Felberg J, Johnson P
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
J Biol Chem. 1998 Jul 10;273(28):17839-45. doi: 10.1074/jbc.273.28.17839.
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.
CD45是一种跨膜双结构域酪氨酸磷酸酶，是淋巴细胞抗原受体启动有效信号转导所必需的。与大多数跨膜双结构域磷酸酶一样，第二个磷酸酶结构域的作用尚不清楚。在本研究中，使用从细菌中纯化的重组CD45胞质结构域蛋白来评估各个磷酸酶结构域的功能。表达CD45膜近端区域、第一个磷酸酶结构域和间隔区的重组蛋白（rD1）具有催化活性，并且发现主要以二聚体形式存在。与此相反，表达CD45间隔区、第二个磷酸酶结构域和羧基末端的重组蛋白（rD2）以单体形式存在，并且对任何测试底物均无催化活性。将rD1与表达CD45整个胞质结构域的重组蛋白（rD1/D2）进行比较表明，rD1/D2的催化活性高2至3倍，更耐热，并且主要以单体形式存在。对rD1/D2进行有限的胰蛋白酶消化，为N端27 kDa片段和C端53 kDa片段之间的非共价结合提供了证据，表明存在分子内相互作用。此外，在体外结合试验中发现rD1与rD2特异性结合。综上所述，这些数据为CD45胞质结构域中发生的分子内相互作用提供了证据。在缺乏包含第二个磷酸酶结构域的C端区域的情况下，会发生分子间相互作用，导致二聚体形成。
