Mapping the fodrin binding domain in CD45, a leukocyte membrane-associated tyrosine phosphatase.
CD45 belongs to a family of high molecular mass leukocyte glycoproteins. It contains both an intrinsic protein tyrosine phosphatase (PTPase) activity and a cytoskeleton binding site in its cytoplasmic domain. Certain cytoskeletal proteins, such as fodrin (a spectrin-like molecule), are known to play an important role in the regulation of CD45's PTPase activity. In this study we mapped the fodrin binding domain of CD45 by deleting various portions of the cytoplasmic region, followed by the expression of these truncated cDNAs using an in vitro transcription/translation system. The results of these experiments indicate that the CD45 fodrin binding domain resides between amino acids 825 and 939. Construction of a fusion protein encoding the region between amino acids 825 and 939 shows that this particular sequence itself is sufficient for fodrin binding. Further analyses indicate that the sequence (930EENKKKNRN939S) in CD45 has good sequence homology with the spectrin binding domain found in the MSP1 glycoprotein of the malarial parasite. Biochemical studies, using binding competition assays, and a synthetic peptide containing the sequence 930EENKKKNRN939S, support the conclusion that the sequence between amino acids 930 and 939 is a critical part of CD45's fodrin binding domain. Further analyses indicate that this sequence is also involved in the fodrin-induced up-regulation of CD45 PTPase activity. Therefore, we suggest that fodrin binding to this domain is required for the onset of CD45-mediated signal transduction and leukocyte activation.
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Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida 33101.
Lokeshwar
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Bourguignon
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D003593:Cytoplasm / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000378:metabolism*; D007962:Leukocytes / Q000201:enzymology*; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D017384:Sequence Deletion; D013049:Spectrin / Q000378:metabolism; D015854:Up-Regulation
D002352:Carrier Proteins; D017493:Leukocyte Common Antigens; D008840:Microfilament Proteins; D017027:Protein Tyrosine Phosphatases; D013049:Spectrin