Of the 37 classical PTP genes in humans, 21 encode transmembrane, RPTPs. The diversity in the extracellular segments of the RPTPs presumably reflects a similar diversity in the nature of the ligands to which they respond; however, the identity and function of such ligands remains a largely unresolved issue in the field .
A characteristic feature of the extracellular segment of many RPTPs is the presence of Ig-like and fibronectin type III domains. These are motifs that are commonly found in cell adhesion molecules, suggesting a role in regulating processes involving cell–cell contact. Early in the discovery of RPTPs we focused our attention on PTPμ, which comprises an extracellular segment containing a MAM (meprin-A5 antigen-PTPμ) domain, one Ig-like and four fibronectin type III domains, a single transmembrane domain and an intracellular segment containing two PTP domains separated from the transmembrane domain by a juxtamembrane sequence with homology to the intracellular segment of the cadherin superfamily of cell adhesion molecules. In our attempts to identify the ligand for PTPμ, we demonstrated that, when it was expressed in non-adhesive Sf9 cells, the formation of cell–cell aggregates was induced through a homophilic binding mechanism that involved only the extracellular segment of the PTP. Thus, the ligand for PTPμ on one cell is the extracellular segment of another PTPμ molecule on the surface of an adjacent cell . Indeed, it is the Ig domain that is both necessary and sufficient for homophilic binding . Yossi Schlessinger's group noted that when Sf9 cells expressing PTPμ and PTPκ (a close relative of PTPμ displaying approximately 75% overall identity) were mixed, they sorted independently . In other words PTPμ binds to itself but not to PTPκ, suggesting that the homophilic binding interaction is highly specific. Nevertheless, aggregation (i.e. ligand binding to the extracellular segment of PTPμ) had no detectable direct effect on the activity of the intracellular PTP domains. It is likely that these interactions play a role in controlling activity by restricting its distribution in the membrane, perhaps targeting it to interactions with the cadherin–catenin cell adhesion complex [61, 62].
Another perspective on the issue of ligands comes from CD45, which is highly glycosylated and comprises up to 10% of the surface of a haematopoietic cell. In this case, although lectins, such as CD22, have been reported to bind to the extracellular segment, they do not appear to modulate PTP activity . Dimerization of CD45 has been reported to vary according to the glycosylation of the extracellular segment, which is determined by the alternative splicing of three exons encoding sequences at the N-terminus. The larger, more highly glycosylated and sialylated form, which expresses all three exons (RABC), is less efficient at forming dimers than the smallest form (RO). When a CD45-deficient T cell line was reconstituted with physiological levels of the RO and RABC isoforms, RO was found to dimerize more efficiently than RABC and was less effective than RABC at reconstituting signalling through the T cell receptor . This suggests there may be an equilibrium between monomers and dimers of CD45 on the cell surface, with PTP activity determined by the differential dimerization of specific isoforms. Nevertheless, there have been reports of ligands that bind to the extracellular segment of RPTPs and alter directly the activity of the intracellular PTP domains. Perhaps the best characterized example came from the laboratory of Thomas Deuel, who reported that binding of the soluble cytokine pleiotrophin led to inhibition of RPTPζ activity, thereby promoting tyrosine phosphorylation . It is thought that some of the effects of pleiotrophin on the cytoskeleton are mediated via RPTPζ-induced increases in tyrosine phosphorylation of β-catenin and β-adducin [65, 66]. It has also been reported that p190 Rho GAP is a pleiotrophin-modulated substrate of RPTPζ . In an elegant study in Drosophila, David Van Vactor identified both soluble and surface-bound ligands that recognize the Ig domains LAR . A high-affinity interaction between LAR on neurones and syndecan (Sdc), a transmembrane heparin sulphate proteoglycan on muscle, serves to promote PTP function, whereas the interaction with the glycosylphosphatidylinositol-anchored protein Dallylike (Dlp) is inhibitory . These ligands compete to regulate a pathway that integrates the effects of LAR with the ABL PTK via changes in the tyrosine phosphorylation of Enabled (Ena), which binds to the cytoplasmic segment of LAR and regulates the actin cytoskeleton and synaptic morphogenesis.
A characteristic feature of 12 of the RPTPs is a tandem arrangement of PTP domains in the intracellular segment . The indication from phylogenetic analyses is that a PTP domain duplication occurred in an ancestral gene before the whole gene duplicated to give rise to other RPTPs . Essentially all of the catalytic activity of these RPTPs resides in the membrane-proximal PTP domain (termed D1). Nevertheless, although the membrane-distal domains (termed D2) are inactive themselves, there are examples in which their structural integrity is required for the enzymatic activity of the PTP as a whole [69, 70]. Although the D2 domains lack intrinsic activity, there is considerable conservation of sequence, as well as secondary and tertiary structure, between domains D1 and D2; in fact, only two point mutations were required to convert LAR D2 into an active enzyme . Nevertheless, there are also structural distinctions that suggest differences in function. For example, all the D2 domains are phylogenetically distinct from domain D1; i.e., D2 sequences do not cluster together with D1 sequences in the phylogenetic tree but define a separate subfamily of PTP domains . Interestingly, within the LAR RPTP subtype, comprising LAR, PTPσ and PTPδ, the sequence similarity between domains D2 is even higher than between the corresponding domains D1 . There have been suggestions that D2 domains serve a binding function . More recently, based upon studies of RPTPα, it has been shown that the D2 domain displays greater sensitivity to oxidation than D1 and may serve as redox sensors . Furthermore, oxidation induces a conformational change in D2 that can be transmitted to the extracellular segment of the receptor PTP [75, 76]. Nevertheless, elucidation of the functions of RPTP D2s remains an issue to be resolved in the PTP field.
The mechanism by which ligand binding to an RPTP may modulate the catalytic activity of the intracellular D1 domain remains a hot button issue in the field. The solution of the crystal structure of the membrane proximal PTP domain of RPTPα by Joe Noel's group was an important development . Within the crystal, the PTP D1 domains are organized in symmetrical dimers, in which an inhibitory helix-turn-helix ‘wedge’ motif from one domain occludes the active site of the partner domain. This led to the proposal of a mechanism by which ligand binding may directly modulate PTP activity. If ligand binding was to induce dimerization, as it does for the PTKs, then the catalytic activity of RPTPs may be attenuated in a dimeric state by reciprocal occlusion of the active sites; it is notable that the effects on activity would be in contrast to RPTKs, which are stimulated by ligand-induced dimerization and trans-phosphorlyation . This has proven to be a controversial model and the issue remains to be fully resolved. The first problem is that this structure describes only the membrane-proximal D1 domain in isolation. In a similar study of PTPμ, which shares 46% sequence identity with the D1 domain of PTPα, we found that the tertiary structures of the two were very similar (rmsd between equivalent Cα-atoms of 1.1 Å); however, although the PTPμ D1 domain was also a dimer in the crystal, the dimer interface was distinct and the active site was present in an open, uninhibited conformation . When both D1 and D2 domains were included in the construct to be crystallized, as reported for LAR  and CD45 , the wedge motif was present in the structure, but there was no evidence of dimerization in the crystal. In addition, the D1 and D2 domains were oriented in such a way that steric hindrance caused by the presence of D2 would prevent wedge-mediated dimerization of D1. These structures were seen in crystals in different space groups with distinct crystallographic contacts between neighbouring molecules. Also, in both LAR and CD45, the D1–D2 domain interfaces were stabilized by short linkers and extensive noncovalent interactions. Of course, within a cell these PTP domains are connected to transmembrane and extracellular segments, such that ligand binding may influence the relative orientations of D1 and D2. Nevertheless, the short linker, coupled with its limited flexibility, suggests that the relative orientations of D1 and D2 may be restricted [71, 79]. There are also conflicting data from cell-based studies. On the one hand, it has been suggested that, in T cell lines, CD45 is monomeric, as are other components of the T cell receptor signalling complex . In contrast, studies in a ‘knock-in’ mouse model highlight the importance of the inhibitory wedge motif in CD45 . Glu613, at the tip of the wedge, was mutated to Arg, in a mutation that would be expected to prevent wedge-mediated inhibition of CD45 in a dimer. It is interesting to note that the consequences of expressing CD45-E613R in the knock-in mice are essentially the opposite of those observed following knockout of the gene for CD45, consistent with the wedge mutation having removed an inhibitory constraint upon CD45 function. Overall, the data clearly demonstrate the regulatory importance of the wedge, but whether or not the mechanism involves dimer-induced inhibition continues to fuel interesting debate.
Although isolated originally as a 37-kDa catalytic domain [1, 2], the cloning of cDNA encoding PTP1B by three separate groups, the laboratories of Ben Neel, Jack Dixon and Dave Hill, revealed a full-length form of the protein that also contains a regulatory segment of approximately 115 residues on the C-terminal side of the catalytic domain [82-84]. The C-terminal 35 residues are predominantly hydrophobic in nature and function in targeting the enzyme to the cytoplasmic face of membranes of the endoplasmic reticulum (ER) . This was similar to the arrangement that had already been reported for the close relative of PTP1B, the 48-kDa form of T-cell enriched PTP (TCPTP) . In the latter case, alternative splicing generates two forms that differ in their extreme C-termini but share a common catalytic domain in the N-terminal portion of the molecule. Whereas the 48 kDa form (TC48) is targeted to the ER, a 45-kDa form (TC45), which lacks the hydrophobic segment, is targeted to the nucleus  and is able to shuttle in and out of the nucleus in response to extracellular stimuli . The C-terminal segment of PTP1B also contains sites of phosphorylation by Ser/Thr kinases  and a site of proteolytic cleavage by calpain, which generates a truncated, soluble form of the enzyme with enhanced activity . This suggests a role for this segment not only in targeting, but also in the direct regulation of PTP1B activity. Such a direct role of the C-terminal segment in suppressing catalytic activity was defined for TCPTP .
Inspection of the sequences of the nontransmebrane PTPs reveals that the situation for PTP1B and TCPTP illustrates a general principle; subcellular targeting is now recognized as an important component of the regulation of PTP function. For example, the presence of SH2 domains in the N-terminal portion of the SHPs targets these PTPs to interact with sites of tyrosine phosphorylation in receptors and scaffolding adaptor proteins . The presence of a FERM domain targets PTPs to interfaces between the plasma membrane and the cytoskeleton . The SEC14 domain functions in lipid binding and membrane targeting . The BRO1 domain has been implicated in targeting proteins to endosomes . As Jack Dixon put it in his review in 1994 , these regulatory motifs function as zip-codes to direct the PTPs to the correct cellular address. Nevertheless, it is important to stress that the PTPs are not simply a collection of nonspecific enzymes for which the activity is regulated indirectly by tethering. There is clear evidence of gene duplication in the nontransmembrane PTPs, giving rise to PTP1B and TCPTP, SHP1 and SHP2, as well as PTPD1 and PTPD2 ; these pairs have a high degree of sequence identity but distinct, nonredundant functions, consistent with specificity. There are examples of intrinsic specificity within the PTP catalytic domains themselves [97-99]. Furthermore, the regulatory sequences that flank the catalytic domain can also influence specificity, such as the kinase interaction motif (KIM) domain directing striatal-enriched phosphatase (STEP) and haematopoietic PTP (HePTP) to dephosphorylate mitogen-activated protein kinases (MAPKs)  and the poly-Pro sequences in PTP-PEST, which influence its interaction with p130cas .
As illustrated for TCPTP , in addition to subcellular targeting, these noncatalytic segments of the PTPs may regulate activity directly. A clear example of this is the SH2 domain-containing PTP SHP2 . Crystallographic analysis revealed that SHP2 exists in a low activity state under basal conditions because the active site is occluded by an intramolecular interaction with residues of the N-terminal SH2 domain, located on the opposite side to its pTyr binding site. Engagement of the SH2 domains by appropriate pTyr ligands induces a conformational change that releases the autoinhibitory interaction and creates a form of the phosphatase in which the active site is now open and can dephosphorylate substrates. Thus, SHP2 becomes activated once it has been recruited into the correct signalling complex (Fig. 5). On the basis of this structure, Ben Neel designed mutants of SHP2 (D61A and E76A) that resulted in a constitutively active enzyme and found that they triggered FGF signalling in the absence of growth factor . It is interesting to note that gain of function mutations in SHP2 have been identified in human disease, initially in Noonan syndrome. This includes mutations in residues in and around the N-SH2 domain, which may facilitate activation by pTyr ligands, and in key residues at the interface between the N-SH2 and catalytic domains, which would induce the active conformation in the absence of a stimulus. Particularly striking is the fact that those key residues that Ben Neel chose to mutate on the basis of the structure to create constitutively active forms of SHP2 are actually mutated in Noonan syndrome . Mutations in SHP2 are now also associated with increased risk of certain childhood malignancies, such as juvenile myelomonocytic leukaemia and acute myeloid leukaemia. In fact, SHP2 was recognized as the first PTP oncogene, its positive role attesting further to the importance of PTPs as regulators of signalling in their own right.
Figure 5. Activation of SHP2. In the basal state, the active site of SHP2 is occluded by an intramolecular interaction with the N-terminal SH2 domain. The phosphatase may be activated either by engagement of the SH2 domains by pTyr sequence motifs in an RPTK or scaffolding molecule, or by mutations in either the N-SH2 or PTP domains that disrupt their interaction .
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