Characterization of Recombinant CD45 Cytoplasmic Domain Proteins

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 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 is a transmembrane protein-tyrosine phosphatase (PTP) 1The abbreviations used are: PTP, protein-tyrosine phosphatase; GST, glutathioneS-transferase; pNPP; para-nitrophenyl phosphate; rD1/D2, recombinant 6-His-tagged CD45 cytoplasmic domain protein; rD1, recombinant 6-His-tagged CD45 protein containing the membrane-proximal region, the first phosphatase domain, and the spacer region; rD2, recombinant 6-His-tagged CD45 protein containing part of the spacer region, the second phosphatase domain, and the carboxy tail; rD2.2, recombinant 6-His-tagged CD45 protein containing the membrane-proximal region, the second phosphatase domain, and the carboxy tail; PTP-D1, the first phosphatase domain; PTP-D2, the second phosphatase domain; RPTP, receptor-like protein-tyrosine phosphatase; CD, circular dichroism; NTA, nitrilotriacetic acid. 1The abbreviations used are: PTP, protein-tyrosine phosphatase; GST, glutathioneS-transferase; pNPP; para-nitrophenyl phosphate; rD1/D2, recombinant 6-His-tagged CD45 cytoplasmic domain protein; rD1, recombinant 6-His-tagged CD45 protein containing the membrane-proximal region, the first phosphatase domain, and the spacer region; rD2, recombinant 6-His-tagged CD45 protein containing part of the spacer region, the second phosphatase domain, and the carboxy tail; rD2.2, recombinant 6-His-tagged CD45 protein containing the membrane-proximal region, the second phosphatase domain, and the carboxy tail; PTP-D1, the first phosphatase domain; PTP-D2, the second phosphatase domain; RPTP, receptor-like protein-tyrosine phosphatase; CD, circular dichroism; NTA, nitrilotriacetic acid. that is expressed on all nucleated hematopoietic cells. Its phosphatase activity is required for efficient signal transduction initiated by lymphocyte antigen receptors, which is essential for T cell development and T and B cell activation (reviewed in Refs. 1Trowbridge I.S. Thomas M.L. Annu. Rev. Immunol. 1994; 12: 85-116Crossref PubMed Scopus (659) Google Scholar and 2Johnson P. Maiti A. Ng D.H.W. Herzenberg L.A. Weir D.M. Herzenberg L.A. Blackwell C. Weir's Handbook of Experimental Immunology. 5th Ed. II. Blackwell Science Inc., Malden, MA1997: 62.1-62.16Google Scholar). In T cells, CD45 has been shown to dephosphorylate p56lck and p59fyn in vitro and to be responsible for their dephosphorylationin vivo (3Ostergaard H.L. Shackelford D.A. Hurley T.R. Johnson P. Hyman R. Sefton B.M. Trowbridge I.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8959-8963Crossref PubMed Scopus (414) Google Scholar, 4Mustelin T. Coggeshall K.M. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6302-6306Crossref PubMed Scopus (408) Google Scholar, 5Mustelin T. Altman A. Oncogene. 1990; 5: 809-813PubMed Google Scholar, 6Mustelin T. Pessa-Morikawa T. Autero M. Gassmann M. Andersson L.C. Gahmberg C.G. Burn P. Eur. J. Immunol. 1992; 22: 1173-1178Crossref PubMed Scopus (159) Google Scholar, 7Shiroo M. Goff L. Biffen M. Shivnan E. Alexander D. EMBO J. 1992; 11: 4887-4897Crossref PubMed Scopus (165) Google Scholar). The role of CD45 in the dephosphorylation of p56lck and p59fyn is likely to be a necessary prerequisite step for their effective participation in T cell receptor-mediated signaling events. In vitro, CD45 can dephosphorylate several artificial and phosphopeptide substrates, yet only a limited number of substrates have been identified for CD45in vivo. How substrate specificity is achieved in the cell is not yet understood. However, it is of interest to note that CD45 can associate with p56lck, a major substrate for CD45 in T cells, both in vitro (8Autero M. Saharinen J. Pessa-Morikawa T. Soula-Rothhut M. Oetken C. Gassmann M. Bergman M. Alitalo K. Burn P. Gahmberg C.G. Mustelin T. Mol. Cell. Biol. 1994; 14: 1308-1321Crossref PubMed Scopus (118) Google Scholar, 9Ng D.H.W. Watts J.D. Aebersold R. Johnson P. J. Biol. Chem. 1996; 271: 1295-1300Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) and in T cells, albeit with low stoichiometry (10Schraven B. Kirchgessner H. Gaber B. Samstag Y. Meuer S. Eur. J. Immunol. 1991; 21: 2469-2477Crossref PubMed Scopus (91) Google Scholar, 11Koretzky G.A. Kohmetscher M. Ross S. J. Biol. Chem. 1993; 268: 8958-8964Abstract Full Text PDF PubMed Google Scholar). It is also not known how the phosphatase activity of CD45 is regulated. By analogy with receptors possessing tyrosine kinase activity, it is possible that receptor tyrosine phosphatases may be regulated by dimerization. It has been suggested by cross-linking studies in T cells that CD45 may exist in dimeric form together with CD45AP, a 30-kDa CD45-associated protein (12Takeda A. Wu J.J. Maizel A.L. J. Biol. Chem. 1992; 267: 16651-16659Abstract Full Text PDF PubMed Google Scholar). Induced dimerization of an epidermal growth factor receptor-CD45 chimera with epidermal growth factor has been shown to inhibit the restoration of T cell signaling events (13Desai D.M. Sap J. Schlessinger J. Weiss A. Cell. 1993; 73: 541-554Abstract Full Text PDF PubMed Scopus (244) Google Scholar), suggesting the possibility that CD45 activity may be regulated by dimerization. In addition, the first phosphatase domain (PTP-D1) of a related phosphatase, RPTPα, crystallized as a dimer, with the membrane-proximal region of one PTP domain inserted into the catalytic region of the second PTP domain. It was predicted that if such an interaction occurred under more physiological conditions, phosphatase activity would be inhibited (14Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar). However, to date, no direct evidence has been obtained for the formation of CD45 RPTPα, to the of protein-tyrosine most of the two-domain phosphatase the of catalytic activity has been shown to in and the function of the second phosphatase domain is unclear. of has been shown to catalytic activity, albeit Y. EMBO J. 1991; PubMed Scopus Google Scholar). In transmembrane no phosphatase activity has been for PTP-D2, which in in M. H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: PubMed Scopus Google Scholar, M. T. M. H. EMBO J. 1990; PubMed Scopus Google Scholar, P. H.L. C. Trowbridge I.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, 1993; PubMed Scopus Google Scholar and in Refs. D. Biol. PubMed Scopus Google Scholar, Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Sci. 1996; 21: Full Text PDF PubMed Scopus Google Scholar). In these a role for has been and in two-domain the of has been shown to be required for activity Y. EMBO J. 1991; PubMed Scopus Google Scholar, M. T. M. H. EMBO J. 1990; PubMed Scopus Google Scholar, 1993; PubMed Scopus Google Scholar). data as to CD45 is an active phosphatase in the absence of M. T. M. H. EMBO J. 1990; PubMed Scopus Google Scholar, P. H.L. C. Trowbridge I.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). data that CD45 is active to a protein is not active the is D. PubMed Scopus Google Scholar). data as to CD45 is an active catalytic activity has been to CD45 vitro as a recombinant protein P. H.L. C. Trowbridge I.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) the essential in has been to a and vitro M. H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: PubMed Scopus Google Scholar, M. T. M. H. EMBO J. 1990; PubMed Scopus Google Scholar, P. H.L. C. Trowbridge I.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). of the in PTP-D1, not in PTP-D2, the restoration of T cell signaling events D.M. Sap J. Schlessinger J. Weiss A. EMBO J. 1994; PubMed Scopus Google Scholar), that phosphatase activity in PTP-D1, and not PTP-D2, was essential for this However, in cells of an active, form of CD45 the catalytic in has to that has phosphatase activity J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). the function of of CD45 is unclear. the function of the individual PTP of CD45 and to the role of these in the function of recombinant CD45 cytoplasmic domain proteins were and It was found that rD1 of CD45 was an active protein-tyrosine phosphatase in the absence of and was as a dimer. In no phosphatase activity was for rD2 of CD45. In the two-domain CD45 cytoplasmic domain the of the of the and was required for binding of rD1 to rD2 and the association of and C-terminal of rD1/D2 provided evidence for an intramolecular interaction occurring in In the absence of the C-terminal region PTP-D2, intermolecular interactions occur, to the formation of rD1 was from and were from was from and were from and of was and purified as D.H.W. Watts J.D. Aebersold R. Johnson P. J. Biol. Chem. 1996; 271: 1295-1300Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and was from was from and trypsin was from The was from M. of and binding domain protein was from of 6-His-tagged CD45 cytoplasmic domain rD1/D2, containing as in M.L. A. Y. Trowbridge I.S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and the catalytically with a of to were as D.H.W. Maiti A. Johnson P. PubMed Scopus Google Scholar). A (rD1) to containing the membrane-proximal region, PTP-D1, and the spacer region, was P. H.L. C. Trowbridge I.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar) and into the D.H.W. Maiti A. Johnson P. PubMed Scopus Google Scholar). (rD2) which part of the spacer region, PTP-D2, and the carboxy was by a fragment and a to a A second the membrane-proximal region, PTP-D2, and carboxy tail was by fragment containing to and a to containing rD2 and in were into D.H.W. Maiti A. Johnson P. PubMed Scopus Google Scholar), and rD2 was also into were expressed in J.J. 1990; PubMed Scopus Google Scholar) E. was expressed in with a T. C. T. J. T. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). rD1/D2, the rD1, and rD2 were purified as D.H.W. Maiti A. Johnson P. PubMed Scopus Google Scholar) with the were and all were were in to were with and and in and The was a in A B in and from a a of to B. was purified as of on a it was a in used for protein and of and recombinant proteins were the from was to and proteins were and were purified as and to was to and and were the as substrates were as D.H.W. Johnson P. J. Immunol. PubMed Scopus Google Scholar). recombinant proteins were in of phosphatase and with of phosphopeptide to of recombinant protein in phosphatase was to of to to the The in was a in a A of was used to the of were obtained by phosphatase activity of of rD1 and rD1/D2 from to as substrate in the for to for and and for to were rD1/D2 and rD1 were from to for phosphatase activity was by of to of was as D.H.W. Maiti A. P. K. T. B. Johnson P. J. PubMed Scopus Google Scholar). of rD1 rD1/D2 was to of to the The was by in a were on a and to and the of substrate was by with as the and Inc., as the The was and to from was to of rD1/D2 in a of of substrate of and The was for to and by the of of of was on a and was on a under of rD1/D2 was with trypsin for was for and the was on a and to The was with the 53-kDa and 27-kDa were and the N-terminal was obtained from the of A was with The was with protein from and from rD1, rD2, rD2.2, and rD1/D2 were to the in from to for rD1, rD2 to and rD1/D2 to were on the to and were used for of protein to was with binding and and of 6-His-tagged protein was to the was to a of and the was on for The was with of binding and on a to and with as the and as the were as The was with to the proteins and to 6-His-tagged CD45 cytoplasmic domain rD1, containing the membrane-proximal region, PTP-D1, and the spacer region, rD2, containing part of the spacer region, PTP-D2, and the carboxy tail a second rD2.2, containing the membrane-proximal region, PTP-D2, and the carboxy tail the cytoplasmic domain proteins rD1/D2, and a catalytically in A. proteins were purified from E. rD1 purified as a with both by cytoplasmic domain not suggesting that the is a were for phosphatase activity, and rD1 was found to be catalytically active the phosphopeptide as a it was active rD1/D2 rD2 and had no activity against this of rD1 and rD1/D2 activity and as substrates indicated that the two-domain phosphatase (rD1/D2) was a 2–3-fold more efficient rD1 In contrast to this, the rD2 proteins expressing the membrane-proximal region the spacer region the of the protein not any phosphatase activity for any of the substrates in more was for to with substrates, no activity was The of in the rD1/D2 protein not the substrate specificity of against the substrates indicated in rD1 and rD1/D2 were also to dephosphorylate recombinant Comparison of the of dephosphorylation indicated that the recombinant domain phosphatase, rD1/D2, was more efficient activity of CD45 cytoplasmic domain of rD1/D2, rD1, and and more of rD2 were against a of phosphopeptide to of were and for rD1/D2 and rD1 and and for rD2 and to is activity of recombinant CD45 cytoplasmic domain and rD1 were as a and rD1 were were from of the data and were from to rD1/D2 and rD1 were as a in a activity of rD1/D2 and rD1 recombinant as the of of rD1/D2 rD1 were with of of protein for the were to and to and the of tyrosine in the was by an indicated on the in of dephosphorylation by rD1/D2 and The were to and the of was as rD1/D2 and rD1 were were from of the data and were from to the between the (rD1) and two-domain CD45 phosphatase activity was rD1 and rD1/D2 activity as the substrate and a of activity in the However, in the between rD1/D2 a of activity rD1 that the of a more for an in the catalytic of activity was also by rD1/D2 and rD1 the indicated in for as a rD1 phosphatase activity rD1/D2, that it is of rD1/D2 and of rD1/D2 and rD1 were the indicated for phosphatase activity was with as was The data was an from were obtained the phosphopeptide was used as the substrate not The of the recombinant proteins were by rD1, not rD2, a to rD1/D2, suggesting that the of rD1 was in a dimeric the of rD2 suggested that it was primarily as a monomer. rD1 primarily as a this a of that rD1 is primarily in a dimeric rD1/D2 as a monomer a rD2 was as a rD2.2, containing the membrane-proximal region in of the spacer region, was in and in the not the of a dimeric form of rD1 and of rD1/D2 and rD2, the recombinant proteins were on both and the the of rD1/D2 and rD2 to under However, under conditions, rD1 to a as rD1/D2 with dimer it as a monomer rD1 primarily as a dimer under monomer was not rD1 was to be active against that dimer formation not catalytic However, it was that this was active rD1/D2, which primarily existed as a monomer. The that rD1 a dimer in rD1/D2 not suggested that an intramolecular interaction may be occurring in the cytoplasmic domain of CD45 between and PTP-D2, dimerization. would also to the of on the catalytic activity and of in the rD1/D2 and intramolecular interactions were occurring in rD1/D2, limited trypsin digestion was and the were on both and A that with of rD1/D2 is primarily into major of and these as a on the that both with one of this from the and on an that both were the in this N-terminal was obtained for the N-terminal the 27-kDa fragment was found to the N-terminal of the CD45 cytoplasmic domain to the of the membrane-proximal region and of the first phosphatase domain. The 53-kDa fragment was found to with the and can be to of the of the first phosphatase domain the catalytic the spacer region, the entire second phosphatase domain, and of the carboxy the of a noncovalent association between the and C-terminal of the cytoplasmic domain of CD45. this intramolecular interaction the in rD2, in vitro binding were with rD1 and rD2 rD2 was as a protein to and recombinant 6-His-tagged proteins rD1 and rD2 were As can be in rD1 to not to to In addition, only rD1 and not 6-His-tagged rD2, with a However, only a of rD1 to The for this is may the that rD1 is as a dimer. The of a interaction between rD1 and rD2 evidence for an intramolecular to tail interaction occurring the cytoplasmic domain of CD45. data that of CD45 not the of to be catalytically which not any phosphatase activity from an in vitro and rD1 protein P. H.L. C. Trowbridge I.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). M. T. M. H. EMBO J. 1990; PubMed Scopus Google Scholar) only activity from a rD1 protein from cell and D. PubMed Scopus Google Scholar) found that a recombinant protein expressing CD45 was not active protein was D. PubMed Scopus Google Scholar). In this study, several were which may of activity were for the rD1 the rD1 protein was in and was and purified from the and was to to that protein and such as were which to the of the protein not catalytic activity was for rD2 rD2 was found to be a monomer in was as a the membrane-proximal region of which may have to this the of this region to may have an indicated that this a to that in the rD1/D2 protein not that an active rD1, no activity was for rD2, in a for several that is not catalytically active against any of the substrates expressed as a recombinant data obtained from in vitro and recombinant proteins P. H.L. C. Trowbridge I.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). In addition, the the predicted catalytic region of CD45 is an and that to a role in the (reviewed in Refs. D. Biol. PubMed Scopus Google Scholar, Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Sci. 1996; 21: Full Text PDF PubMed Scopus Google Scholar). of the catalytic in had no on the protein-tyrosine phosphatase activity of CD45 in recombinant and in the restoration of T cell function by a epidermal growth factor phosphatase M. H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: PubMed Scopus Google Scholar, P. H.L. C. Trowbridge I.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google D.M. Sap J. Schlessinger J. Weiss A. EMBO J. 1994; PubMed Scopus Google Scholar), that is not an active phosphatase as a recombinant protein expressed in T cells. However, one CD45 proteins of from cells, which had catalytic activity, and this was to an active J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). In this have that in the absence of any purified recombinant proteins were not catalytically active against any of the substrates tested. that CD45 is to domain phosphatases such as RPTPα, and has been shown to be catalytically active Y. EMBO J. 1991; PubMed Scopus Google Scholar, M. T. M. H. EMBO J. 1990; PubMed Scopus Google Scholar, 1993; PubMed Scopus Google Scholar). the of M. T. M. H. EMBO J. 1990; PubMed Scopus Google Scholar, M. M. H. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar), catalytic activity was with these phosphatases both and were no catalytic activity has been for the second domain of these phosphatases, for RPTPα, the catalytic activity for was that with Y. EMBO J. 1991; PubMed Scopus Google Scholar). In domain phosphatases, to be for catalytic activity, it that these would be active phosphatases (reviewed in Refs. D. Biol. PubMed Scopus Google Scholar, Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Sci. 1996; 21: Full Text PDF PubMed Scopus Google Scholar). the second phosphatase domain in two-domain phosphatases has been shown to have no activity in to their first phosphatase domain, and in these required for activity of the first domain. It has been suggested that of can the substrate specificity of CD45 against artificial substrates M. T. M. H. EMBO J. 1990; PubMed Scopus Google Scholar, Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). In this study, a of substrates, and the protein substrate no evidence was obtained for a major role of in substrate Comparison of rD1 and rD1/D2 indicated that is required for and activity of the first phosphatase domain of CD45. The that the of catalytic activity that can the catalytic of is to the catalytic by the of is not However, this and data an interaction between rD1 and rD2 that and may be in to one of the first phosphatase of transmembrane two-domain tyrosine phosphatases, and have been (14Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar, D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). both crystallized as it was suggested that dimerization of the rD1 was an of as indicated that the protein was a monomer in to of D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of rD1 were by the membrane-proximal region of one rD1 a and binding to the region of the an interaction that was predicted to phosphatase and formation occurred for rD1 between and not for the rD2 protein (14Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar). However, of rD1 of was with it active as a monomer in L. A. den Hertog J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In this study, CD45 rD1 existed primarily as a dimer the the two-domain phosphatase, rD1/D2, existed primarily as a monomer that the of in the dimerization. to the from studies (14Bilwes A.M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar), dimer formation of CD45 rD1 not in an phosphatase; it was 2–3-fold active rD1/D2, the possibility that dimer formation may However, one both of the phosphatase active in the dimer. to the responsible for a intramolecular interaction in rD1/D2 identified a 27-kDa fragment containing the N-terminal region membrane-proximal region and of with a 53-kDa fragment containing the C-terminal region of PTP-D1, the spacer region, PTP-D2, and the carboxy limited trypsin 53-kDa fragment is the fragment identified and purified by J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) that the catalytic activity was from However, in these found this fragment with a 27-kDa fragment from no activity with rD2 found this to be catalytically active not for a noncovalent intramolecular interaction from digestion studies and for a association an interaction between the N-terminal part of the protein containing and the C-terminal part containing It is to that in the rD1/D2 the membrane-proximal region with to the intramolecular interaction and intermolecular dimer resulting in rD1/D2 In the absence of PTP-D2, the membrane-proximal region of rD1 may with rD1 dimer formation. may to dimerization. of this membrane-proximal interaction would in the formation of intermolecular which may to CD45 with this is data from T cells expressing an epidermal growth factor receptor-CD45 epidermal growth factor and dimerization of the epidermal growth factor receptor-CD45 chimera with function in T cell activation (13Desai D.M. Sap J. Schlessinger J. Weiss A. Cell. 1993; 73: 541-554Abstract Full Text PDF PubMed Scopus (244) Google Scholar). that a the membrane-proximal region is required to this R. A.M. Noel J.P. Hunter T. Weiss A. PubMed Scopus Google Scholar). this is with it to be and to an rD1 protein the membrane-proximal region to role in dimerization have not yet a It is that interactions between a protein can as a form of of this can be found in the protein-tyrosine intramolecular interactions between the domain and a region as as the domain the with a to the Nature. PubMed Scopus Google Scholar, J. Nature. PubMed Scopus Google Scholar). an interaction in a that can be to an active dephosphorylation of the tyrosine (reviewed in 1996; PubMed Scopus Google Scholar). the of the of that an intramolecular interaction between the N-terminal domain and the C-terminal PTP domain P. S. S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of this intramolecular interaction is to of the to a more active, U. M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, D. U. U. 1994; PubMed Scopus Google Scholar, S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, intramolecular may also for T the C-terminal domain of T has been shown to phosphatase activity L. T. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). provide evidence for and intermolecular interactions occurring in the cytoplasmic domain of the possibility that CD45 activity may also be regulated by intramolecular interactions by the C-terminal region containing the second phosphatase domain, and for the M. and R. for for and D. Ng for of the