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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL044985-11
Application #
6030606
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1990-07-01
Project End
2000-08-31
Budget Start
1999-07-01
Budget End
2000-08-31
Support Year
11
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Huang, Shu-Ching; Zhou, Anyu; Nguyen, Dan T et al. (2016) Protein 4.1R Influences Myogenin Protein Stability and Skeletal Muscle Differentiation. J Biol Chem 291:25591-25607
Mattagajasingh, Subhendra N; Huang, Shu-Ching; Benz Jr, Edward J (2009) Inhibition of protein 4.1 R and NuMA interaction by mutagenization of their binding-sites abrogates nuclear localization of 4.1 R. Clin Transl Sci 2:102-11
Huang, Shu-Ching; Liu, Eva S; Chan, Siu-Hong et al. (2005) Mitotic regulation of protein 4.1R involves phosphorylation by cdc2 kinase. Mol Biol Cell 16:117-27
Huang, Shu-Ching; Jagadeeswaran, Ramasamy; Liu, Eva S et al. (2004) Protein 4.1R, a microtubule-associated protein involved in microtubule aster assembly in mammalian mitotic extract. J Biol Chem 279:34595-602
Kontrogianni-Konstantopoulos, A; Frye, C S; Benz Jr, E J et al. (2001) The prototypical 4.1R-10-kDa domain and the 4.1g-10-kDa paralog mediate fodrin-actin complex formation. J Biol Chem 276:20679-87
Mattagajasingh, S N; Huang, S C; Hartenstein, J S et al. (2000) Characterization of the interaction between protein 4.1R and ZO-2. A possible link between the tight junction and the actin cytoskeleton. J Biol Chem 275:30573-85
Kontrogianni-Konstantopoulos, A; Huang, S C; Benz Jr, E J (2000) A nonerythroid isoform of protein 4.1R interacts with components of the contractile apparatus in skeletal myofibers. Mol Biol Cell 11:3805-17
Mattagajasingh, S N; Huang, S C; Hartenstein, J S et al. (1999) A nonerythroid isoform of protein 4.1R interacts with the nuclear mitotic apparatus (NuMA) protein. J Cell Biol 145:29-43
Baklouti, F; Huang, S C; Vulliamy, T J et al. (1997) Organization of the human protein 4.1 genomic locus: new insights into the tissue-specific alternative splicing of the pre-mRNA. Genomics 39:289-302
Baklouti, F; Huang, S C; Tang, T K et al. (1996) Asynchronous regulation of splicing events within protein 4.1 pre-mRNA during erythroid differentiation. Blood 87:3934-41

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