Cholinergic receptor-mediated differential cytoskeletal recruitment of actin- and integrin-binding proteins in intact airway smooth muscle

We tested the hypothesis that cholinergic receptor stimulation recruits actin- and integrin-binding proteins from the cytoplasm to the cytoskeleton-membrane complex in intact airway smooth muscle. We stimulated bovine tracheal smooth muscle with carbachol and fractionated the tissue homogenate into pellet (P) and supernatant (S) by ultracentrifugation. In unstimulated tissues, calponin exhibited the highest basal P-to-S ratio (P/S; 2.74 +/- 0.47), whereas vinculin exhibited the lowest P/S (0.52 +/- 0.09). Cholinergic receptor stimulation increased P/S of the following proteins in descending order of sensitivity: alpha-actinin > talin approximate to metavinculin > alpha-smooth muscle actin > vinculin approximate to calponin. Carbachol induced ERK1/2 phosphorylation by 300% of basal value. U0126 (10 muM) completely inhibited carbachol-induced ERK1/2 phosphorylation but did not significantly affect the correlation between alpha-actinin P/S and carbachol concentration. This observation indicates that cytoskeletal/membrane recruitment of alpha-actinin is independent of ERK1/2 mitogen-activated protein kinase activation. Metavinculin and vinculin are splice variants of a single gene, but metavinculin P/S was significantly higher than vinculin P/S. Furthermore, the P/S of metavinculin but not vinculin increased significantly in response to cholinergic receptor stimulation. Calponin and alpha-actinin both belong to the family of calponin homology (CH) domain proteins. However, unlike alpha-actinin, the calponin P/S did not change significantly in response to cholinergic receptor stimulation. These findings indicate differential cytoskeletal/membrane recruitment of actin- and integrin-binding proteins in response to cholinergic receptor stimulation in intact airway smooth muscle. alpha-Actinin, talin, and metavinculin appear to be key cytoskeletal proteins involved in the recruitment process.