Abstract

The specific aim of this project is to define the molecular structure of gelsolin in order to better understand its role in the cell and in the blood. Cytoplasmic gelsolin is a cytoskeletal protein of a variety of human tissues, which in vitro is capable of shortening actin filaments in a calcium dependent manner. It was first found in leukocytes and is thought to provide the means by which the actin filament structure is altered in response to micromolar calcium fluxes intracellularly, thereby allowing movement and secretion. A plasma variant of gelsolin is closely related to cytoplasmic gelsolin in structure and function in vitro. The role of plasma gelsolin is uncertain but it has been shown by an ELISA assay to be present in all humans studied. It binds fibrin and fibronectin, and may be important in coagulation and/or clearing of actin filaments from the circulation released by cell death. Plasma levels of gelsolin appear to be reduced in the Adult Respiratory Distress Syndrome but no other disease examined. A human hepatoma derived cell line, Hep G2, specifically makes both forms of gelsolin. The simultaneous production of a cytoskeletal and closely related secreted protein is unique in mammalian cells.
The aims of this project are as follows. The molecular structure of gelsolin will be determined by a combination of protein structural analysis and gene technology. The primary amino acid sequence will be determined by partially sequencing purified intact gelsolin and peptide fragments of gelsolin, and gelsolin cDNA clones will be isolated and sequenced. Sites of actin-binding and Ca+2 binding within the molecule will be determined by studying proteolytically-derived fragments for these activities. The gelsolin cDNA clones will be used to identify genomic clones to determine the gelsolin gene structure with a view to determining how regulation is exerted over synthesis and movement of each form of gelsolin within the cell. The gelsolin cDNA clone will also be used to assess the tissue distribution of gelsolin and the response of gelsolin synthesis in various tissues to factors such as steroids, cellular differentiation, and transforming agents. The gelsolin cDNA clones will permit comparison of gelsolin with homologous actin-binding proteins including villin.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08HL001582-03
Application #
3081971
Study Section
Research Manpower Review Committee (MR)
Project Start
1985-07-01
Project End
1990-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost

  Institution

Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199

  Publications

Gorlin, J B; Henske, E; Warren, S T et al. (1993) Actin-binding protein (ABP-280) filamin gene (FLN) maps telomeric to the color vision locus (R/GCP) and centromeric to G6PD in Xq28. Genomics 17:496-8
Kwiatkowski, D J; Perman, S (1991) Dinucleotide repeat polymorphism at the GSN locus (9q32-34). Nucleic Acids Res 19:967
Kwiatkowski, D J; Nygaard, T G; Schuback, D E et al. (1991) Identification of a highly polymorphic microsatellite VNTR within the argininosuccinate synthetase locus: exclusion of the dystonia gene on 9q32-34 as the cause of dopa-responsive dystonia in a large kindred. Am J Hum Genet 48:121-8
Kwiatkowski, D J (1991) Dinucleotide repeat polymorphism at the ABL locus (9q34). Nucleic Acids Res 19:967
Youssoufian, H; McAfee, M; Kwiatkowski, D J (1990) Cloning and chromosomal localization of the human cytoskeletal alpha-actinin gene reveals linkage to the beta-spectrin gene. Am J Hum Genet 47:62-72
Kwiatkowski, D J; Aklog, L; Ledbetter, D H et al. (1990) Identification of the functional profilin gene, its localization to chromosome subband 17p13.3, and demonstration of its deletion in some patients with Miller-Dieker syndrome. Am J Hum Genet 46:559-67
Kramer, P L; de Leon, D; Ozelius, L et al. (1990) Dystonia gene in Ashkenazi Jewish population is located on chromosome 9q32-34. Ann Neurol 27:114-20
Kwiatkowski, D J; Janmey, P A; Yin, H L (1989) Identification of critical functional and regulatory domains in gelsolin. J Cell Biol 108:1717-26
Hartwig, J H; Chambers, K A; Hopcia, K L et al. (1989) Association of profilin with filament-free regions of human leukocyte and platelet membranes and reversible membrane binding during platelet activation. J Cell Biol 109:1571-9
Kwiatkowski, D J; Ozelius, L; Schuback, D et al. (1989) The gelsolin (GSN) cDNA clone, from 9q32-34, identifies BclI and StuI RFLPs. Nucleic Acids Res 17:4425

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