In this report, we demonstrate that the first catalytic domain of PTPς binds to the second catalytic domain of PTPδ, an interaction which requires the presence of the wedge sequence of PTPς and which leads to partial inhibition of the catalytic activity of PTPς. The first catalytic domains of other LAR family members can also bind PTPδ-D2, although more weakly, and none of the LAR family D1 domains is able to bind D2 domains other than that of PTPδ.
A recent determination of the tertiary structure of RPTPα-D1 revealed that the domain crystallizes as a homodimer. This D1-D1 dimerization is mediated by an ~30-aa helix-turn-helix (wedge) sequence located at the N terminus of RPTPα-D1 which is tucked into the active site of the opposing partner of the dimer (3
). Based on this structure, it was predicted that such dimerization would inhibit catalytic activity, because the active site is occupied by the wedge sequence. Our PTPς-D1–PTPδ-D2 heterodimerization results provide a variation on this theme, but with a fundamental difference; we believe that the wedge sequence of PTPς-D1 indeed binds to the “pseudoactive” site of PTPδ-D2 (i.e., homologous to the active site of D1), which, like other LAR family D2 domains, is catalytically inactive (11a
). The D2 domains of LAR family RPTPs (and other RPTPs) do not possess an N-terminal wedge sequence, and moreover, all of the PTPδ-D2 sequences that we isolated in the yeast two-hybrid screens did not contain their N termini. Thus, the observed inhibition of PTPς-D1 catalytic activity suggests either the existence of a downstream inhibitory region(s) in the D2 domain of PTPδ which may bind to and inhibits the active site of PTPς-D1 or that binding of PTPδ-D2 to the wedge sequence of PTPς-D1 somehow distorts the active site of PTPς-D1 or, alternatively, interferes with substrate accessibility. Determination of the tertiary structure of D2 domains alone or in complex with D1 domains, not yet available, should help in the identification of the exact mode of D1-D2 interactions and D2-mediated inhibition of D1 catalytic activity. Whatever the mechanism(s) of binding, the observation of partial inhibition of the PTP activity of a D1 domain by a D2 domain could have important biological implications (see below). More importantly, it may provide an explanation for the long-standing observation that the D2 domains of many RPTPs are inactive; our work suggests that the role of these D2 domains is to regulate the activity of the D1 domains. In LAR family members, this regulation is likely mediated by intermolecular interactions between these closely related phosphatases, although we cannot preclude the possibility of a weak intermolecular association between the two catalytic domains of PTPδ. Based on our lack of PTPς D1-D1 binding, we believe that the observed D1-D1 homodimerization of RPTPα (3
) may represent a different mode of regulation of that phosphatase; indeed, unlike most receptor PTPs, both catalytic domains of RPTPα are catalytically active (47
An alternative possibility to explain our data, although less likely, is that the PTPς-D1–PTPδ-D2 association somehow induces PTPς D1-D1 dimerization which was undetected by our yeast two-hybrid binding assays.
The second catalytic domains of LAR, PTPδ, and PTPς are very similar in sequence, with only minor substitutions, mostly in nonconserved amino acids (Fig. ). It is therefore difficult to explain the vast difference between PTPδ-D2 and the D2 domain of PTPς or LAR in the ability to bind to PTPς-D1 (or other LAR family D1 domains), demonstrated here by yeast two-hybrid binding assays and coprecipitations from mammalian cells. Detailed mutation analysis is required to sort out the source of this specificity.
The ectodomains of LAR, PTPδ, and PTPς are composed of Ig and FNIII repeats, resembling the cell adhesion molecules N-CAM, fasciclin, and L1. CAMs such as N-CAM or Ng-CAM have been demonstrated to aggregate through homophilic interactions (14
). Indeed, recent studies have demonstrated that PTPκ, PTPμ, and PTPλ, a subfamily of phosphatases closely resembling LAR, can each aggregate via its extracellular domain in a homophilic, but not heterophilic, manner (4
); such interactions, however, have no effect on the catalytic activity of these PTPs (5
). So far, homophilic interactions of LAR, PTPδ, and PTPς have not been demonstrated, raising the possibility that the ectodomains of these PTPs interact either with other extracellular components (e.g., extracellular matrix proteins) or, possibly, with each other in a heterophilic manner. Our results described here demonstrate that these LAR family PTPs can form heterocomplexes via their intracellular domains. Moreover, such putative heterodimerization is likely to inhibit the catalytic activity of at least one of the binding partners. Although it is not known whether these LAR family members are coexpressed in the same cells, this is likely, since recent reports have demonstrated expression of these PTPs in the same types of neuronal and epithelial tissues or cells. For example, both PTPς and PTPδ have been shown to be expressed in the hippocampus, especially in the pyramidal cell layer and granular layer of dentate gyrus (23
), and we and others have found that LAR, PTPς, and PTPδ are expressed in fetal alveolar epithelial cells (11a
). In addition, a recent report has demonstrated colocalization of PTPς and LAR in adhesion plaques of A431 cells (1
The physiological substrates for most PTPs, including LAR family members, are not known. Several proteins that interact with LAR family members have been described recently, but unlike the PTPδ-D2 described here, none seem to affect PTP activity. A coiled-coil phosphoserine called LAR-interacting protein was shown to bind to the second catalytic domains of LAR, PTPδ, and PTPς and appears to localize LAR to focal adhesions (29
). Recently, it was demonstrated that LAR family members can associate with the β-catenin–cadherin complex and can dephosphorylate β-catenin in vitro (1
). The cadherin–α-, β-, or γ-catenin complex is associated with the cytoskeleton and is found in regions of cell-cell contact. The presence of these phosphatases in such regions suggests that the interactions may regulate tyrosine dephosphorylation of β-catenin, thus affecting the integrity of the cadherin–α-, β-, or γ-catenin complex and therefore that of cell adhesion. This could potentially have major implications for tissue development, particularly for events associated with neurite outgrowth and epithelial differentiation. Several drosophila receptor PTPs, including DLAR, have been shown to be expressed in a subset of the developing axons and pioneer neurons in the central nervous system (41
) and were recently demonstrated to be necessary for motor axon guidance in the Drosophila
). This suggests that LAR or its other family members may have a parallel role in vertebrates as well. Indeed, PTPς, PTPδ, and LAR were previously shown to be strongly expressed during development in selected regions within the central nervous system and the peripheral nervous system, as well as in other epithelial and neuroepithelial cells (17
), and a recent gene knockout of LAR has demonstrated a reduction in the size of cholinergic neurons and defects in hippocampal cholinergic innervation (51
The biological meaning of our observed association between LAR family members, especially between PTPς-D1 and PTPδ-D2, and the resultant inhibition of PTP activity, is not known. It is possible, however, that such an association keeps one or both binding partners in an inactive state, perhaps analogous to the intramolecular interactions recently identified in src family members which keep the kinase domain inactive (33
). We speculate that upon arrival of the appropriate (high-affinity) tyrosine-phosphorylated substrate, the D1-D2 intermolecular complex is likely to dissociate, allowing substrate dephosphorylation (Fig. ). The identification of a biological substrate(s) for PTPς, the role that this PTP and other LAR family members play in neuronal and epithelial morphogenesis and development, and the possible inhibitory role of PTPδ in these processes, are important questions that now need to be addressed.
FIG. 8 Model of D1-D2 heterodimerization of LAR family PTPs. Under resting conditions, the first catalytic domain (D1) of PTPς (or possibly other LAR family PTPs [in parentheses]) is associated with the second catalytic domain (D2) of (more ...)