Dimerization of Receptor Protein-Tyrosine Phosphatase alpha in living cells

In order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPα, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPα -CFP and -YFP fusion proteins, and thus that RPTPα dimerized in living cells. RPTPα dimerization was constitutive, extensive and specific. RPTPα dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization.We demonstrate here that RPTPα dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.Protein phosphorylation on tyrosine residues is one of the most important eukaryotic cell signalling mechanisms, and cellular protein phosphotyrosine (pTyr) levels are regulated by the antagonistic activities of the protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs) [1]. Our insight into the function of PTPs - in contrast to the PTKs - is limited. However, in recent years evidence is emerging that PTPs play important and specific roles in biological processes [2,3,4]. Transmembrane PTPs, tentatively called receptor PTPs, RPTPs, are interesting, because their diverse extracellular domains may function as ligand binding domains. Recently, Pleiotrophin was identified as a ligand of RPTPβ/ζ, and binding of Pleiotrophin led to inactivation of RPTPβ/ζ activity, resulting in increased tyrosine phosphorylation of its substrate, β-Catenin [5]. The mechanism underlying ligand-induced modulation of RPTP activity remains to be determined.Dimerization is a well-established regulatory mechanism f