TGFβ Regulates the Expression of Gαi2 via an Effect on the Localization of Ras

S. M. Ward, A. P. Gadbut, D. Tang, A. G. Papageorge, L. Wu, G. LI, J. V. Barnett and J. B. Galper. TGFβ Regulates the Expression of Gαi2 via an Effect on the Localization of Ras. Journal of Molecular and Cellular Cardiology (2002) 34, 1217–1226. The negative chronotropic response of the heart to parasympathetic stimulation is mediated via the interaction of M2 muscarinic receptors, Gαi2 and the G-protein coupled inward rectifying K+ channel, GIRK1. Here TGFβ1 is shown to decrease the expression of Gαi2 in cultured chick atrial cells in parallel with attenuation of the negative chronotropic response to parasympathetic stimulation. The response to the acetylcholine analogue, carbamylcholine, decreased from a 95±2% (±SEM, n=8) inhibition of beat rate in control cells to 18±2% (±SEM,n =8) in TGFβ1 treated cells. Data support the conclusion that TGFβ regulation of Gαi2 expression was mediated via an effect on Ras. TGFβ1 inhibited Gαi2 promoter activity by 56±6% (±SEM, n=4) compared to control. A dominant activating Ras mutant reversed the effect of TGFβ on Gαi2 expression and stimulated Gαi2 promoter activity 1.7 fold above control. A dominant negative Ras mutant mimicked the effect of TGFβ1 on Gαi2 promoter activity. TGFβ had no effect on the ratio of GDP/GTP bound Ras, but markedly decreased the level of membrane associated Ras and increased the level of cytoplasmic Ras compared to control. Furthermore, farnesol, a precursor to farnesylpyrophosphate, the substrate for the farnesylation of Ras, not only reversed TGFβ1 inhibition of Ras localization to the membrane, but also reversed TGFβ1 inhibition of Gαi2promoter activity. FTI-277, a specific inhibitor of the farnesylation of Ras, mimicked the effect of TGFβ1 on Ras localization and Gαi2 promoter activity. These data suggest a novel relationship between TGFβ signaling, regulation of Ras function and the autonomic response of the heart.