| Summary: | <p>CD2AP is a member of the CD2AP/CIN85 family of adaptors and involved in several cellular processes, such as kidney podocyte development, actin mediated membrane trafficking and T cell activation. It contains three SH3 domains whose binding properties and interaction partners remain largely unexplored.</p> <p>The CD2AP SH3 interaction with the novel partner Rab5-activating GEF RIN3 was studied extensively by isothermal titration calorimetry (ITC), peptide scanning arrays, mutagenesis and X-ray crystallography. Mapping of the interaction regions showed that human RIN3 contains two binding sites for the CD2AP SH3 domains. From these studies, the CD2AP SH3 recognition motif P-x-P/A-x-x-R emerged. Two crystal structures (1.65 Å and 1.2 Å) of the SH3 1 and SH3-2 domains in complex with RIN3 epitopes 1 and 2 respectively revealed that these residues serve as anchoring points. With the aid of bioinformatics tools, this motif was used to conduct a peptide array-based screen for additional signalling partner candidates. One of the hits was the Arf-GAP ARAP1. ITC data indicate that the three SH3 domains differentially recognise three ARAP1 epitopes, with the first ARAP1 epitope binding to SH3-2 in the nanomolar range. A crystal structure (1.6 Å) of the SH3-2 domain in complex with the first ARAP1 epitope implicates two additional anchoring residues that extend beyond the PPII helical region of the canonical motif. The CD2AP/ARAP1 interaction was confirmed in podocytes and cancer cells at the endogenous protein level. Even though RIN3 and ARAP1 are involved in membrane trafficking, a direct link to CD2AP had not been reported before. Other candidates from the peptide array analyses were also investigated by ITC.</p> <p>In conclusion, this study led to the elucidation of the CD2AP SH3-1 and SH3-2 domain binding signatures and the identification of putative novel binding partners for all three SH3 domains. Lastly, insight was gained into the binding preferences of CD2AP SH3-3.</p>
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