The transmembrane domain mediates RPTPα dimerization. (A) Schematic representation of RPTPα and deletion mutants. RPTPα fused to XFP (RPTPα-XFP) and deletion mutants lacking D2 (RPTPα-516-XFP), D1 and D2 (RPTPα-250-XFP), the entire cytoplasmic domain (RPTPα-200-XFP), or the cytoplasmic domain and the extracellular domain (RPTPα-200 ED-XFP) are depicted. RPTPα-EGFR-200-XFP is identical to RPTPα-200-XFP, except that the transmembrane and a short part of the intracellular domain were replaced by the corresponding regions of the human EGFR (depicted on the right). XFP (X) represents either YFP or CFP. The RPTPα extracellular domain (ED) is indicated, as well as the transmembrane domain (RPTPα, black box; EGFR, open box), the wedge (triangle) and the two PTP domains (D1 and D2). The EGFR ligand binding domain (LBD) and tyrosine kinase (TK) domain are indicated on the right. (B) Deletion mutants of RPTPα, fused to CFP and YFP (depicted in A), were co-transfected as indicated, and F(440)/F(490) was determined in single living cells. Averages of at least 10 individual cells from at least two independent experiments are depicted with error bars indicating the standard deviation. The dashed lines indicate theoretical minimal F(440)/F(490) ratio (bottom dashed line), and the FRET threshold level (upper dashed line). According to a two-tailed student's t-test, all values except for RPTPalpha-200-YFP/ RPTPalpha-EGFR-200-CFP are significantly different from the negative control, RPTPalpha-EGFR-200-CFP/ -YFP (p<0.003). (C) Expression of the constructs that were used in (B) was monitored by SDS-polyacrylamide gel electrophoresis using different gels (7.5 - 12.5%, depending on the size of the construct), and immunoblotting, using an anti-HA-tag antibody as a probe, to verify that CFP and YFP were expressed at similar levels. The molecular weights of the fusion proteins are: RPTPα-XFP, 160 kDa; RPTPα-516-XFP, 130 kDa; RPTPα-250-XFP, 110 kDa; RPTPα-200-XFP, 104 kDa; RPTPα-200Δ ED-XFP, 50 kDa; RPTPα-EGFR-200-XFP, 104 kDa.