Four major components of the erythrocyte cortical cytoskeleton (spectrin, actin, protein 4.1, and ankyrin) are now understood in considerable detail. Nearly a score of inherited diseases with erythrocyte instability have also been linked to specific molecular defects in one of these four proteins. However, our understanding of the role of other erythrocyte cytoskeletal proteins such as adducin or protein 4.9, or even how many additional proteins there are (eg, that bind to spectrin), or what diseases result from their dysfunction, remains rudimentary. The proposed studies seek to learn how specific interactions between proteins of the cytoskeleton individually and collectively contribute in vivo to the processes of cytoskeletal assembly, surface receptor segregation during erythropoiesis, and membrane stabilization in mature erythrocytes. Building on our previous studies, three complimentary approaches will be pursued: i) identification of new proteins that bind to spectrin using deletional analysis and in vitro binding assays. Novel proteins that bind to spectrin will be identified and cloned using powerful and recently established techniques to screen reticulocyte cDNA libraries for specific non-covalent protein-protein interactions. ii) Examination of the role of selected functional domains in spectrin, based on their ability to alter receptor segregation and the assembly of the nascent cytoskeleton in cultured mouse erythroleukemia cells (MEL). Recombinant functional domains are expressed in differentiating MEL cells under a globin promoter and enhancer. iii) Characterization of the phenotype that results in vivo from specific mutations in functionally active regions of selected cytoskeletal proteins. One approach will be to examine patients with inherited disorders of rbc shape or stability, using PCR followed by DNA sequencing of the active binding domains. A second more direct approach will involve the production of transgenic mice expressing spectrin or other cytoskeletal proteins with inactivating mutations in selected functional domains. This latter approach will allow the erythrocyte phenotype of dominant negative mutations involving the functional domains of spectrin to be directly evaluated, and will aid the search for patients with naturally occuring diseases involving such functional domains. Collectively, these studies will enhance our understanding of the spectrin cytoskeleton under in vivo conditions, identify the molecular basis of new inherited diseases, and extend the generality and significance of the erythrocyte paradigm for the study of more complex cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL028560-10
Application #
3339932
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1983-05-01
Project End
1996-04-30
Budget Start
1992-05-01
Budget End
1993-04-30
Support Year
10
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
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Kim, Jung H; Kwon, Soojung J; Stankewich, Michael C et al. (2016) Reactive protoplasmic and fibrous astrocytes contain high levels of calpain-cleaved alpha 2 spectrin. Exp Mol Pathol 100:1-7
Stankewich, Michael C; Cianci, Carol D; Stabach, Paul R et al. (2011) Cell organization, growth, and neural and cardiac development require ?II-spectrin. J Cell Sci 124:3956-66
La-Borde, Penelope J; Stabach, Paul R; Simonovic, Ivana et al. (2010) Ankyrin recognizes both surface character and shape of the 14-15 di-repeat of beta-spectrin. Biochem Biophys Res Commun 392:490-4
Cairo, Christopher W; Das, Raibatak; Albohy, Amgad et al. (2010) Dynamic regulation of CD45 lateral mobility by the spectrin-ankyrin cytoskeleton of T cells. J Biol Chem 285:11392-401
Stankewich, Michael C; Gwynn, Babette; Ardito, Thomas et al. (2010) Targeted deletion of betaIII spectrin impairs synaptogenesis and generates ataxic and seizure phenotypes. Proc Natl Acad Sci U S A 107:6022-7
Stabach, Paul R; Simonovi?, Ivana; Ranieri, Miranda A et al. (2009) The structure of the ankyrin-binding site of beta-spectrin reveals how tandem spectrin-repeats generate unique ligand-binding properties. Blood 113:5377-84
Stabach, Paul R; Devarajan, Prasad; Stankewich, Michael C et al. (2008) Ankyrin facilitates intracellular trafficking of alpha1-Na+-K+-ATPase in polarized cells. Am J Physiol Cell Physiol 295:C1202-14
Glantz, Susan B; Cianci, Carol D; Iyer, Rathna et al. (2007) Sequential degradation of alphaII and betaII spectrin by calpain in glutamate or maitotoxin-stimulated cells. Biochemistry 46:502-13
Simonovic, Miljan; Zhang, Zhushan; Cianci, Carol D et al. (2006) Structure of the calmodulin alphaII-spectrin complex provides insight into the regulation of cell plasticity. J Biol Chem 281:34333-40

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