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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cardiovascular System / growth & development
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Cardiovascular System / metabolism
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Endothelial Cells / metabolism
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Endothelial Cells / physiology
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Gene Expression
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Genetic Vectors
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HEK293 Cells
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Humans
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Intercellular Junctions / genetics*
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Intercellular Junctions / metabolism
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KRIT1 Protein
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Microtubule-Associated Proteins / chemistry
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Microtubule-Associated Proteins / genetics
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Microtubule-Associated Proteins / metabolism*
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Microtubules / genetics
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Microtubules / metabolism
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Monomeric GTP-Binding Proteins / chemistry
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Monomeric GTP-Binding Proteins / genetics
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Monomeric GTP-Binding Proteins / metabolism*
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Muscle Proteins / genetics
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Muscle Proteins / metabolism*
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Muscle Proteins / physiology
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Protein Conformation
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Protein Structure, Tertiary
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Proto-Oncogene Proteins / chemistry
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism*
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RNA, Small Interfering
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Signal Transduction
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Structure-Activity Relationship
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Umbilical Veins / cytology
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Zebrafish / genetics
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Zebrafish / growth & development*
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Zebrafish / metabolism
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Zebrafish Proteins / chemistry
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Zebrafish Proteins / genetics
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Zebrafish Proteins / metabolism*
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Zebrafish Proteins / physiology
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rap1 GTP-Binding Proteins / chemistry
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rap1 GTP-Binding Proteins / genetics
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rap1 GTP-Binding Proteins / metabolism*
Substances
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KRIT1 Protein
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KRIT1 protein, human
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Microtubule-Associated Proteins
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Muscle Proteins
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Proto-Oncogene Proteins
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RNA, Small Interfering
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Zebrafish Proteins
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krit1 protein, zebrafish
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Monomeric GTP-Binding Proteins
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rap1 GTP-Binding Proteins
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terf2ip protein, zebrafish