Membrane-associated guanylate kinase homologs (MAGuKs), MPP3 and Dlg, link CADM1 with p85 by forming a multi-protein complex.

(A) Schematic representation of the structures of CADM1, MPP3, and Dlg proteins. In amino acid sequences of the cytoplasmic domain of CADM1, consensus sequences of 4.1- and class II PDZ-binding motifs are highlighted by grey and black, respectively. The GST-fusion protein of CADM1 with an entire cytoplasmic fragment (GST-CADM1-C) and a mutant form lacking a class II PDZ-binding motif (GST-CADM1- CΔ4) are schematically represented below. N-terminal fragments of MPP3 and Dlg were purified as His-fusion proteins and used for an in vitro binding assay. (B) Interaction of GST-CADM1 with His-MPP3 and/or His-Dlg was examined by GST pull-down assay. Binding proteins were detected by Western blotting using anti-His tag antibodies, whereas GST-fusion proteins were detected by staining the membrane with Coomassie Brilliant Blue (CBB). (C) Localization of CADM1, MPP3, Dlg, and p85 in confluent Caco-2 cells. Confluent Caco-2 cells were fixed and stained with anti-CADM1 antibodies (green) and anti-MPP3 (upper), anti-Dlg (middle), or anti-p85 (lower) antibodies (red). Merged images are shown in the right panel. Bars: 20 μm. (D) Lysates of Caco-2 cells expressing MPP3-HA were immunoprecipitated (IP) with control rabbit IgG and anti-CADM1 (left) or anti-Dlg (right) antibodies and analyzed by Western blotting with antibodies against HA, Dlg, p85, and CADM1, as indicated. Black arrowheads indicate signals found in both the input and immunoprecipitates, whereas white arrowheads indicate signals only found in the input. Asterisks show non-specific bands.