In this proposal, the principal investigator intends to do detailed molecular biology and biochemical studies of human erythrocyte ankyrin and human erythrocyte adducin. Studies on erythrocyte ankyrin will include structural analysis of the 89 kDa domain and its constituient 33-residue repeats. Defined regions of the 89 k Da domain will be expressed in bacteria and the minimal number of repeats required for native secondary structure as monitored by circular dichroism will be determined. Crystals of the 89 k Da domain will be prepared for analysis of structure by X-ray diffraction. The binding sites for erythrocyte band 3 within the 33 residue repeat region of this domain will be mapped and the minimal number of repeats and unique repeats required for binding determined. Mapping for sites of tubulin and Na/K ATPase binding within the 89 k Da domain will be performed. Domains of other proteins with homologous 33 residue repeats (Drosophila Notch and C. elegans lin12 gene products) will be expressed and they will be used to identify possible common binding specificity for tubulin and other macromolecules with periodic structures (nucleic acids, phospholipids). The spectrin binding sites of red cell and brain ankyrins will be mapped and the minimum regions competent for expressing spectrin-binding activities determined. The regions within these ankyrins that provide specificity for red cell spectrin, and brain ankyrin for brain spectrin, will be determined. The hypothesis that the region of ankyrin deleted in protein 2.2, a product of alternatively spliced mRNA, functions as a pseudosubstrate and blocks potential binding sites will be explored. Proteins in kidney microsomes that have been demonstrated to recognize protein 2.2 and not unspliced ankyrin will be identified. The second major component of the study relates to human erythrocyte adducin. Complete cloning and sequencing of cDNA encoding aplha and beta subunits of human erythrocyte adducin are proposed. Boths subunits will be expressed and recombined to form heterodimers and possibly homodimers. Active sites for interaction with calmodulin, actin, spectrin/actin, subunit interactions to heterodimers and associations between heterodimers to form tetramers and higher oligomers will be determined using mapping techniques. Sites of phosphorylation by protein kinases A and C will also be determined.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK029808-19
Application #
2684112
Study Section
Special Emphasis Panel (NSS)
Project Start
1987-09-01
Project End
2000-03-31
Budget Start
1998-04-01
Budget End
2000-03-31
Support Year
19
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Duke University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Freeman, J L; Pitcher, J A; Li, X et al. (2000) alpha-Actinin is a potent regulator of G protein-coupled receptor kinase activity and substrate specificity in vitro. FEBS Lett 473:280-4
Michaely, P; Kamal, A; Anderson, R G et al. (1999) A requirement for ankyrin binding to clathrin during coated pit budding. J Biol Chem 274:35908-13
Zhang, X; Bennett, V (1998) Restriction of 480/270-kD ankyrin G to axon proximal segments requires multiple ankyrin G-specific domains. J Cell Biol 142:1571-81
Lambert, S; Davis, J Q; Bennett, V (1997) Morphogenesis of the node of Ranvier: co-clusters of ankyrin and ankyrin-binding integral proteins define early developmental intermediates. J Neurosci 17:7025-36
Kuhlman, P A; Hughes, C A; Bennett, V et al. (1996) A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments. J Biol Chem 271:7986-91
Schengrund, C L; DasGupta, B R; Hughes, C A et al. (1996) Ganglioside-induced adherence of botulinum and tetanus neurotoxins to adducin. J Neurochem 66:2556-61
Kordeli, E; Lambert, S; Bennett, V (1995) AnkyrinG. A new ankyrin gene with neural-specific isoforms localized at the axonal initial segment and node of Ranvier. J Biol Chem 270:2352-9
Hu, R J; Moorthy, S; Bennett, V (1995) Expression of functional domains of beta G-spectrin disrupts epithelial morphology in cultured cells. J Cell Biol 128:1069-80
Li, Z P; Burke, E P; Frank, J S et al. (1993) The cardiac Na+-Ca2+ exchanger binds to the cytoskeletal protein ankyrin. J Biol Chem 268:11489-91
Michaely, P; Bennett, V (1993) The membrane-binding domain of ankyrin contains four independently folded subdomains, each comprised of six ankyrin repeats. J Biol Chem 268:22703-9

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