title: Expression and subcellular distribution of filamin isotypes in endothelial cells and pericytes.
abstract: Two principal forms of the actin binding protein, filamin, are expressed in mammalian cells: nonmuscle and muscle isotypes (FLN-1 and FLN-2). A protein that copurifies with an alpha-naphthyl acetate hydrolyzing esterase from human omentum microvessel endothelial cells (EC) is isolated by nondenaturing electrophoresis, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and electroblotting. The purified protein is subjected to in situ trypsin cleavage, reversed-phase high performance liquid chromatography (HPLC) and automated Edman degradation. Six peptide fragments from the protein are identified to have 60-66% identity with nonmuscle filamin (ABP-280). Two of these peptides are 100% identical to a previously sequenced human muscle filamin fragment. Polyclonal antibody is produced using a 16-residue synthetic peptide corresponding to a structural beta-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 vein EC and at least three times the amount expressed in human omentum microvessel and bovine pulmonary artery EC. Differential detergent fractionation 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 microfilament organization in pericytes may be controlled in part by the two filamin isotypes, which in turn may contribute to pericyte contractility.
authors.affiliation: Microvascular Research Laboratory, Biological Science Center, Boston University, MA, USA.
authors.forename: N
authors.initials: N
authors.lastname: Shojaee
authors.forename: W F
authors.initials: WF
authors.lastname: Patton
authors.forename: N
authors.initials: N
authors.lastname: Chung-Welch
authors.forename: Q
authors.initials: Q
authors.lastname: Su
authors.forename: H B
authors.initials: HB
authors.lastname: Hechtman
authors.forename: D
authors.initials: D
authors.lastname: Shepro
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002478:Cells, Cultured; D003285:Contractile Proteins / Q000276:immunology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D004950:Esterases / Q000302:isolation & purification; D064448:Filamins; D006801:Humans; D008840:Microfilament Proteins / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009852:Omentum; D011817:Rabbits; D012160:Retina / Q000378:metabolism; D017421:Sequence Analysis; D013347:Subcellular Fractions
chemical_list: D000906:Antibodies; D003285:Contractile Proteins; C578663:FLNA protein, human; D064448:Filamins; D008840:Microfilament Proteins; D004950:Esterases