Investigation of binding preferences and identification of novel binding partners for the SH3 domains of the multifunctional adaptor protein CD2AP
<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...
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| Format: | Thesis |
| Language: | English |
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2014
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| _version_ | 1797109931767234560 |
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| author | Rouka, E |
| author2 | Feller, S |
| author_facet | Feller, S Rouka, E |
| author_sort | Rouka, E |
| collection | OXFORD |
| description | <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> |
| first_indexed | 2024-03-07T07:48:05Z |
| format | Thesis |
| id | oxford-uuid:2eb39788-1916-4995-bd2c-b910d06a5a9b |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:48:05Z |
| publishDate | 2014 |
| record_format | dspace |
| spelling | oxford-uuid:2eb39788-1916-4995-bd2c-b910d06a5a9b2023-06-27T08:15:05ZInvestigation of binding preferences and identification of novel binding partners for the SH3 domains of the multifunctional adaptor protein CD2APThesishttp://purl.org/coar/resource_type/c_db06uuid:2eb39788-1916-4995-bd2c-b910d06a5a9bOncologyBiology (medical sciences)EnglishOxford University Research Archive - Valet2014Rouka, EFeller, S<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> |
| spellingShingle | Oncology Biology (medical sciences) Rouka, E Investigation of binding preferences and identification of novel binding partners for the SH3 domains of the multifunctional adaptor protein CD2AP |
| title | Investigation of binding preferences and identification of novel binding partners for the SH3 domains of the multifunctional adaptor protein CD2AP |
| title_full | Investigation of binding preferences and identification of novel binding partners for the SH3 domains of the multifunctional adaptor protein CD2AP |
| title_fullStr | Investigation of binding preferences and identification of novel binding partners for the SH3 domains of the multifunctional adaptor protein CD2AP |
| title_full_unstemmed | Investigation of binding preferences and identification of novel binding partners for the SH3 domains of the multifunctional adaptor protein CD2AP |
| title_short | Investigation of binding preferences and identification of novel binding partners for the SH3 domains of the multifunctional adaptor protein CD2AP |
| title_sort | investigation of binding preferences and identification of novel binding partners for the sh3 domains of the multifunctional adaptor protein cd2ap |
| topic | Oncology Biology (medical sciences) |
| work_keys_str_mv | AT roukae investigationofbindingpreferencesandidentificationofnovelbindingpartnersforthesh3domainsofthemultifunctionaladaptorproteincd2ap |