Figure 5. Ce-DAB-1 associates with Ce-LRP-1 and Ce-LRP-2 and with Golgi components. (A,B) The Ce-DAB-1 PTB domain (residues 1-252) shows interaction with the intracellular domains of both Ce-LRP-1 (residues 4595-4753) and Ce-LRP-2 (C-terminal 81 residues) by yeast two-hybrid assay. β-Galactosidase activity (replicates; A) and growth in the absence of histidine (B). (C) Potential binding sites in Ce-LRP-1 (underlined amino acids indicate those that match the consensus binding site for the PTB domains of mammalian Dab1 and Dab2). (D) The Ce-DAB-1 PTB domain interacts with an FXNPXY motiffrom residues 4653-4658. Substitution of Tyr 4658 with alanine reduces interaction between the Ce-DAB-1 PTB domain and the intracellular domain of Ce-LRP-1. (E) Subcellular localization of Ce-DAB-1 expressed in tissue culture cells. NIH3T3 (WGA) and HeLa cells (gamma-adaptin) were transfected to express Ce-DAB-1 and processed for indirect immunofluorescence with antibodies to gamma-adaptin subunit AP-1, Ce-DAB-1, and directly labeled WGA. Note that there are two HeLa cells in the image shown. (F) Ce-DAB-1::GFP is present in punctate structures within P6.p daughters. Indirect immunofluorescence of qaEx4003 [
Ce-dab-1::Ce-DAB-1::GFP;
unc-119(+)];
unc-119(
e2498) animals with antibodies to LET-23 (left) and Ce-DAB-1::GFP (anti-GFP; center) and merged images (right) are shown. Images are single 0.2-μm confocal planes through P6.p after one division.