Rap1 - Krit1 Regulation of Endothelial Permeability

Rap1 - Krit1 内皮渗透性调节

• 批准号: 7660417
• 负责人: REBECCA A STOCKTON
• 金额: $ 11.58万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7660417
• 关键词: Actin-Binding Protein; Actins; Acute; Adhesions; Animals; Arterial Fatty Streak; Atherosclerosis; Binding; Binding Sites; Biological Assay; Biology; Biosensor; Blood; Blood Vessels; CCM1 gene; Cardiovascular Pathology; Cardiovascular system; Cavernous Malformation; Cell Adhesion; Cell Line; Cell-Cell Adhesion; Cells; Cerebrum; Co-Immunoprecipitations; Cultured Cells; Cytoplasmic Tail; Cytoskeleton; Disease; Ectopic Expression; Endothelial Cells; Endothelium; Extracellular Matrix; Faculty; Family; Focal Adhesions; Funding; Genetic; Goals; Guanosine Triphosphate Phosphohydrolases; Hemostatic function; Image; Immunofluorescence Microscopy; In Vitro; Inflammation; Inflammatory Response; Integrin Binding; Integrin beta Chains; Integrins; Intercellular Junctions; Kinetics; Label; Lesion; Leukocytes; Link; Liquid substance; Lung; Mediating; Mentors; Methodology; Molecular; Monomeric GTP-Binding Proteins; Movement; Mutant Strains Mice; Mutate; Mutation; Nuclear; Nuclear Translocation; PTB Domain; Pathology; Peptide Signal Sequences; Permeability; Pharmacological Treatment; Phosphotransferases; Physiology; Play; Positioning Attribute; Principal Investigator; Process; Protein Binding; Protein Binding Domain; Proteins; Recruitment Activity; Regulation; Reperfusion Injury; Research; Role; Scientist; Signal Transduction; Signaling Molecule; Site; Small Interfering RNA; Stress Fibers; Structure; Swelling; Talin; Tertiary Protein Structure; Testing; Thrombin; Time; Transgenic Mice; Vascular Permeabilities; Work; abstracting; angiogenesis; career; cerebral cavernous malformations; ezrin; in vivo; interstitial; macromolecule; migration; moesin; novel; programs; protein function; radixin protein; receptor; response; rho; rho GTP-Binding Proteins; scaffold; skills; therapeutic target

DESCRIPTION (provided by applicant): PROJECT SUMMARY: Candidate's immediate career plans are advancement to Asst Project Scientist with several years' mentored project work to gain skills in transgenic mouse methodology/genetics. Long-term goal: acquire independent RO1 funding and a faculty position with independent research in cardiovascular biology. This proposal aims to identify signaling molecules promoting endothelial barrier integrity. Endothelial cell-cell junction disruption plus contractile stress fiber formation increase vessel permeability. Dysregulation of endothelial barrier function contributes to vessel leak in cardiovascular pathology. Rap1 GTPase stabilizes endothelial junctions. KRIT1 and CCM2 mutations produce "Cerebral Cavernous Malformations" disease characterized by vascular lesions with abnormal endothelium. Preliminary work identified KRIT1 as a Rap1 effectors having beta-integrin binding site, and demonstrated KRIT1 and CCM2 targeting to endothelial junctions that was associated with stabilized cortical actin and decreased permeability. HYPOTHESIS: KRIT1 is a Rap1 effector recruiting ICAP1 modulating beta-integrins and/or CCM2, regulating f-actin mediated barrier function through Rho-GTPases. SPECIFIC AIMS are to: 1) test whether KRIT1 localization and function are mediated by ICAP1 and CCM2 binding. 2) Evaluate KRIT1 PERM domain interactions with beta-integrins in regulating junctional stability. 3) Examine Rac- and Rho-GTPase interactions with KRIT1 in modulating actin cytoskeleton associated with barrier function. Molecular interactions in cultured cells will be tested with in vitro protein binding assays and co-immunoprecipitations, using siRNA depletion and ectopic expression of mutated binding domain proteins. Permeability will be tested in vivo with pulmonary vascular leak assays using beta-integrin, KRIT1, and CCM2 mutant mice; and in vitro using the animal's cultured endothelial cells in Tran swell filters. Molecular localization will be imaged using confocal immunofluorescence microscopy, plus real-time micro imaging to visualize kinetic localization of labeled ectopic proteins and fluorescent Rho-GTPase biosensors. RELEVANCE: This project will increase understanding of vascular permeability in general and CCM disease in particular. It will potentially identify target molecules for pharmacological treatment of pathological leak, and genetic treatment of CCM2 disease. (End of Abstract)

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