The aim of these studies is to define the physiological function of dematin in mature erythrocytes. We wish to identify the proteins(s) to which dematin binds on eh erythroid plasma membrane and to understand how these interactions are regulated by phosphorylation. The identification of dematin binding protein(s) on the membrane, and the understanding of the regulation of dematin-membrane interactions by phosphorylation, may elucidate a new mechanism by which spectrin-actin complexes are attached to the plasma membrane. Since immunoreactive homologs of dematin exist in many non-erythroid cells, the results of the proposed studies may have general implication well beyond red cell biology. In order to achieve these goals: (1) We propose to determine the number and affinity of dematin binding protein(s) on human erythrocyte inside- out-vesicles, and to identify the individual protein(s) using chemical crosslinking, immunoprecipitation, affinity chromatography, and gel filtration techniques. Then using defined cDNA constructs expressed in vitro, we will test whether dematin binding sites include the cytoplasmic domains of band 3 and glycophorin. Finally, we will examine whether tyrosine phosphorylation of dematin regulates its interaction with the plasma membrane. (2) To test the hypothesis that dematin may function as an adaptor protein that links spectrin-actin complexes to the plasma membrane, we will carry out a detailed characterization of the dematin- spectrin interaction. The studies will include quantification of the dematin-spectrin interaction by sedimentation equilibrium assays, the mapping of dematin and beta-spectrin binding site using cDNA constructs, and the regulation of dematin-spectrin interaction by tyrosine phosphorylation.
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