Characterization of recombinant CD45 cytoplasmic domain proteins. Evidence for intramolecular and intermolecular interactions.
CD45 is a transmembrane two-domain tyrosine phosphatase required for efficient signal transduction initiated by lymphocyte antigen receptors. As with most transmembrane two-domain phosphatases, the role of the second phosphatase domain is unclear. In this study, recombinant CD45 cytoplasmic domain proteins purified from bacteria were used to evaluate the function of the individual phosphatase domains. A recombinant protein expressing the membrane-proximal region, first phosphatase domain, and spacer region of CD45 (rD1) was catalytically active and found to exist primarily as a dimer. In contrast to this, a recombinant protein expressing the spacer region, the second phosphatase domain and the carboxy tail of CD45 (rD2) existed as a monomer and had no catalytic activity against any of the substrates tested. Comparison of rD1 with the recombinant protein expressing the entire cytoplasmic domain of CD45 (rD1/D2) indicated that rD1/D2 was 2-3-fold more catalytically active, was more thermostable, and existed primarily as a monomer. Limited trypsin digestion of rD1/D2 provided evidence for a noncovalent association between an N-terminal 27-kDa fragment and a C-terminal 53-kDa fragment, suggesting an intramolecular interaction. Furthermore, rD1 was found to specifically associate with rD2 in an in vitro binding assay. Taken together, these data provide evidence for an intramolecular interaction occurring in the cytoplasmic domain of CD45. In the absence of the C-terminal region containing the second phosphatase domain, intermolecular interactions occur, resulting in dimer formation.
Felberg
J
J
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
Johnson
P
P
D000595:Amino Acid Sequence; D002384:Catalysis; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012996:Solutions
D011994:Recombinant Proteins; D012996:Solutions; D017493:Leukocyte Common Antigens