Abstract
Receptor-type protein tyrosine phosphatases (RPTPs) of the R3 subgroup play key roles in the immune, vascular and nervous systems. They are characterised by a large ectodomain comprising multiple FNIII-like repeats, a transmembrane domain, and a single intracellular phosphatase domain. The functional role of the extracellular region has not been clearly defined and potential roles in ligand interaction, dimerization, and regulation of cell-cell contacts have been reported. Here bimolecular fluorescence complementation (BiFC) in live cells was used to examine the molecular basis for the interaction of VE-PTP with VE-cadherin, two proteins involved in endothelial cell contact and maintenance of vascular integrity. The potential of other R3-PTPs to interact with VE-cadherin was also explored using this method. Quantitative BiFC analysis, using a VE-PTP construct expressing only the ectodomain and transmembrane domain, revealed a specific interaction with VE-cadherin, when compared with controls. Controls were sialophorin, an unrelated membrane protein with a large ectodomain, and a membrane anchored C-terminal Venus-YFP fragment, lacking both ectodomain and transmembrane domains. Truncation of the first 16 FNIII-like repeats from the ectodomain of VE-PTP indicated that removal of this region is not sufficient to disrupt the interaction with VE-cadherin, although it occurs predominantly in an intracellular location. A construct with a deletion of only the 17th domain of VE-PTP was, in contrast to previous studies, still able to interact with VE-cadherin, although this also was predominantly intracellular. Other members of the R3-PTP family (DEP-1, GLEPP1 and SAP-1) also exhibited the potential to interact with VE-cadherin. The direct interaction of DEP-1 with VE-cadherin is likely to be of physiological relevance since both proteins are expressed in endothelial cells. Together the data presented in the study suggest a role for both the ectodomain and transmembrane domain of R3-PTPs in interaction with VE-cadherin.
Highlights
The VEcadherin antibody detected a protein of approximately 100 kDa, which is larger than the vascular endothelial protein tyrosine phosphatase (VE-protein tyrosine phosphatases (PTPs)) ectodomain interactions predicted size of 82 kDa
Co-expression of VE-PTP-VN and VE-cadherin-VC in HEK-293T cells resulted in a fluorescence complementation signal detectable by confocal microscopy which was stronger 24 hours post-transfection (Fig 2B), when compared with experiments conducted after 48 hours
The molecular basis for the interaction of VE-PTP with VE-cadherin has previously been studied using co-immunoprecipitation experiments; it has not to-date been studied in live cells, and it is not known whether other R3 subgroup Receptor-type protein tyrosine phosphatases (RPTPs) have the potential to interact with VE-cadherin
Summary
The large family of human protein tyrosine phosphatases (PTPs) catalyse dephosphorylation and their activity and specificity is tightly regulated by a range of mechanisms [3,4,5]. PTPs have been divided into transmembrane receptor-type PTPs (RPTPs, subgroups R1-R8) and intracellular non-transmembrane PTPs (subgroups NT1-NT9), based on sequence similarity and the presence of similar structural and functional domains. The receptor-type PTPs have highly-variable ectodomains, a single transmembrane spanning region, and an intracellular region which may contain either one or two phosphatase domains [6]. Much is known about the structure, function and substrate specificity of the phosphatase domain [7], the function of the extracellular region within many RPTPs is only beginning to be uncovered. Roles have been described for this region in binding a diverse range of ligands either in trans or in cis, mediating cell-cell contact, oligomerization, and steric exclusion of the phosphatase from an immunological synapse [8,9,10,11,12,13,14]
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