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 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.
Zhang
Huanchun
H
Department of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
Wang
Dongsheng
D
Sun
Hong
H
Hall
Randy A
RA
Yun
C Chris
CC
D045744:Cell Line, Tumor; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism*; D051058:Oncogene Protein v-akt / Q000378:metabolism; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods; D049368:Receptors, Lysophosphatidic Acid / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D008246:Lysophospholipids; C496469:MAGI3 protein, human; D008565:Membrane Proteins; D049368:Receptors, Lysophosphatidic Acid; D051058:Oncogene Protein v-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D020742:rhoA GTP-Binding Protein