Galphai-2 is required for carbachol-induced stress fiber formation in human airway smooth muscle cells.

Abstract

To determine which heterotrimeric G protein couples muscarinic receptors to stress fiber formation [measured by an increase in the filamentous (F)- to monomeric (G)-actin ratio] in human airway smooth muscle (ASM) cells, cultured human ASM cells expressing the M2 muscarinic receptor were grown to confluence. Cells were exposed for 6 days to 10 microM antisense oligonucleotides designed to specifically bind to the mRNA encoding Galphai-2, Galphai-3, or Gqalpha. A randomly scrambled oligonucleotide served as a control. F- to G-actin ratios were measured with dual-fluorescence labeling after 5 min of carbachol exposure, which is known to increase the F- to G-actin ratio. Cells in parallel wells were harvested for immunoblot analysis of G protein alpha-subunit expression. Oligonucleotide antisense treatment decreased protein expression of the respective G protein alpha-subunit. Antisense depletion of the Galphai-2 protein but not of Galphai-3 or Gqalpha protein blocked the carbachol-induced increase in the F- to G-actin ratio. These results show that the Galphai-2 protein couples muscarinic receptors to stress fiber formation in ASM.