Participation of calmodulin in immunoglobulin capping.

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Abstract
When mouse B lymphocytes are incubated with antibodies against their surface Ig, patching and capping occur in a process that involves the action of the actomyosin cytoskeleton and the mobilization of cell Ca. Calmodulin (CaM), which plays a central role in the Ca2+ regulation of many cellular structures and processes, including the cytoskeleton and membrane-bound enzymes, was investigated for its role in capping. CaM was isolated from mouse lymphocytes by affinity chromatography on fluphenazine-Sepharose. Lymphocyte CaM co-migrates with calf brain CaM on SDS [sodium dodecyl sulfate] polyacrylamide gels, where its Rf is Ca2+-dependent. It stimulates the activity of the CaM-dependent cAMP phosphodiesterase (PDE) of bovine heart. Several phenothiazine and thioxanthene compounds and the drugs W7 [N-(6-aminohexyl)-5-cloro-1-napthalene-sulfonamide], W5 [N-(6-aminohexyl)-1-napthalene-sulfonamide] and R24571 inhibit CaM in in vitro enzyme assays with ID50 of from 1 .mu.M to > 1 mM. These were tested for their effects on capping of Ig and were found to inhibit capping in dose-dependent fashions with ID50 that corresponded to their anti-CaM potencies. The drugs also disrupted preformed caps and were all reversible. CaM was localized in lymphocytes by staining with a highly fluorescent trifluoperazine derivative (TFP) produced by photo-oxidation. TFP staining was diffuse in untreated lymphocytes but stained under caps in uropods in cells capped with anti-Ig antibodies. Staining of cells with antibodies against calf brain and rat testis calmodulin gave similar staining patterns. Staining of patched cells with either antibodies or TFP showed patched distributions of CaM, but submembranous CaM patches did not map one-on-one with respect to Ig patches. Calmodulin apparently participates in the latter stages of ligand-induced Ig redistribution probably by regulating the interaction of the cytoskeleton with the membrane.