[Frontiers in Bioscience 2, d207-221, May 1, 1997]

	[Frontiers in Bioscience 2, d207-221, May 1, 1997] Reprints PubMed CAVEAT LECTOR
	RECENT ADVANCES IN LYMPHOCYTE SIGNALING AND REGULATION Chun Kung and Matthew L. Thomas Department of Pathology, HHMI, Washington University, 660 S. Euclid Avenue, St. Louis, MO 63110 Received 4/4/97; Accepted 4/24/97; On-line 5/1/97 4. The protein tyrosine phosphatase, CD45 CD45 is a single chain transmembrane glycoprotein with two cytoplasmic phosphatase domains, of which the second domain appears to be inactive. This protein exists in various isoforms of molecular weights 180-220kDa, as a result of alternative splicing between exons 4, 5 and 6 (or A, B and C). These exons encode for the amino-terminal extracellular O-linked glycosylated region (70-73). Thus, these alternatively spliced isoforms differ in the lengths of their extracellular domains. Furthermore, they are differentially expressed on T cell subsets and resting or activated T cells, and their expression is dependent on cell differentiation and activation. CD45 is expressed by all hematopoietic cells except mature erythrocytes and platelets. 4.1 CD45 regulates protein tyrosine kinases CD45 functions to regulate Src kinase activity. Evidence for this has been obtained from T and B cells deficient in CD45. In these cells, the negative regulatory domains of Lck and Fyn are hyperphosphorylated. As a consequence antigen-mediated signal transduction is compromised (74-76). CD45 does not regulate Lck and Fyn equally. (75,77). In T cells deficient in CD45, Lck tyrosine phosphorylation increases 8-10 fold over wild type compared to a 2-3 fold increase for Fyn, despite equal expression of the Src kinases. Deletion of the SH4 domain from Lck or replacement of it with the analogous domain from Fyn results in a 5 fold increase in tyrosine phosphorylation of the negative regulatory domain (78). This suggests that there are mechanisms that mediate CD45 interaction with specific Src kinases. In B cells, Src kinases are also regulated by CD45. Lyn is hyperphosphorylated at its negative regulatory domain and autophosphorylation site in chicken DT40 B cells deficient in CD45 (79). Accordingly, BCR signaling in these cells is severely compromised. In comparison, DT40 B cells deficient in Csk and in the resting state exhibit a constitutively activated Lyn, whose autophosphorylation site is hyperphosphorylated but its negative regulatory domain is unphosphorylated (80). This implies that dephosphorylation of the carboxyl-terminal negative regulatory domain tyrosine by CD45 is a prerequisite for Lyn activity during BCR signaling. Consequently CD45 is an important positive regulator of Lyn activity and may also participate in dephosphorylating the autophosphorylation site of Lyn. Lck and Fyn are hyperphosphorylated at the negative regulatory domain, and exhibit hyperactivity in Yac-1 T cells deficient in CD45 (74). Phosphopeptide studies on Lck from these cells demonstrate that Lck is hyperphosphorylated at both the autophosphorylation site and the negative regulatory domain, although to a greater degree at the latter site (38). Tyrosine to phenylalanine mutations of Lck at the autophosphorylation site (Y394) and negative regulatory domain (Y505) establish that the autophosphorylation site is more dominant in affecting Lck activity. In cells expressing mutations at both Y505F and Y394F, no kinase activity is observed. Furthermore, in vitro assays using the phosphatase region of CD45 and active Lck in its native conformation demonstrates that CD45 can dephosphorylate the autophosphorylation site of Lck. Thus CD45 is responsible for dephosphorylating both regulatory phosphorylation sites on Lck (38). These findings point to CD45 as a negative and positive regulator of Src kinase activity. 4.2 The extracellular domain of CD45 affects antigen-mediated signal transduction The extracellular region of CD45 is modified by alternative splicing of exons 4, 5 and 6 (or A, B and C), which code for O-linked glycosylation and thus govern the amount of O-linked glycosylation present (70-73). As a result, CD45 isoforms are highly regulated and differentially expressed on the various lymphocyte subsets. Results from CD45 chimeric experiments indicate that the cytoplasmic domain of CD45 is sufficient for supporting TCR signaling (81,82). However, CD45 isoform expression has been associated with lymphocyte maturation and activation. This argues that CD45 isoforms, and in particular the extracellular domain of CD45, may influence lymphocyte function. Studies expressing the individual CD45 isoforms in transgenic mice or T cell lines demonstrate that each CD45 isoform affects TCR signaling differently (83-85). In fact, cells expressing the low molecular weight isoform of CD45 appear to be the most effective in TCR signaling. Not only is TCR signaling affected by which CD45 isoforms are present but also the tyrosine phosphorylation pattern of intracellular proteins (86,87). Adapter proteins such as Vav and SLP-76 show differential phosphorylation and varying degrees of physical association, with higher levels of each in the presence of the largest CD45 isoform (86). The regulatory effect of the CD45 isoforms may result from selective interactions of the particular isoform with cell surface molecules. The extracellular domain of CD45 is important in mediating interactions with other membrane surface proteins which are involved in T cell activation. CD2, a 55-60 kDa glycoprotein, is involved in T cell activation. However, signal transduction via CD2 stimulation requires CD2 interactions with other signaling molecules, such as CD3eta and zeta chain (88-90). Moreover, the protein tyrosine kinases, Lck and Fyn associate with the signaling complex formed by CD2 and zeta chain (91,92). In addition, CD45 interacts with CD2 to modulate its activation of T cells (93). Studies using CD2 chimeras with CD4, CD28 and CD58 show that CD45/CD2 complexes are primarily governed by extracellular domain interactions and to a lesser extent by cytoplasmic associations. Apparently, the cytoplasmic domain of CD2 associates mainly with the zeta chain of the TCR complex. These findings point to CD45 involvement in regulating CD2 activation of T cells. CD4, a co-stimulatory protein, associates with Lck and is involved in the antigen recognition process. CD45 isoform studies demonstrate that low molecular weight and not high molecular weight isoforms of CD45 preferentially interact with CD4 and TCR, and this association affects antigen recognition (94). Furthermore, the interaction between CD4 and CD45 is dependent upon the external domains of the CD45 isoforms but independent of the cytoplasmic domains. These results point to CD45's role in regulating antigen receptor signaling and also CD4 function in antigen recognition. In addition, CD45 interaction with CD4 may regulate Lck function and activity. 4.3 Effects of CD45 on lymphocyte development Mice deficient in CD45 exhibit defects in T cell development and impaired B cell signaling (95-98). Two separate gene targeted mice have been described in which either exon 6 or exon 9 was replaced with a neomycin cassette (95,98). The phenotype of deficient mice developed from either targeted exon is similar. T cell development is severely inhibited at two distinct stages: development of DP thymocytes from DN thymocytes is reduced twofold and the maturation of DP in to SP is decreased fivefold. In addition, TCR induced apoptosis of thymocytes is impaired whereas non-TCR stimulated apoptosis is unaffected. Altogether, these results demonstrate that CD45 is required for T cell development and is consistent with the observation that CD45 is necessary for efficient signaling through the TCR. Exon 6 targeted mice have normal numbers of B cells, which are responsive to lipopolysaccharrides (95,96). However, IgM stimulation fails to induce B cell proliferation. Furthermore while extracellular Ca²⁺ influx is abrogated, intracellular Ca²⁺ mobilization is normal upon anti-Ig induction. In mice with the exon 9 mutation, B cell development is unaffected but no BCR signaling is observed when stimulated by anti-IgM or anti-IgD (98). However stimulation through CD40 (anti-CD40) is unaffected compared to reduced signaling through CD38 (anti-CD38). Altogether, CD45 plays an important role in Ig mediated-BCR signaling and in some aspects of CD38 signaling. It also may be important for extracellular Ca²⁺ influx. As noted above, immature B cells undergo selection during maturation to determine the competency of the BCR. In mice deficient in CD45, this selection process is altered due to changes in antigen receptor signaling (97). The threshold signal required for selection is abnormally lowered compared to wild type, eliminating B cells which normally would be selected. Clearly, the signal generated here from antigen receptor stimulation is recognized by the deficient B cell as being improper for B cell maturation. These results demonstrate that antigen signaling is a requirement for normal mature B cell accumulation and the degree of signaling regulates proper selection. Accordingly, CD45 appears to act as a positive regulator of the signaling threshold required for B cell maturation. During lymphocyte development, the level of CD45 expression is important for antigen receptor signaling. CD45 expression is up regulated during T cell maturation particularly during the positive selection of SP thymocytes (99). CD45 levels are low on DP thymocytes but increase when cells differentiate to CD4⁺ or CD8⁺ SP, in conjunction with increased levels of TCR-CD3 complex. Consequently greater than 90% of the positively selected thymocytes display a CD45^high phenotype in contrast to a CD45^low phenotype for non-selected thymocytes. Similarly CD45 expression is drastically increased during the developmental period which correlates BCR up regulation with B cell maturation (99). As a result, CD45 expression during lymphocyte development is tightly regulated with those for TCR and BCR complexes. 4.4 Regulation of CD45 activity Currently, only a limited amount of information is known about the mechanism and participants involved in the regulation of CD45 activity. Recent studies propose that CD45 activation and function could be regulated through phosphorylation by Csk, a negative regulator of Src kinase activity (37). Cotransfection of Csk and CD45 into COS-1 cells reveal that CD45 is phosphorylated on two tyrosines, and upon phosphorylation, CD45 exhibits increased phosphatase activity. Therefore, Csk may up regulate CD45 activity and down regulate Src kinase activity. CD45 dimerization may also be involved in regulating CD45 activity. EGF receptor chimeras containing phosphatase domain of CD45 dimerize in the presence of EGF (100). Dimer formation neutralizes CD45 function and TCR activity. However, the addition of a EGF receptor with its cytoplasmic tail truncated restores both activities. These findings suggest that CD45 dimer formation can potentially regulate CD45 activity via inactivation of phosphatase activity. Moreover CD45 is related to receptor protein tyrosine phosphatase alpha (RPTPalpha). The crystal structure of the RPTPalpha membrane-proximal catalytic domain has been solved (101). The deduced structure shows that a dimer is formed from two catalytic domains, with the N-terminal region of one monomer wedged into the active site of the other monomer. This association blocks the active site of one catalytic domain, making it inaccessible to substrate. As a result dimer formation by RPTPalpha could play a role in regulating phosphatase function, and as such a similar event may also be important in regulating CD45 function. 4.5 CD45 is involved in cell adhesion T cell activation requires both antigen presentation and cellular adhesion with the antigen presenting cell. TCR activation concomitantly stimulates integrin-mediated cell adhesion (102). In general, cell adhesion is mediated by integrin stimulation and the formation of focal adhesions. During this process tyrosine phosphorylation of intracellular proteins occurs. Focal adhesion kinase (FAK), a protein tyrosine kinase, is phosphorylated in response to integrin cross-linking and has been implicated in the Ras-MAPK pathway (1,2). Phosphorylation of FAK provides binding sites for Src kinases, Grb2 and paxillin. Integrin stimulation and cell adhesion induce the phosphorylation of paxillin, a cytoskeletal protein involved in transducing signals to the nucleus (1,2). Both of these proteins are major components of focal adhesions. Apart from CD45's involvement in signal transduction, CD45 is involved in regulating the phosphorylation of paxillin and FAK in B cells (87). The phosphorylation of FAK and paxillin is dependent on the presence of CD45 in both stimulated and unstimulated B cells. Apparently, stimulation of B cells decreases FAK phosphorylation. B cells deficient in CD45 exhibit no phosphorylation of either FAK or paxillin, regardless of stimulation. These results suggest an involvement for CD45 in regulating cytoskeletal functions and cell adhesion. CD45 influences homotypic cell adhesion of T and B cells (103-105). For T cells, only activated T cells can be induced via CD45 ligation to aggregate. Antibodies to the extracellular domain of certain CD45 isoforms are able to induce homotypic adhesion, whereas others inhibit adhesion. This adhesion can be blocked using antibodies against LFA-1, ICAM-1 and ICAM-3, suggesting that LFA-1/ICAM-1 and LFA-1/ICAM-3 pathways are involved. Typically, CD45 is found to co-localize with LFA-1 at the cell-cell contacts after induction of cell aggregation via CD45 ligation. Antibodies to CD45 which block adhesion alter tyrosine phosphorylation of intracellular proteins induced by adhesion-activating antibodies to ICAM-3 or LFA-1. These results indicate that CD45 is an important component in mediating LFA-1 induced cell-cell aggregation. CD45 also associates with CD100, a disulfide-linked dimer involved in T cell proliferation and this interaction increases during T cell activation (106). The expression pattern of CD100 is similar to that for CD45. Epitope-dependent antibody coupling of CD45 down regulates CD100 expression at the cell surface and induces shedding of a soluble 120kDa form of CD100. Homotypic adhesion of T cells stimulated by antibodies against CD45 is enhanced by antibodies against CD100. However the CD100 antibody does not induce homotypic adhesion. Therefore CD45 modulates CD100 function in cell aggregation and proliferation. The association of CD45 with other membrane-associated proteins during cell adhesion may be mediated through CD45AP. Monomeric and dimeric forms of CD45 interact with the putative adapter protein CD45AP, a 36kDa phosphoprotein also known as the lymphocyte phosphatase associated protein (LPAP) (107-110). CD45AP expression in T and B cells correlates with that for CD45 (111). Cells deficient in CD45 show no surface expression of CD45AP although normal levels of its mRNA are present. Transfection of CD45 into these cells restores CD45AP expression. Therefore complex formation between CD45 and CD45AP prevents CD45AP from proteolytic degradation. Both molecules interact mainly through their transmembrane domains. Interestingly, the cytoplasmic domain of CD45AP is marked by a putative WW domain, which functionally resembles SH3 domains and may bind proline rich sequences (112). As a result, CD45AP can potentially act as an adapter protein for CD45 substrates.

[Frontiers in Bioscience 2, d207-221, May 1, 1997]
Reprints
PubMed
CAVEAT LECTOR

RECENT ADVANCES IN LYMPHOCYTE SIGNALING AND REGULATION

Chun Kung and Matthew L. Thomas

Department of Pathology, HHMI, Washington University, 660 S. Euclid Avenue, St. Louis, MO 63110

Received 4/4/97; Accepted 4/24/97; On-line 5/1/97

4. The protein tyrosine phosphatase, CD45

CD45 is a single chain transmembrane glycoprotein with two cytoplasmic phosphatase domains, of which the second domain appears to be inactive. This protein exists in various isoforms of molecular weights 180-220kDa, as a result of alternative splicing between exons 4, 5 and 6 (or A, B and C). These exons encode for the amino-terminal extracellular O-linked glycosylated region (70-73). Thus, these alternatively spliced isoforms differ in the lengths of their extracellular domains. Furthermore, they are differentially expressed on T cell subsets and resting or activated T cells, and their expression is dependent on cell differentiation and activation. CD45 is expressed by all hematopoietic cells except mature erythrocytes and platelets.

4.1 CD45 regulates protein tyrosine kinases

CD45 functions to regulate Src kinase activity. Evidence for this has been obtained from T and B cells deficient in CD45. In these cells, the negative regulatory domains of Lck and Fyn are hyperphosphorylated. As a consequence antigen-mediated signal transduction is compromised (74-76). CD45 does not regulate Lck and Fyn equally. (75,77). In T cells deficient in CD45, Lck tyrosine phosphorylation increases 8-10 fold over wild type compared to a 2-3 fold increase for Fyn, despite equal expression of the Src kinases. Deletion of the SH4 domain from Lck or replacement of it with the analogous domain from Fyn results in a 5 fold increase in tyrosine phosphorylation of the negative regulatory domain (78). This suggests that there are mechanisms that mediate CD45 interaction with specific Src kinases.

In B cells, Src kinases are also regulated by CD45. Lyn is hyperphosphorylated at its negative regulatory domain and autophosphorylation site in chicken DT40 B cells deficient in CD45 (79). Accordingly, BCR signaling in these cells is severely compromised. In comparison, DT40 B cells deficient in Csk and in the resting state exhibit a constitutively activated Lyn, whose autophosphorylation site is hyperphosphorylated but its negative regulatory domain is unphosphorylated (80). This implies that dephosphorylation of the carboxyl-terminal negative regulatory domain tyrosine by CD45 is a prerequisite for Lyn activity during BCR signaling. Consequently CD45 is an important positive regulator of Lyn activity and may also participate in dephosphorylating the autophosphorylation site of Lyn.

Lck and Fyn are hyperphosphorylated at the negative regulatory domain, and exhibit hyperactivity in Yac-1 T cells deficient in CD45 (74). Phosphopeptide studies on Lck from these cells demonstrate that Lck is hyperphosphorylated at both the autophosphorylation site and the negative regulatory domain, although to a greater degree at the latter site (38). Tyrosine to phenylalanine mutations of Lck at the autophosphorylation site (Y394) and negative regulatory domain (Y505) establish that the autophosphorylation site is more dominant in affecting Lck activity. In cells expressing mutations at both Y505F and Y394F, no kinase activity is observed. Furthermore, in vitro assays using the phosphatase region of CD45 and active Lck in its native conformation demonstrates that CD45 can dephosphorylate the autophosphorylation site of Lck. Thus CD45 is responsible for dephosphorylating both regulatory phosphorylation sites on Lck (38). These findings point to CD45 as a negative and positive regulator of Src kinase activity.

4.2 The extracellular domain of CD45 affects antigen-mediated signal transduction

The extracellular region of CD45 is modified by alternative splicing of exons 4, 5 and 6 (or A, B and C), which code for O-linked glycosylation and thus govern the amount of O-linked glycosylation present (70-73). As a result, CD45 isoforms are highly regulated and differentially expressed on the various lymphocyte subsets. Results from CD45 chimeric experiments indicate that the cytoplasmic domain of CD45 is sufficient for supporting TCR signaling (81,82). However, CD45 isoform expression has been associated with lymphocyte maturation and activation. This argues that CD45 isoforms, and in particular the extracellular domain of CD45, may influence lymphocyte function. Studies expressing the individual CD45 isoforms in transgenic mice or T cell lines demonstrate that each CD45 isoform affects TCR signaling differently (83-85). In fact, cells expressing the low molecular weight isoform of CD45 appear to be the most effective in TCR signaling.

Not only is TCR signaling affected by which CD45 isoforms are present but also the tyrosine phosphorylation pattern of intracellular proteins (86,87). Adapter proteins such as Vav and SLP-76 show differential phosphorylation and varying degrees of physical association, with higher levels of each in the presence of the largest CD45 isoform (86). The regulatory effect of the CD45 isoforms may result from selective interactions of the particular isoform with cell surface molecules.

The extracellular domain of CD45 is important in mediating interactions with other membrane surface proteins which are involved in T cell activation. CD2, a 55-60 kDa glycoprotein, is involved in T cell activation. However, signal transduction via CD2 stimulation requires CD2 interactions with other signaling molecules, such as CD3eta and zeta chain (88-90). Moreover, the protein tyrosine kinases, Lck and Fyn associate with the signaling complex formed by CD2 and zeta chain (91,92). In addition, CD45 interacts with CD2 to modulate its activation of T cells (93). Studies using CD2 chimeras with CD4, CD28 and CD58 show that CD45/CD2 complexes are primarily governed by extracellular domain interactions and to a lesser extent by cytoplasmic associations. Apparently, the cytoplasmic domain of CD2 associates mainly with the zeta chain of the TCR complex. These findings point to CD45 involvement in regulating CD2 activation of T cells.

CD4, a co-stimulatory protein, associates with Lck and is involved in the antigen recognition process. CD45 isoform studies demonstrate that low molecular weight and not high molecular weight isoforms of CD45 preferentially interact with CD4 and TCR, and this association affects antigen recognition (94). Furthermore, the interaction between CD4 and CD45 is dependent upon the external domains of the CD45 isoforms but independent of the cytoplasmic domains. These results point to CD45's role in regulating antigen receptor signaling and also CD4 function in antigen recognition. In addition, CD45 interaction with CD4 may regulate Lck function and activity.

4.3 Effects of CD45 on lymphocyte development

Mice deficient in CD45 exhibit defects in T cell development and impaired B cell signaling (95-98). Two separate gene targeted mice have been described in which either exon 6 or exon 9 was replaced with a neomycin cassette (95,98). The phenotype of deficient mice developed from either targeted exon is similar. T cell development is severely inhibited at two distinct stages: development of DP thymocytes from DN thymocytes is reduced twofold and the maturation of DP in to SP is decreased fivefold. In addition, TCR induced apoptosis of thymocytes is impaired whereas non-TCR stimulated apoptosis is unaffected. Altogether, these results demonstrate that CD45 is required for T cell development and is consistent with the observation that CD45 is necessary for efficient signaling through the TCR.

Exon 6 targeted mice have normal numbers of B cells, which are responsive to lipopolysaccharrides (95,96). However, IgM stimulation fails to induce B cell proliferation. Furthermore while extracellular Ca²⁺ influx is abrogated, intracellular Ca²⁺ mobilization is normal upon anti-Ig induction. In mice with the exon 9 mutation, B cell development is unaffected but no BCR signaling is observed when stimulated by anti-IgM or anti-IgD (98). However stimulation through CD40 (anti-CD40) is unaffected compared to reduced signaling through CD38 (anti-CD38). Altogether, CD45 plays an important role in Ig mediated-BCR signaling and in some aspects of CD38 signaling. It also may be important for extracellular Ca²⁺ influx.

As noted above, immature B cells undergo selection during maturation to determine the competency of the BCR. In mice deficient in CD45, this selection process is altered due to changes in antigen receptor signaling (97). The threshold signal required for selection is abnormally lowered compared to wild type, eliminating B cells which normally would be selected. Clearly, the signal generated here from antigen receptor stimulation is recognized by the deficient B cell as being improper for B cell maturation. These results demonstrate that antigen signaling is a requirement for normal mature B cell accumulation and the degree of signaling regulates proper selection. Accordingly, CD45 appears to act as a positive regulator of the signaling threshold required for B cell maturation.

During lymphocyte development, the level of CD45 expression is important for antigen receptor signaling. CD45 expression is up regulated during T cell maturation particularly during the positive selection of SP thymocytes (99). CD45 levels are low on DP thymocytes but increase when cells differentiate to CD4⁺ or CD8⁺ SP, in conjunction with increased levels of TCR-CD3 complex. Consequently greater than 90% of the positively selected thymocytes display a CD45^high phenotype in contrast to a CD45^low phenotype for non-selected thymocytes. Similarly CD45 expression is drastically increased during the developmental period which correlates BCR up regulation with B cell maturation (99). As a result, CD45 expression during lymphocyte development is tightly regulated with those for TCR and BCR complexes.

4.4 Regulation of CD45 activity

Currently, only a limited amount of information is known about the mechanism and participants involved in the regulation of CD45 activity. Recent studies propose that CD45 activation and function could be regulated through phosphorylation by Csk, a negative regulator of Src kinase activity (37). Cotransfection of Csk and CD45 into COS-1 cells reveal that CD45 is phosphorylated on two tyrosines, and upon phosphorylation, CD45 exhibits increased phosphatase activity. Therefore, Csk may up regulate CD45 activity and down regulate Src kinase activity.

CD45 dimerization may also be involved in regulating CD45 activity. EGF receptor chimeras containing phosphatase domain of CD45 dimerize in the presence of EGF (100). Dimer formation neutralizes CD45 function and TCR activity. However, the addition of a EGF receptor with its cytoplasmic tail truncated restores both activities. These findings suggest that CD45 dimer formation can potentially regulate CD45 activity via inactivation of phosphatase activity. Moreover CD45 is related to receptor protein tyrosine phosphatase alpha (RPTPalpha). The crystal structure of the RPTPalpha membrane-proximal catalytic domain has been solved (101). The deduced structure shows that a dimer is formed from two catalytic domains, with the N-terminal region of one monomer wedged into the active site of the other monomer. This association blocks the active site of one catalytic domain, making it inaccessible to substrate. As a result dimer formation by RPTPalpha could play a role in regulating phosphatase function, and as such a similar event may also be important in regulating CD45 function.

4.5 CD45 is involved in cell adhesion

T cell activation requires both antigen presentation and cellular adhesion with the antigen presenting cell. TCR activation concomitantly stimulates integrin-mediated cell adhesion (102). In general, cell adhesion is mediated by integrin stimulation and the formation of focal adhesions. During this process tyrosine phosphorylation of intracellular proteins occurs. Focal adhesion kinase (FAK), a protein tyrosine kinase, is phosphorylated in response to integrin cross-linking and has been implicated in the Ras-MAPK pathway (1,2). Phosphorylation of FAK provides binding sites for Src kinases, Grb2 and paxillin. Integrin stimulation and cell adhesion induce the phosphorylation of paxillin, a cytoskeletal protein involved in transducing signals to the nucleus (1,2). Both of these proteins are major components of focal adhesions.

Apart from CD45's involvement in signal transduction, CD45 is involved in regulating the phosphorylation of paxillin and FAK in B cells (87). The phosphorylation of FAK and paxillin is dependent on the presence of CD45 in both stimulated and unstimulated B cells. Apparently, stimulation of B cells decreases FAK phosphorylation. B cells deficient in CD45 exhibit no phosphorylation of either FAK or paxillin, regardless of stimulation. These results suggest an involvement for CD45 in regulating cytoskeletal functions and cell adhesion.

CD45 influences homotypic cell adhesion of T and B cells (103-105). For T cells, only activated T cells can be induced via CD45 ligation to aggregate. Antibodies to the extracellular domain of certain CD45 isoforms are able to induce homotypic adhesion, whereas others inhibit adhesion. This adhesion can be blocked using antibodies against LFA-1, ICAM-1 and ICAM-3, suggesting that LFA-1/ICAM-1 and LFA-1/ICAM-3 pathways are involved. Typically, CD45 is found to co-localize with LFA-1 at the cell-cell contacts after induction of cell aggregation via CD45 ligation. Antibodies to CD45 which block adhesion alter tyrosine phosphorylation of intracellular proteins induced by adhesion-activating antibodies to ICAM-3 or LFA-1. These results indicate that CD45 is an important component in mediating LFA-1 induced cell-cell aggregation.

CD45 also associates with CD100, a disulfide-linked dimer involved in T cell proliferation and this interaction increases during T cell activation (106). The expression pattern of CD100 is similar to that for CD45. Epitope-dependent antibody coupling of CD45 down regulates CD100 expression at the cell surface and induces shedding of a soluble 120kDa form of CD100. Homotypic adhesion of T cells stimulated by antibodies against CD45 is enhanced by antibodies against CD100. However the CD100 antibody does not induce homotypic adhesion. Therefore CD45 modulates CD100 function in cell aggregation and proliferation.

The association of CD45 with other membrane-associated proteins during cell adhesion may be mediated through CD45AP. Monomeric and dimeric forms of CD45 interact with the putative adapter protein CD45AP, a 36kDa phosphoprotein also known as the lymphocyte phosphatase associated protein (LPAP) (107-110). CD45AP expression in T and B cells correlates with that for CD45 (111). Cells deficient in CD45 show no surface expression of CD45AP although normal levels of its mRNA are present. Transfection of CD45 into these cells restores CD45AP expression. Therefore complex formation between CD45 and CD45AP prevents CD45AP from proteolytic degradation. Both molecules interact mainly through their transmembrane domains. Interestingly, the cytoplasmic domain of CD45AP is marked by a putative WW domain, which functionally resembles SH3 domains and may bind proline rich sequences (112). As a result, CD45AP can potentially act as an adapter protein for CD45 substrates.