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 has a region homologous to the phosphoinositide binding region of RasGRP1. The objective of this study was to explore the hypothesis that this region of RasGRP3, like that of RasGRP1, indeed contributes to phosphoinositide binding. For these studies, we prepared full length RasGRP1/3, RasGRP1/3 C1 domain, and truncated (C1-SuPT-PT) RasGRP1 and RasGRP3 green fluorescent protein labeled constructs to transfect human prostate adenocarcinoma (LNCaP) and human cervical carcinoma (HeLa) cells. In HeLa cells, both RasGRP1 and RasGRP3 translocated to the endonuclear membranes, most probably to the Gogli in response to phorbol ester (PMA) and other agents. In the LNCaP cells, which are a PTEN mutant cell line with markedly elevated phosphoinositide levels, RasGRP1 translocates to the plasma membrane. In contrast, the full length RasGRP3 does not translocate to the plasma membrane even though it has a PT (plasma membrane-targeting) domain with a basic/hydrophobic cluster of amino acids (BHC) similar to that of RasGRP1. In parallel, we examined the translocation of the C1 domains of RasGRP1/3 and of the truncated RasGRP1/3 constructs, as well as the corresponding constructs with amino acid substitutions in the predicted phosphoinositide binding domain of RasGRP3. The RasGRP1 translocated to the plasma membrane in response to PMA. The C1 domain of RasGRP3 was insufficient for targeting RasGRP3 to the plasma membrane. However, truncated RasGRP3, which contains the PT domain, showed stronger translocation. Substituting amino acids (Asp647, Lys648 and Ala651) in the PT domain of full length RasGRP3 triggered the plasma membrane translocation in the LNCaP cells. We conclude that the differences in the BHC section of the PT domain of RasGRP3 play an important role in the membrane translocation of RasGRP3 and will reduce its sensitivity to membrane phosphoinositide levels. Citation Format: Agnes Czikora, Noemi Kedei, Peter M. Blumberg. Comparison of structural elements in RasGRP3 and RasGRP1 controlling membrane interaction. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4450. doi:10.1158/1538-7445.AM2014-4450