Figure 1.

Identification of an interaction of NCAM with D₂R. A, The sequence of a peptide selected by screening a phage library using the recombinant intracellular domain of NCAM180 (NCAM180-ICD) as a bait shows significant similarity to a sequence in the third intracellular domain of the dopamine D₂ receptor (IC3-D₂R). Identical amino acids (|) are shown. Numbers designate amino acid positions. B, Increasing amounts of NCAM180-ICD (1–10 μg/ml) were incubated with GST-IC3-D₂R, followed by pull down with glutathione beads. Incubation of 10 μg/ml NCAM180-ICD with GST alone was served as control. Pulled down proteins were detected by NCAM antibody 5B8 and GST antibody. C, Schematic representation of NCAM180-ICD structure. The gray-marked sequence highlights N-terminal truncated fragment of NCAM140-ICD (140-ICDΔN, lacking amino acids 730-772). The dashed line indicates exon 18 encoded sequence of NCAM. Numbers designate amino acid positions. D, GST pull-down assay was performed with recombinant His-tagged NCAM180-ICD, exon 18 encoded protein, and NCAM140-ICDΔN with GST-IC3-D₂R or GST as control. Precipitated proteins were detected by anti-Penta His antibody. E, GST pull-down assay with NCAM180-ICD and GST-IC3-D₂R in the presence of NCAM peptide 1 (P1) and peptide 2 (P2). The minus sign (−) designates no application of NCAM peptides. F, Schematic representation of D₂R structure. The line highlights the D₂R peptide that shows similarity to the peptide identified by phage library screening, and numbers designate amino acid positions. Pull down was performed with NCAM180-ICD and GST-IC3-D₂R in the presence of increasing concentrations of D₂R peptide (2 and 8 μg/ml). The minus sign (−) designates no application of D₂R peptide. G, GST pull-down assay was performed by incubation NCAM180-ICD with GST-IC3-D₂R or mutated GST-IC3-D₂R (S311C).