Abstract
Axin, a negative regulator of the Wnt signaling pathway, contains a canonical regulator of G protein signaling (RGS) core domain. Herein, we demonstrate both in vitro and in cells that this domain interacts with the α subunit of the heterotrimeric G protein G12 but not with the closely related Gα13 or with several other heterotrimeric G proteins. Axin preferentially binds the activated form of Gα12, a behavior consistent with other RGS proteins. However, unlike other RGS proteins, that of axin (axinRGS) does not affect intrinsic GTP hydrolysis by Gα12. Despite its inability to act as a GTPase-activating protein, we demonstrate that in cells, axinRGS can compete for Gα12 binding with the RGS domain of p115RhoGEF, a known G12-interacting protein that links G12 signaling to activation of the small G protein Rho. Moreover, ectopic expression of axinRGS specifically inhibits Gα12-directed activation of the Rho pathway in MDA-MB 231 breast cancer cells. These findings establish that the RGS domain of axin is able to directly interact with the α subunit of heterotrimeric G protein G12 and provide a unique tool to interdict Gα12-mediated signaling processes.
Footnotes
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This work was supported by National Institutes of Health grant CA100869 (to P.J.C.) and the National Institutes of Health Clinical Scientist Development Award DK62833 (to T.A.F.). L.N.S. and T.A.F. contributed equally to this work.
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ABBREVIATIONS: GPCR, G protein-coupled receptor; RGS, regulator of G protein signaling; APC, adenomatous polyposis coli; RhoGEF, Rho guanine nucleotide exchange factor; QL, mutationally activated Gα form; WT, wild-type; GFP, green fluorescent protein; DTT, dithiothreitol; GST, glutathione transferase; HEK, human embryonic kidney; LPX, polyoxyethylene 10 laurel ether; PAGE, polyacrylamide gel electrophoresis; GTPγS, guanosine 5′-O-(3-thio)triphosphate; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); PHEM, PIPES/HEPES/EGTA/MgSO4; PBS, phosphate-buffered saline; GAP, GTPase-activating protein.
- Received February 21, 2006.
- Accepted July 25, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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