Participation of calmodulin in immunoglobulin capping

When mouse b lymphocytes are incubated with antibodies against their surface immunoglobulin (Ig), patching and capping occur in a process that involves the action of the actomyosin cytoskeleton and the mobilization of cell calcium. Calmodulin (CaM) plays a central role in the Ca(++) regulation of many cellular structures and processes, including the cytoskeleton and membrane-bound enzymes, and therefore 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 polyacrylamide gels, where its R(f) is Ca(++)-dependent. It stimulates the activity of the CaM-dependent cyclic AMP phosphodiesterase (PDE) of bovine heart. Several phenothiazine and thioxanthene compounds as well as the drugs W7, W5, and R24571 inhibit CaM in in vitro enzyme assays with ID(50)’s of from 1 muM to more than 1 mM. These were tested for their effects on capping of Ig and were found to inhibit capping in dose- dependent fashions with ID(50)’s 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 and 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. These observations suggest that calmodulin participates in the latter stages of ligand-induced Ig redistribution probably by regulating the interaction of the cytoskeleton with the membrane.