EXPRESSION AND SUBCELLULAR-DISTRIBUTION OF FILAMIN ISOTYPES IN ENDOTHELIAL-CELLS AND PERICYTES

Two principal forms of the actin binding protein, filamin, are express ed in mammalian cells: nonmuscle and muscle isotypes (FLN-1 and FLN-2) . A protein that copurifies with an a-naphthyl acetate hydrolyzing est erase from human omentum microvessel endothelial cells (EC) is isolate d by nondenaturing electrophoresis, sodium dodecyl sulfate (SDS)-polya crylamide gel electrophoresis and electroblotting. The purified protei n is subjected to in situ trypsin cleavage, reversed-phase high perfor mance liquid chromatography (HPLC) and automated Edman degradation. Si x peptide fragments from the protein are identified to have 60-66% ide ntity with nonmuscle filamin (ABP-280). Two of these peptides are 100% identical to a previously sequenced human muscle filamin fragment. Po lyclonal antibody is produced using a 16-residue synthetic peptide cor responding to a structural P-sheet region of muscle filamin. Compared with a variety of vascular cells evaluated, retinal pericytes express an abundance of both muscle and non-muscle filamin isotypes. Pericytes contain at least 10 times more muscle filamin than human umbilical ve in EC and at least three times the amount expressed in human omentum m icrovessel and bovine pulmonary artery EC. Differential detergent frac tionation indicates that both filamin isotypes are primarily localized in the cytosol and membrane/organelle fractions of pericytes. Another actin crosslinking protein, alpha-actinin, is primarily found in the cytosol and cytoskeletal fractions. The dynamic regulation of actin mi crofilament organization in pericytes may be controlled in part by the two filamin isotypes, which in turn may contribute to pericyte contra ctility.