Here, we provide evidence that RPTPα dimerized in living cells. We have successfully used FRET to detect RPTPα dimers and dimerization was consistent with cross-linking experiments. Our results provide strong support for the model that dimerization is involved in regulation of RPTPs.
FRET was detected between RPTPα-CFP and RPTPα-YFP using three different techniques: dual wavelength excitation (Fig. ), SPIM (Fig. ) and FLIM (Fig. ). The excitation-ratio method for determining FRET suffers from uncertainties when the CFP:YFP expression ratios, R(CFP/YFP), are not exactly known. Simulations show that high excitation ratios can be caused either by FRET or by an unfavorable (high) R(CFP/YFP) (Fig. ). Although for the entire cell population R(CFP/YFP) expression ratios were close to 1 (Fig. ), one cannot determine the expression ratio independently from FRET at the single cell level. Using the SPIM-method, FRET can be mimicked by unfavorable CFP/YFP expression ratios, but, in contrast to the excitation-ratio method, only when the R(CFP/YFP) is very low. The most reliable (but less sensitive) technique is FLIM. In conclusion, ratiometric analysis of FRET by dual wavelength excitation is a sensitive method for detection of FRET, which is reliable when similar levels of the two fluorophores are expressed.
We also demonstrated that RPTPα dimerized by chemical cross-linking, a technique that is fundamentally different from FRET analysis. BS3
-mediated chemical cross- linking is a two-step chemical reaction, leading to a covalent bond, while FRET is only dependent on the distance between the two fluorophores. Dimerization, detected by chemical cross-linking appeared to be much less efficient than with FRET. However, this apparent difference is caused by the relatively low efficiency of the cross-linker, BS3
. Only ~ 10% of RPTPα-P137C was detected in dimeric form following BS3
-mediated cross-linking, like wild type RPTPα (data not shown), while ~ 80% of RPTPα-P137C dimerized according to non-denaturing gels (Fig. ). According to the FRET analysis dimerization was extensive, since similar FRET levels were detected in wild type RPTPα as in constitutively dimeric RPTPα-P137C (Fig. ). Taken together, both FRET and cross-linking experiments indicate that dimerization of RPTPα is extensive. Subtle changes, for instance in the wedge, did not affect dimerization detected by FRET, but did lead to detection of reduced dimerization according to the cross-linking experiments [24
]. This discrepancy may be explained by the difference in detection of dimerization. The extracellular domain may dimerize, without an interaction intracellularly. Therefore, extracellular cross-linkers may allow detection of these dimers, while FRET analysis does not, due to the topology of the two fluorophores in the intracellular domains. Importantly, the requirements for detection of dimerization are more stringent for cross-linkers than for FRET, since the linker between the two reactive groups in BS3
is only ~ 12Å, while FRET allows distances up to ~ 60Å between the two fluorophores. It is noteworthy that a distance of 60Å or less between the two fluorophores is only achieved when the two proteins are in a protein complex, not when they merely colocalize subcellularly. Taken together, both cross-linking and FRET analysis show that RPTPα dimerizes extensively.
FRET analysis demonstrated that the transmembrane domain was sufficient to drive dimerization of RPTPα which is consistent with cross-linking experiments [24
] (Fig. ). Other regions in RPTPα may be involved in dimerization of the full length protein as well. For instance, cross-linking experiments demonstrated that the extracellular domain dimerized by itself [24
]. By contrast, the extracellular domain of RPTPα fused to the EGFR transmembrane domain did not show FRET (Fig. ), suggesting that this fusion protein did not dimerize. This may be explained by the difference between cross-linking and FRET, as described above. Yet other regions in RPTPα may be involved in dimerization as well. We have evidence that RPTPα-D2 binds to RPTPα-D1 [40
]. Similarly, CD45 and RPTPμ may be engaged in intra-or intermolecular interactions [41
]. In addition, we and others found heterodimerization between RPTP-D1s and RPTP-D2s from different RPTPs, suggesting cross-talk between RPTPs [40
]. It is noteworthy that the wedge is not required for dimerization, since deletion of the wedge did not abolish dimerization (Fig. , data not shown). Nevertheless, the wedge may be involved in stabilization of the dimer, since mutations in the wedge decreased the cross-linking efficiency [24
]. Moreover, whereas the EGFR transmembrane domain by itself was not sufficient to mediate dimerization (Fig. ), introduction of the juxtamembrane domain and D1 of RPTPα (residues 200-516) induced dimerization (data not shown). Taken together, multiple regions in RPTPα contribute to dimerization.
Here, we demonstrate for the first time that RPTPα dimerizes constitutively in living cells. Previously, indirect detection of protein-protein interactions using chemical cross-linkers demonstrated that CD45 homodimerizes [44
]. In addition, RPTPα elutes from gel filtration columns as a large protein complex, suggesting dimerization or multimerization [45
]. Regulation of dimerization is ill-understood. Like dimerization of RPTKs, dimerization of RPTPs may be regulated by ligand binding. RPTPs have diverse extracellular domains, and several RPTPs bind ligands [46
]. Interestingly, GPI-linked Contactin binds laterally to the extracellular domain of RPTPα, i.e. in cis
on the same cell [50
]. Whether Contactin functions as a ligand, or as a ligand-binding moiety remains to be determined. Pleiotrophin, a ligand of RPTPβ/ζ inactivates RPTPβ/ζ activity [5
]. Whether ligand binding affects dimerization of any of these RPTPs remains to be determined. RPTPα dimerization was constitutive and extensive. The transmembrane domain of RPTPα by itself was sufficient to drive dimerization, suggesting that ligands are not required for dimerization. However, ligand binding may modulate the extent of RPTPα dimerization. Analysis of RPTP dimerization using FRET may provide a powerful means to screen for factors that modulate dimerization or monomerization, and FRET may facilitate analysis of the dynamics of RPTP dimerization.
Dimerization of RPTPs may negatively regulate their activity. Ligand-induced dimerization of EGFR/CD45 led to functional inactivation [15
]. Mutation of a single residue in the wedge of CD45 abolished ligand-induced inactivation [16
], strongly supporting the model that dimerization leads to wedge-mediated occlusion of the catalytic sites. We have demonstrated that constitutively dimeric RPTPα-P137C was inactive, since it failed to dephosphorylate and activate c-Src in vivo
. Mutation of the wedge rendered RPTPα-P137C active, while it did not affect dimerization, demonstrating that inactivation of RPTPs by dimerization was dependent on the wedge [23
]. RPTPα-F135C with a disulfide bridge at position 135 dimerized constitutively, like RPTPα-P137C, but was still active, like wild type RPTPα. Apparently, dimerization-induced inactivation requires a specific rotational coupling, i.e. the monomers in the inhibited dimer need to be oriented in a specific geometry with respect to each other [23
]. Ligand binding to the extracellular domain may induce rotation of the monomers, relative to each other, thus leading to activation or inactivation of RPTPα, similar to ligand-induced rotation that has been suggested to activate RPTKs [51
Previously, we found that phorbol ester treatment of cells led to activation of RPTPα, and to phosphorylation of RPTPα Ser180 and Ser204 [53
]. These serine phosphorylation sites are localized close to the wedge, suggesting that phosphorylation of these sites may interfere with interactions of the wedge. Phosphorylation of Ser180 and Ser204 may not affect dimerization per se
, but still may lead to opening up of the catalytic site and thus to activation of RPTPα.
Since the crystal structures of RPTPμ-D1 and LAR did not show dimers like RPTPα-D1 [9
], the model that RPTPs are regulated by dimerization has been the subject of debate. The transmembrane domain of RPTPα was sufficient to drive dimerization. It will be interesting to see whether the transmembrane domains of other RPTPs, including RPTPμ and LAR, drive dimerization as well. We propose that dimerization via the transmembrane domain of RPTPα provides a conformational basis for regulation by dimerization. Whether the dimer is inactive depends on the exact topology of the intracellular domain, which may be regulated by phosphorylation, by interaction with other proteins, or by rotational coupling for instance as a result of ligand binding to the extracellular domain. Here we demonstrate that RPTPα dimerized in living cells for the first time, providing strong support for the model that dimerization is involved in regulation of RPTP activity.