title: A mechanism of Rap1-induced stabilization of endothelial cell--cell junctions.
abstract: Activation of Rap1 small GTPases stabilizes cell--cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1-Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ~40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell-cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell--cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Jian J
authors.initials: JJ
authors.lastname: Liu
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Stockton
authors.forename: Alexandre R
authors.initials: AR
authors.lastname: Gingras
authors.forename: Ararat J
authors.initials: AJ
authors.lastname: Ablooglu
authors.forename: Jaewon
authors.initials: J
authors.lastname: Han
authors.forename: Andrey A
authors.initials: AA
authors.lastname: Bobkov
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ginsberg
mesh_terms: D000818:Animals; D002319:Cardiovascular System / Q000254:growth & development / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism / Q000502:physiology; D015870:Gene Expression; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D007365:Intercellular Junctions / Q000235:genetics* / Q000378:metabolism; D000075927:KRIT1 Protein; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000235:genetics / Q000378:metabolism; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014471:Umbilical Veins / Q000166:cytology; D015027:Zebrafish / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020827:rap1 GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000075927:KRIT1 Protein; C107928:KRIT1 protein, human; D008869:Microtubule-Associated Proteins; D009124:Muscle Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D029961:Zebrafish Proteins; C514537:krit1 protein, zebrafish; D020559:Monomeric GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins; C581234:terf2ip protein, zebrafish