Abstract 1126: Importance of the REM (Ras exchange) motif for membrane interactions by RasGRP3

Abstract

Abstract RasGRP comprises a family of guanine nucleotide exchange factors that activate small GTPases, regulating the dissociation of GDP from Ras GTPases to enhance the formation of the active GTP-bound form. RasGRP3 is a complex protein with REM (Ras exchange motif), GEF (catalytic domain), EF-hand, C1, SuPT, and PT (plasma membrane-targeting) domains, each of which can affect the cellular membranes to which RasGRP1/3 localizes. The C1 domain was the first membrane localizing domain identified in RasGRP1/3. It binds to the lipid second messenger diacylglycerol (DAG) and has the potential to mediate translocation of RasGRP1/3. The catalytic GEF domain and REM domain are both required for membrane localization of RasGRP1, affecting both endomembrane and plasma membrane targeting of RasGRP1. The objective of this study was to explore the importance of these two domains in translocation of RasGRP3. For these studies, we prepared full length RasGRP1/3, RasGRP1/3 C1 domains, and truncated RasGRP1/3 green fluorescent protein labeled constructs to transfect human prostate adenocarcinoma (LNCaP) cells. In the LNCaP cells, which are a PTEN mutant cell line with markedly elevated phosphoinositide levels, RasGRP1 translocates to the plasma membrane in response to PMA. In contrast, the full length RasGRP3 does not translocate to the plasma membrane even though it has a PT (plasma membrane-targeting) domain. In parallel, we examined the translocation of the C1 domain of RasGRP1/3 and of the truncated RasGRP1/3 constructs. The C1 domain of RasGRP3 was insufficient for targeting RasGRP3 to the plasma membrane. However, truncated RasGRP3s, which contain the PT domain and are missing the REM motif, showed stronger translocation. To better understand differences in the translocation pattern of these two proteins we compared RasGRP1 (“template”) and RasGRP3 (“target”) domains using Swiss-Model (homology or comparative modelling methods) and amino acid sequence alignment (NCBI/ BLAST). The percentage of identities showed remarkable differences in the structure of the REM and SuPT-PT domains. Based on these findings we prepared chimeras where we exchanged either the N or C terminus of RasGRP1/3. Exchange of the REM motif between RasGRP1 and RasGRP3 enhanced the translocation of RasGRP3, while it does not affect the translocation of RasGRP1. Exchange of C termini (SuPT-PT domain) of both proteins did not cause any changes in plasma membrane translocation. Our results clearly show that the N terminus (REM motif) of RasGRP3 is a critical element contributing to the difference between RasGRP1 and RasGRP3 in their ability to translocate to the plasma membrane. This difference in membrane translocation, in turn, should imply important differences in their regulation and, potentially, in their ligand selectivity in the living cell. Citation Format: Agnes Czikora, Noemi Kedei, Peter M. Blumberg. Importance of the REM (Ras exchange) motif for membrane interactions by RasGRP3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1126.