Endothelial Cytoprotective Signaling by Activated Protein C/Protease-activated Receptor-1

激活蛋白 C/蛋白酶激活受体 1 的内皮细胞保护信号传导

• 批准号: 10816153
• 负责人: Joann Trejo
• 金额: $ 4.98万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10816153
• 关键词: Anti-Inflammatory Agents; Apoptosis; Apoptotic; Arr2; Arrestins; Binding; CRISPR/Cas technology; Caveolae; Cell membrane; Cells; Cytoprotection; Endothelial Cells; Endothelium; Exhibits; Future; G-Protein-Coupled Receptors; GRK5 gene; Goals; Human; Inflammatory Response; Injury; Knock-out; Mediating; Mediator; Membrane Microdomains; Morbidity - disease rate; PAR-1 Receptor; Pathway interactions; Phosphorylation; Physiological; Research; SPHK1 enzyme; Sepsis; Signal Induction; Signal Pathway; Signal Transduction; System; Testing; Therapeutic; Thrombin; Transducers; Vascular Diseases; activated Protein C; arrestin 2; caveolin 1; desensitization; endothelial dysfunction; improved; innovation; mortality; new therapeutic target; novel; receptor; response; therapeutic development

Summary/Abstract There are currently limited treatment options for improving endothelial dysfunction in vascular diseases such as sepsis, resulting in high morbidity and mortality. Endothelial dysfunction results in endothelial cell activation, disruption of endothelial barrier function and sensitivity to apoptosis. The long-term goal of this proposal is to delineate the pathways by which the endothelium can resist injury and disruption to facilitate the advancement of new targets for therapeutic development. Activated protein C (APC) is a promising therapeutic and exhibits multiple beneficial effects including stabilization of endothelial barriers and anti-apoptotic activities. Protease- activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR), is the central mediator of APC cellular signaling, which requires caveolin-1 (Cav1) and compartmentalization in caveolae. We discovered that APC- activated PAR1 signals primarily through -arrestin-2 (-arr2) to promote endothelial barrier protection, and not heterotrimeric G proteins like thrombin (Th)-activated PAR1. The overall objective of this proposal is to develop a mechanistic understanding of how APC/PAR1 generates -arr2 transducer bias to promote endothelial cytoprotection. We hypothesize that distinct GRK5 determinants and co-receptors facilitate APC/PAR1-induced -arr2 transducer bias to promote endothelial cytoprotection through pathways enabled by Cav1 phosphorylation. We propose three specific aims. Aim 1: To delineate the mechanisms that enable GRK5 to distinctly regulate APC- vs. Th-induced biased signaling. GRK5 is required for APC-stimulated signaling and desensitization of Th-induced signaling. However, the mechanisms that enable distinct GRK5 functions are not known. We will determine if distinct GRK5 functions are regulated by localization to discrete plasma membrane microdomains such as caveolae using human cultured endothelial cells, a native system that permits the study of endogenous PAR1 and GRK5 and HEK293 CRISPR/Cas9 knockout cells. Aim 2: To determine the mechanisms by which APC vs. thrombin control -arrestin transducer bias. It is not known how -arrestin transducer bias (signaling vs. desensitization) is induced by APC- vs. Th-activated PAR1 nor how APC/PAR1 promotes two distinct -arr2-mediated cytoprotective signaling pathways: dishevelled2 (Dvl2)-Rac1 controls endothelial barrier protection whereas sphingosine kinase 1 (SphK1)-Akt regulates anti-apoptotic activities. We will determine if distinct determinants of -arrestin and GPCR co-receptors control different -arr2 binding modes and functions induced by APC vs. thrombin. Aim 3: To define the mechanisms by which APC/PAR1 regulates Cav1 function to promote cytoprotection. APC/PAR1 stimulates Cav1 phosphorylation but how this modulates Cav1 function and is integrated into the cytoprotective pathway is not known and will be determined. The proposed research is innovative because it will test novel hypotheses to explain how GRK5, -arr2 and Cav1 drive APC/PAR1 endothelial cytoprotective responses in a physiologically relevant context and will help advance the status of new targets as future therapeutics for the treatment of endothelial dysfunction.

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