The goal of the proposed investigation is to characterize the array of isoforms of protein 4.1 in selected cells to discern tissue distribution, intracellular localization, binding partners, and physiologic function, with the expectation that these studies will provide new information about the cellular organization and role of 4.1 isoforms. After the patterns of endogenous isoform expression are defined (using various strategies including RT-PCR, nuclease protection assays, immunoprecipitation and blotting using motif-specific monoclonals), three Specific Aims will be addressed.
Specific Aim 1 will examine the 135 kD isoform having the """"""""headpiece"""""""" extension. Its location will be identified by immunoprecipitation/blotting of subcellular preparations and by confocal and electron microscopy, and potential binding partners will be examined by co-precipitation, affinity chromatography and the yeast-2 hybrid system (and, inevitably, extension of this strategy to a mammalian system). To complement this, similar studies will be done using transfection of epitope-tagged isoforms to track localization/binding. Functional studies will involve (a) forced expression of 4.1 isoforms in mammalian cells, (b) loss of function in nonerythroid cells induced using isoform-specific hammerhead ribozymes or antisense vectors, and (c) substitution of isoforms using these approaches in combination. The second Specific Aim will assess the functional importance of alternate splicing of the SAB domain by expression of epitope-tagged recombinant isoforms representing the eight alternate combinations of SAB. Similar methods will be used to identify location and binding partners. In addition, cell-free binding assays will examine binding to non-erythroid spectrin (S), fodrin (F), myosin, actin (A), S/A, or F/A. Expression of alternate isoforms in various cell lines will be used to assess effect on isoform localization, cell shape, and migration. The third Specific Aim will similarly extend these observations to the 30 kDa end-terminal domain. Binding partners will be identified using soluble systems, and similar strategies to those above will be employed as indicated. A fourth Specific Aim is mentioned (preparation of a 4.1 knock-out mouse), but it is also specifically stated that this is a collaborative effort with other groups and support will be obtained from other sources.
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