ICMT (Dai et al., 1998) as polytopic membrane proteins restricted to the ER demonstrated that CAAX processing of RAS and related proteins, begun in the cytosol by FTase, is completed on the cytosolic face of the ER. This has raised the question of how RAS proteins are transferred from the ER to the PM. After CAAX processing, NRAS and HRAS are palmitoylated on the Golgi apparatus and then transferred to the PM via vesicular trafficking (Choy et al., 1999; Apolloni et al., 2000). In contrast, KRAS4B does not visit the Golgi and is not palmitoylated. The mechanism through which nascent KRAS4B traffics from the cytosolic face of the ER to the PM after CAAX processing has not been clearly defined. There is evidence for both rapid, fluidphase transfer (Silvius et al., 2006) and transfer by vesicular trafficking (Lu et al., 2009; Schmick et al., 2014). Transfer through the cytosol may be facilitated by prenyl-binding proteins such as the δ subunit of phosphodiesterase type 6 (PDE6δ; Chandra et al., 2011; Tsai et al., 2015). FTase, RCE1, ICMT, and PDE6δ have all served as targets for anticancer drug discovery, but only FTase inhibitors have made it to the clinic, where they failed to show efficacy (Cox et al., 2015). This has stimulated renewed efforts to more fully elucidate the details of RAS trafficking and prompted us to take an unbiased approach to identify previously unrecognized The product of the KRAS oncogene, KRAS4B, promotes tumor growth when associated with the plasma membrane (PM). PM association is mediated, in part, by farnesylation of KRAS4B, but trafficking of nascent KRAS4B to the PM is incompletely understood. We performed a genome-wide screen to identify genes required for KRAS4B membrane association and identified a G protein–coupled receptor, GPR31. GPR31 associated with KRAS4B on cellular membranes in a farnesylation-dependent fashion, and retention of GPR31 on the endoplasmic reticulum inhibited delivery of KRAS4B to the PM. Silencing of GPR31 expression partially mislocalized KRAS4B, slowed the growth of KRAS-dependent tumor cells, and blocked KRAS-stimulated macropinocytosis. Our data suggest that GPR31 acts as a secretory pathway chaperone for KRAS4B. The G protein–coupled receptor GPR31 promotes membrane association of KRAS