Fig. 5. Mapping of the DRAL/FHL-2-binding site in the N2B region of titin. Five different GFP-tagged deletion constructs of the N2B region were assayed for their interaction with DRAL/FHL-2 in pull-down assays. (A) Schematic representation of the employed constructs. (B) Investigation of the interaction between deletion constructs of the N2B region and DRAL by a GST pull-down assay. Only constructs that contain the residues between 3749 and 4019 of the unique sequence 3 are able to bind to DRAL/FHL-2-GST. The putative DRAL/FHL-2-binding site is indicated by a dotted line in the schematic drawing of the titin N2B region. (C) The putative DRAL/FHL-2-binding site alone shows proper targeting to the sarcomere, as shown in NRC double-transfected with GFP-N2B5 (a) and DRAL/FHL-2-FLAG (a'). Arrowheads in the insets point at the doublet flanking the Z-disc. (D) Coimmunoprecipitation of GFP-N2B5 and DRAL/FHL-2-FLAG. COS cells were cotransfected with GFP-N2B5 and DRAL/FHL-2-FLAG, immunprecipitation was carried out with a monoclonal anti-FLAG antibody followed by immunoblotting with a peroxidase-conjugated polyclonal rabbit anti-GFP antibody. The domain nomenclature and amino acid numbering correspond to that of the human cardiac titin sequence [X90568 (Labeit and Kolmerer, 1995)]. Bar, 10 µm.

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