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

Abstract

Lysophosphatidic acids (LPA) exert multiple biological effects through specific G protein-coupled receptors. The LPA-activated receptor subtype LPA(2) 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 LPA(2), the LPA(2) carboxyl-terminus was used to screen a proteomic array of PDZ domains. In addition to the previously identified NHERF2, several additional LPA(2)-interacting PDZ domains were found. These included MAGI-2, MAGI-3 and neurabin. In the present work, we demonstrate the specific interaction between LPA(2) and MAGI-3, and the effects of MAGI-3 in colon cancer cells using SW480 as a cell model. MAGI-3 specifically bound to LPA(2), but not to LPA(1) and LPA(3). This interaction was mediated via the fifth PDZ domain of MAGI-3 interacting with the carboxyl-terminal 4 amino acids of LPA(2), and mutational alteration of the carboxyl-terminal sequences of LPA(2) severely attenuated its ability to bind MAGI-3. LPA(2) 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 Galpha(12/13) - as well as Galpha(q)-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 LPA(2) and regulates the ability of LPA(2) to activate Erk and RhoA.

Human papillomaviruses, cervical cancer and cell polarity

“…Their known binding partners include b-catenin (Dobrosotskaya and James, 2000) and mNET-1 (Dobrosotskaya, 2001). MAGI-3 has been reported to bind the lysophosphatidic activated receptor LPA(2) to enhance Erk and RhoA activation (Zhang et al, 2007a), with potential downstream effects on cell survival signalling. Most interestingly, perhaps, they, like Dlg, have been reported to interact with the PTEN tumour suppressor (Adey et al, 2000;Wu et al, 2000a, b;Kotelevets et al, 2005).…”

Distinct Phospholipase C-β Isozymes Mediate Lysophosphatidic Acid Receptor 1 Effects on Intestinal Epithelial Homeostasis and Wound Closure

“…RhoA activity was determined as described previously (18). Activation of Rac1 was determined using a Rac1 activation assay kit (Cytoskeleton Inc., Denver, CO).…”

LPAR2 (lysophosphatidic acid receptor 2)

“…In the distal region, there are several serine and threonine residues that can be phosphorylated by G protein-coupled receptor kinases (GRKs) and may be involved in β-arrestin binding and receptor internalization. The last four amino acids of this region (DSTL) contains a class I PDZ-binding motif and mediates interactions with a number of proteins such as Na+/H+ exchanger regulatory factor 2 (NHERF2) (Oh et al, 2004;Yun et al, 2005), PDZ-RhoGEF and LARG (Yamada et al, 2005), and MAGI-3 (Zhang et al, 2007).…”