Specific and constitutive dimerization of RPTPα, as assessed by FRET. (A) Schematic representation of the constructs that were used. RPTPα-D2 was replaced by CFP or YFP in RPTPα-516-XFP. Pro137 was mutated to Cys in RPTPα-P137C-XFP and RPTPα-P137C, leading to the formation of a disulfide bridge, and thus to constitutive dimerization. In EGFR-XFP, the C-terminus of the EGFR was replaced by CFP or YFP. (B) Theoretical values of F(440)/F(490), calculated as described in the Experimental Procedures section for no FRET and/or dimerization (bottom trace), or significant dimerization (E*α = 0.5, upper trace). The dashed lines indicate the F(440)/F(490) levels under conditions of no FRET and/or dimerization with R(CFP/YFP) = 0.5 (lower dashed line), or R(CFP/YFP) = 1.5 (upper dashed line). F(440)/F(490) levels close to the lower dashed line indicate that no FRET and/or dimerization occurs. F(440)/F(490) values above the upper dashed line are most likely due to FRET. F(440)/F(490) values between the two dashed lines are ambiguous, since F(440)/F(490) values may indicate high CFP:YFP expression ratios and no FRET and/or dimerization, or low CFP:YFP expression ratios and little FRET and dimerization. (C) SK-N-MC cells were transiently transfected with combinations of CFP- and YFP-fusion proteins, fused to RPTPα-516, to the EGFR, or to constitutively dimeric RPTPα-P137C. The F(440)/F(490) ratios were determined in at least 10 single living cells from at least two independent experiments, and the error bars indicate the standard deviation. The dashed lines indicate the theoretically minimal F(440)/F(490) ratio (bottom dashed line), and the FRET threshold level (upper dashed line). (D) Expression of the fusion proteins in (C) was monitored by immunoblotting to verify that similar levels of fusion proteins were expressed, using an anti-GFP antibody (left two panels), or an anti-HA-tag antibody (right panel). (E) Wild type RPTPα and RPTPα-P137C were transfected into SK-N-MC cells. Lysates of these cells were run on 5% SDS-PAGE gels under reducing (+β-mercaptoethanol, β-ME) or non-reducing conditions (-β-ME). Immunoblots, probed with anti-RPTPα antibodies are depicted with the molecular weight of marker proteins that were co-electrophoresed with the samples on the left (in kDa). The position of monomeric (M) and dimeric (D) RPTPα is depicted on the right.