The long range goal of this proposal is to define in detail the protein associations of isoforms of erythrocyte membrane structural proteins, spectrin and ankyrin, that have been discovered to be general components of cell plasma membranes, and to elucidate cellular functions of these protein linkages. The approach will be to analyse quantitatively the intermolecular associations of purified, radiolabeled brain spectrin, brain ankyrin, and defined proteolytic domains of these proteins, with proteins in membrane and cytoplasmic fractions. These measurements will a) provide assays for identification and purification of binding proteins; b) allow evaluation of regulation of the protein interactions; c) be used to screen for toxins and mutations that perturb specific protein associations. Specific goals are in the following areas: (1) studies of protein associations of brain spectrin and defined domains of brain spectrin with proteins in membranes and the cytosol. Newly identified spectrin-binding proteins will be purified and characterized. Regulation of spectrin-protein association will be studied with Ca++, calmodulin, and various protein kinases. (2) Brain ankyrin. This protein will be purified and characterized regarding modification by phosphorylation/dephosphorylation and its cellular localization. (3) Protein associations of brain ankyrin will be defined and binding proteins purified as described with brain spectrin. (4) Functions of integral membrane proteins that bind to ankyrin. (5) Cellular functions of protein associations described above will be evaluated by a) using defined protein domains or antibody against against these domains as competitive antagonists of protein associations in living cells, b) screening toxins for modulation of protein associations with the goal of identifying a specific inhibitor, c) screening mutant mice for alteration in function or expression of spectrin, ankyrin or any binding proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033996-03
Application #
3284311
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1984-07-01
Project End
1989-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Davis, L H; Otto, E; Bennett, V (1991) Specific 33-residue repeat(s) of erythrocyte ankyrin associate with the anion exchanger. J Biol Chem 266:11163-9
Nehls, V; Drenckhahn, D; Joshi, R et al. (1991) Adducin in erythrocyte precursor cells of rats and humans: expression and compartmentalization. Blood 78:1692-6
Hu, R J; Bennett, V (1991) In vitro proteolysis of brain spectrin by calpain I inhibits association of spectrin with ankyrin-independent membrane binding site(s). J Biol Chem 266:18200-5
Kordeli, E; Davis, J; Trapp, B et al. (1990) An isoform of ankyrin is localized at nodes of Ranvier in myelinated axons of central and peripheral nerves. J Cell Biol 110:1341-52
Bennett, V (1990) Spectrin-based membrane skeleton: a multipotential adaptor between plasma membrane and cytoplasm. Physiol Rev 70:1029-65
Bennett, V (1990) Spectrin: a structural mediator between diverse plasma membrane proteins and the cytoplasm. Curr Opin Cell Biol 2:51-6
Steiner, J P; Walke Jr, H T; Bennett, V (1989) Calcium/calmodulin inhibits direct binding of spectrin to synaptosomal membranes. J Biol Chem 264:2783-91
Davis, J; Davis, L; Bennett, V (1989) Diversity in membrane binding sites of ankyrins. Brain ankyrin, erythrocyte ankyrin, and processed erythrocyte ankyrin associate with distinct sites in kidney microsomes. J Biol Chem 264:6417-26
Bennett, V (1989) The spectrin-actin junction of erythrocyte membrane skeletons. Biochim Biophys Acta 988:107-21
Kaiser, H W; O'Keefe, E; Bennett, V (1989) Adducin: Ca++-dependent association with sites of cell-cell contact. J Cell Biol 109:557-69

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