MAGI-3 regulates LPA-induced activation of Erk and RhoA

Lysophosphatidic acids (LPA) exert multiple biological effects through specific G protein-coupled receptors. The LPA-activated receptor subtype LPA2 contains a carboxyl-terminal motif that allows interaction with PDZ domain-containing proteins, such as NHERF2 and PDZ-RhoGEF. To identify additional interacting partners of LPA2, the LPA2 carboxyl-terminus was used to screen a proteomic array of PDZ domains. In addition to the previously identified NHERF2, several additional LPA2-interacting PDZ domains were found. These included MAGI-2, MAGI-3 and neurabin. In the present work, we demonstrate the specific interaction between LPA2 and MAGI-3, and the effects of MAGI-3 in colon cancer cells using SW480 as a cell model. MAGI-3 specifically bound to LPA2, but not to LPA1 and LPA3. This interaction was mediated via the fifth PDZ domain of MAGI-3 interacting with the carboxyl-terminal 4 amino acids of LPA2, and mutational alteration of the carboxyl-terminal sequences of LPA2 severely attenuated its ability to bind MAGI-3. LPA2 also associated with MAGI-3 in cells as determined by co-affinity purification. Overexpression of MAGI-3 in SW480 cells showed no apparent effect on LPA-induced activation of Erk and Akt. In contrast, silencing of MAGI-3 expression by siRNA drastically inhibited LPA-induced Erk activation, suggesting that the lack of an effect by overexpression was due to the high endogenous MAGI-3 level in these cells. Previous studies have shown that the cellular signaling elicited by LPA results in activation of the small GTPase RhoA by Gα12/13 — as well as Gαq-dependent pathways. Overexpression of MAGI-3 stimulated LPA-induced RhoA activation, whereas silencing of MAGI-3 by siRNA resulted in a small but statistically significant decrease in RhoA activation. These results demonstrate that MAGI-3 interacts directly with LPA2 and regulates the ability of LPA2 to activate Erk and RhoA.