The adaptor disabled-2 binds to the third psi xNPxY sequence on the cytoplasmic tail of megalin.
The cytoplasmic tail (CT) of megalin possesses several functional motifs likely to participate in protein-protein interactions within the proximal tubular epithelial cell (PTEC) of the kidney. One such interaction is with the phosphotyrosine interaction domain (PID) of the adaptor protein disabled-2 (Dab2), a mitogen-responsive phosphoprotein, which interacts via its PID with Psi xNPxY (where Psi represents a hydrophobic residue) motifs on its binding partners. Megalin CT has three such motifs; it has been established that there is no interaction of Dab2 with the first (from N to C) (Biochem. J. 3 (2000) 613). Here, we analyse in real-time the binding of recombinant megalin CT, and of synthetic peptide sequences encompassing the second and third Psi xNPxY motifs, to Dab2PID in real-time using surface plasmon resonance (SPR). We report a binding affinity of DabPID for megalin CT of K(D) = 2.6 x 10(-7) +/- 5.3 x 10(-8). Direct binding and competition studies indicate that this interaction is with the third Psi xNPxY motif. The dissociation of Dab2 from the third Psi xNPxY peptide was significantly slower than that from the second (k(off) (mean +/- S.E.M.) (per s) = 0.002 +/- 0.002 vs. 0.007 +/- 0.002, P < 0.05). Synthetic peptide sequences encompassing the third Psi xNPxY but not the second inhibited Dab2PID binding both to intact megalin CT and to the third Psi xNPxY motif. Tyrosine phosphorylation of either motif did not exert a major effect upon competition efficacy. We further demonstrate for the first time the presence of Dab2 expression in primary human PTEC.
Gallagher
Hugh
H
SW Thames Institute for Renal Research, St. Helier Hospital, Carshalton, Surrey SM5 1AA, UK. hugh@joco.demon.co.uk
Oleinikov
Andrew V
AV
Fenske
Christiane
C
Newman
David J
DJ
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D051017:Apoptosis Regulatory Proteins; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D015870:Gene Expression; D016147:Genes, Tumor Suppressor; D006801:Humans; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000737:chemistry* / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D025521:Tumor Suppressor Proteins
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051017:Apoptosis Regulatory Proteins; C102885:DAB2 protein, human; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D025521:Tumor Suppressor Proteins; D019000:Phosphotyrosine