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Structurally RhoGAP6 contains a central catalytic GAP domain surrounded by large, disordered N- and C-termini of unknown function. Sequence analysis revealed three conserved consecutive overlapping di-tryptophan motifs close to the RhoGAP6 C-terminus which were predicted to bind to the mu homology domain (MHD) of \u03b4-COP, a component of the COPI vesicle complex. We confirmed an endogenous interaction between RhoGAP6 and \u03b4-COP in human platelets using GST-CD2AP which binds an N-terminal RhoGAP6 SH3 binding motif. Next, we confirmed that the MHD of \u03b4-COP and the di-tryptophan motifs of RhoGAP6 mediate the interaction between both proteins. Each of the three di-tryptophan motifs appeared necessary for stable \u03b4-COP binding. Proteomic analysis of other potential RhoGAP6 di-tryptophan motif binding partners indicated that the RhoGAP6\/\u03b4-COP interaction connects RhoGAP6 to the whole COPI complex. 14-3-3 was also established as a RhoGAP6 binding partner and its binding site was mapped to serine 37. We provide evidence of potential cross-regulation between 14-3-3 and \u03b4-COP binding, however, neither \u03b4-COP nor 14-3-3 binding to RhoGAP6 impacted RhoA activity. Instead, analysis of protein transport through the secretory pathway demonstrated that RhoGAP6\/\u03b4-COP binding increased protein transport to the plasma membrane, as did a catalytically inactive mutant of RhoGAP6. Overall, we have identified a novel interaction between RhoGAP6 and \u03b4-COP which is mediated by conserved C-terminal di-tryptophan motifs, and which might control protein transport in platelets.<\/jats:p>","DOI":"10.1042\/bcj20230013","type":"journal-article","created":{"date-parts":[[2023,7,6]],"date-time":"2023-07-06T08:57:56Z","timestamp":1688633876000},"page":"1109-1127","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["RhoGAP6 interacts with COPI to regulate protein transport"],"prefix":"10.1042","volume":"480","author":[{"given":"Lorna","family":"O'Donoghue","sequence":"first","affiliation":[{"name":"1UCD School of Medicine, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield Dublin 4, Ireland"},{"name":"2Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin D02 YN77, Ireland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shane P.","family":"Comer","sequence":"additional","affiliation":[{"name":"1UCD School of Medicine, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield Dublin 4, Ireland"},{"name":"2Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin D02 YN77, Ireland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dishon W.","family":"Hiebner","sequence":"additional","affiliation":[{"name":"2Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin D02 YN77, Ireland"},{"name":"3UCD School of Chemical & Bioprocess Engineering, Engineering & Materials Science Centre, University College Dublin, Belfield Dublin 4, Ireland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ingmar","family":"Schoen","sequence":"additional","affiliation":[{"name":"2Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin D02 YN77, Ireland"},{"name":"4School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland (RCSI), 123 St Stephen's Green, Dublin D02 YN77, Ireland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alex","family":"von Kriegsheim","sequence":"additional","affiliation":[{"name":"5Edinburgh Cancer Research UK Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South, Edinburgh EH4 2XR, U.K."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9210-9406","authenticated-orcid":false,"given":"Albert","family":"Smolenski","sequence":"additional","affiliation":[{"name":"1UCD School of Medicine, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield Dublin 4, Ireland"},{"name":"2Irish Centre for Vascular Biology, Royal College of Surgeons in Ireland, 123 St Stephen's Green, Dublin D02 YN77, Ireland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"288","published-online":{"date-parts":[[2023,7,19]]},"reference":[{"key":"2023071916502898000_BCJ-2023-0013C1","doi-asserted-by":"publisher","first-page":"1054","DOI":"10.1182\/blood-2011-08-372193","article-title":"Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis","volume":"119","year":"2012","journal-title":"Blood"},{"key":"2023071916502898000_BCJ-2023-0013C2","doi-asserted-by":"publisher","first-page":"820945","DOI":"10.3389\/fcvm.2021.820945","article-title":"Turning platelets off and on: role of RhoGAPs and RhoGEFs in platelet activity","volume":"8","year":"2021","journal-title":"Front. 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