CD45 is a receptor- like PTP (RPTP) expressed on all nucleated hematopoietic cells. Consistent with a critical role for CD45 in T and B cell signal transduction, CD45-deficient T and B cell lines fail to respond to antigen receptor stimulation, and patients with CD45 deficiency have a severe combined immunodeficiency phenotype. CD45-deficient mice likewise have a profound block in T and B cell development and function (refs.16
and references therein). One key function of CD45 is to serve as a positive regulator of SFK by opposing Csk function and by dephosphorylating the negative regulatory C-terminal tyrosine of SFKs. Dephosphorylation of this site maintains SFKs in a primed, or signal-competent, state capable of full activation upon receptor stimulation. Consistent with this hypothesis, SFKs have decreased kinase activity and are hyperphosphorylated at the negative regulatory tyrosine in most CD45-deficient cells. Moreover, expression of a constitutively active Lck Y505F mutant in CD45-deficient mice largely rescues T cell development (18
). CD45 may also dephosphorylate the autocatalytic tyrosine in SFKs, albeit less efficiently. Dephosphorylation of the C-terminal tyrosine leaves the SFKs in an unphosphorylated primed conformation, ready for rapid activation. As discussed below, other PTPs have also been implicated in the dephosphorylation of this site.
A recent report suggests that CD45 may also be involved in the reciprocal regulation of a second family of PTKs, the Janus kinases (JAKs) (20
). JAKs function as positive regulators of cytokine-mediated signal transduction by phosphorylating the members of the signal transducer and activator of transcription (STAT) family of transcription factors. The STATs then translocate to the nucleus, where they regulate the expression of genes involved in cytokine and chemokine responses. Recent experiments employing CD45-deficient cell lines and mice suggest that CD45 acts as a negative regulator of JAKs (20
). CD45-deficient mice have increased cytokine-dependent myelopoiesis and erythropoiesis and corresponding hyperphosphorylation of JAK2 and STATs 3 and 5. CD45 deficiency also protects mice from the lethal cardiomyopathy that results from Coxsackie virus B3 (CVB3) infection. While intriguing, a role for CD45 as a JAK phosphatase must be viewed cautiously, since the phenotype observed could also be attributed to inhibition of SFK activity in the CD45-deficient mice. However, it is becoming increasingly clear that SFKs activate STATs, either in parallel with or downstream of JAKs (21
). The observation that Lck-deficient mice are also protected from CVB3 infection provides support for the notion that some effects of CD45 ascribed to JAK kinase effects may be mediated by its effects on SFKs (22
). Additional work is needed to clarify the role of CD45 in regulation of JAK function.
Given the critical role of CD45 function in T cell biology, it is not surprising that its regulation has become an area of intense research. Current data suggest two potential mechanisms for CD45 regulation: localization and receptor dimerization. Although it is clear that CD45 plays a positive role in T cell activation, other studies indicate it can function as a negative regulator in other contexts (refs.17
and references therein). CD45-deficient macrophages and T cells are abnormally adherent, a phenotype that is reversed with the addition of wild-type CD45 (17
). This effect has been ascribed to negative regulation of SFKs by CD45 during integrin-mediated signal transduction. Surprisingly, despite the fact that the negative regulatory tyrosine of the SFKs is hyperphosphorylated in the CD45-deficient macrophages, kinase activity is actually enhanced, due to hyperphosphorylation of the autocatalytic site. Thus, the distribution of SFK phosphorylation may account for the increased adhesiveness of these cells, despite the seemingly paradoxical positive and negative regulation of SFKs by CD45. Apparently, CD45’s effect depends upon its localization in relation to its substrate, such that physical separation of CD45 from the TCR during antigen recognition at the immunological synapse results in a net positive effect, whereas access to its substrate during integrin-mediated adhesion results in a negative effect. The biochemical and microscopic evidence that CD45, but not Lck, is excluded from lipid rafts and the immunological synapse (8
) is consistent with this model. In resting T cells, CD45 may counteract the negative regulation of Csk by dephosphorylating the negative regulatory, and to a lesser extent the autocatalytic, tyrosine in SFKs, providing a pool of signal-competent Lck. The latter kinase would then be available to phosphorylate the TCR ITAMs and bound ZAP-70 whenever foreign antigen is encountered. During antigen recognition, TCR clustering in the central region of the immunologic synapse, a process that evolves over time, would result in functional segregation of CD45 from its substrate and would sustain Lck activity during initiation of signal transduction cascades (Figure , a and b).
Receptor dimerization may represent a second means of regulating CD45 function. Like most members of the RPTP family, CD45 consists of an extracellular domain, a single transmembrane domain, and a cytoplasmic domain containing tandemly duplicated PTP domains (23
). CD45 exists as multiple isoforms due to alternative splicing of exons 4, 5, and 6, which encode a portion of the extracellular domain. The alternatively spliced exons encode multiple sites of O-linked glycosylation, which are variably modified by sialic acid. Thus, the extracellular domain of high–molecular weight isoforms (CD45RA+) differs in structure and overall charge from the low–molecular weight isoform (CD45RO) lacking these exons. Isoform expression is highly regulated in a cell- and activation state–specific manner. For example, in T cells, alternative splicing of CD45 is regulated such that naive T cells predominantly express CD45RA isoforms containing exons 4 and 5 or 5 and 6. Upon activation, alternative splicing machinery is induced, in a Ras-dependent manner, to favor the exclusion of exons 4, 5, and 6, resulting in the expression of the lower–molecular weight CD45RO isoform (24
). The tight control of CD45 isoform expression supports the notion that the extracellular domain could play a central role in regulation of CD45 function by facilitating isoform-specific differential interaction with a ligand and/or differential homodimerization.
A definitive ligand for CD45 has not been established. However, several lines of evidence suggest dimerization may regulate CD45 function. First, dimeric forms of CD45 have been identified (25
). Second, work with a chimeric protein consisting of the extracellular and transmembrane domains of the EGF-R fused to the cytoplasmic domain of CD45 (27
) suggests that dimerization directly suppresses CD45 activity. This chimera is sufficient to restore TCR-mediated signal transduction in a CD45-deficient T cell line, but its activity is lost when the EGF is provided to induce dimerization. Hence, dimerization of the native CD45 — either induced by some as-yet unidentified ligand or occurring spontaneously — would be expected to silence TCR-mediated signal transduction.
A molecular explanation for this inhibition was provided by the crystal structure of the juxtamembrane and proximal catalytic domain of a related phosphatase, RPTPα (28
). This protein fragment forms a symmetrical dimer in which the catalytic site of one molecule is blocked by specific contacts with a wedge formed by the juxtamembrane region of the dimer partner. The dimeric PTP is thus inhibited by mutual occlusion of the active sites. The homologous region within the juxtamembrane domain of CD45 is remarkably conserved phylogenetically, suggesting a preserved evolutionary function. This region of CD45 could be modeled into a wedge-like structure, based on the RPTPα structure. Mutation of a highly conserved acidic amino acid that is predicted to reside at the tip of the CD45 wedge domain significantly blunts the inhibitory effect of dimerization in the experiments using the EGF-R–CD45 chimera (29
We recently tested the physiologic significance of this model by generating an analogous mutation (E613R) in vivo by homologous recombination (26
). In mice carrying the CD45
E613R mutation, T and B cell development is initially normal, but at 12–16 weeks of age, homozygous mutant mice develop a lymphoproliferative disorder with polyclonal expansion of activated T and B cells, autoantibody production, and severe autoimmune nephritis. These animals die prematurely, and their phenotype is consistent with hyperactivity of SFK-mediated pathways and suggests that dimerization plays a critical negative regulatory role in CD45 function in vivo.
The identification of dimeric forms of CD45 and RPTPα in the absence of exogenously added protein suggests that spontaneous homodimerization occurs. This finding has led us to question the need for a putative endogenous CD45 ligand that might drive dimerization of this phosphatase. We hypothesize that differential isoform expression may regulate CD45 homodimerization and function. The extensive O-linked glycosylation and sialylation of the alternatively spliced exons in CD45RA+ impart on the protein a strong negative charge that may form an electrostatic repulsive barrier to homodimerization. The lack of these modifications in the CD45RO isoform may allow more efficient homodimerization. We propose that the CD45RA+-to-CD45RO switch would allow for more efficient dimer formation, downregulating CD45 PTP activity and contributing to the cessation of the primary T cell response (Figure c). The wedge mutation in the E613R mice eliminates the negative regulatory effect of CD45RO homodimerization, resulting in persistent T cell activation and autoimmune disease.
Genetic support for the importance of the RA-to-RO isoform switch during the primary immune response comes from identification of a single nucleotide polymorphism, silent at the protein level, in exon 4 of the human CD45
gene. This alteration prevents the switch from RA- to RO-encoding splicing forms. According to the above model, individuals with this mutation would be unable to regulate CD45 by dimerization and could be predisposed to autoimmune disease. Consistent with this hypothesis, population studies have recently demonstrated that this mutation is associated in a small subset of German individuals with the development of multiple sclerosis (30
). While intriguing, a causal role for this mutation in disease development awaits further analysis.