The adaptor disabled-2 binds to the third ΨxNPxY sequence on the cytoplasmic tail of megalin

Abstract

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 ΨxNPxY (where Ψ 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 ΨxNPxY motifs, to Dab2PID in real-time using surface plasmon resonance (SPR). We report a binding affinity of DabPID for megalin CT of KD = 2.6 × 10–7 ± 5.3 × 10–8. Direct binding and competition studies indicate that this interaction is with the third ΨxNPxY motif. The dissociation of Dab2 from the third ΨxNPxY peptide was significantly slower than that from the second (koff (mean ± S.E.M.) (per s) = 0.002 ± 0.002 vs. 0.007 ± 0.002, P < 0.05). Synthetic peptide sequences encompassing the third ΨxNPxY but not the second inhibited Dab2PID binding both to intact megalin CT and to the third ΨxNPxY 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.