Angiotensin II Receptors And Signaling Mechanisms

血管紧张素 II 受体和信号传导机制

• 批准号: 7594117
• 负责人: Kevin J Catt
• 金额: $ 67.21万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594117
• 关键词: 1-Phosphatidylinositol 3-Kinase; Agonist; Aldosterone; Angiotensin II; Angiotensin II Receptor; Angiotensin-Converting Enzyme Inhibitors; Angiotensins; Australia; Basal Cell; Benign Prostatic Hypertrophy; Binding; Binding Sites; Blood Vessels; Calcium; Cancer Cell Growth; Cardiac; Caveolae; Cell Line; Cell Proliferation; Cell Surface Receptors; Cell membrane; Cell physiology; Cell surface; Cells; Chinese Hamster Ovary Cell; Cholesterol; Co-Immunoprecipitations; Collaborations; Complex; Coupled; Cytoplasm; Cytoplasmic Tail; DU145; Data; Diabetes Mellitus; EGF gene; Embryo; Endoplasmic Reticulum; Epidermal Growth Factor Receptor; Epithelial Cells; Equilibrium; Etiology; Evaluation; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits; GTP-Binding Proteins; Generations; Gleason Grade for Prostate Cancer; Gonadotropin Hormone Releasing Hormone; Growth; Helix (Snails); Hepatic; Hepatocyte; Human; Impairment; Infiltration; Inositol Phosphates; Kidney; Kidney Diseases; LNCaP; LY294002; Laboratories; Localized; MAP Kinase Gene; Malignant - descriptor; Malignant Epithelial Cell; Malignant neoplasm of prostate; Mediating; Membrane; Membrane Microdomains; Metabolic; Molecular; Molecular Conformation; Monomeric GTP-Binding Proteins; Movement; Mutagenesis; Mutation; NADPH Oxidase 1; PC3 cell line; PLC gamma1; PTPN11 gene; Pathway interactions; Phagocytes; Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide; Phospholipase D; Phosphorylation; Positioning Attribute; Premalignant; Process; Production; Proliferating; Property; Prostate; Prostate-Specific Antigen; Prostatic Intraepithelial Neoplasias; Protein Kinase C; Proteins; Reactive Oxygen Species; Receptor Activation; Receptor Signaling; Regulation; Relative (related person); Research; Rest; Role; Screening procedure; Side; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Site-Directed Mutagenesis; Sodium; Staining method; Stains; Structure; Superoxides; Surface; Tail; Transmembrane Domain; United States National Institutes of Health; Yeasts; Zona Glomerulosa; base; beta-arrestin; blood pressure regulation; cDNA Library; cancer cell; cell growth; cell type; computerized data processing; density; ear helix; hormone regulation; human RIPK1 protein; indexing; invariant chain; kinase inhibitor; molecular modeling; mutant; novel; peptide hormone; phospholipase C beta; programs; protein protein interaction; receptor; reconstitution; response; scaffold; three-dimensional modeling; trafficking; tumorigenesis; wortmannin; yeast two hybrid system

Caveolin1 (Cav1) is an important component of the plasma-membrane microdomains, such as caveolae and lipid rafts, that are associated with AT1 and EGF receptors in certain cell types. An analysis of the interactions between Cav1 and other signaling molecules that mediate AT1R function was performed in Ang II- and EGF-stimulated hepatic C9 cells. This study demonstrated that cholesterol-rich domains mediate the actions of early upstream signaling molecules such as Src and intracellular calcium in cells stimulated by Ang II, but not by EGF, and that Cav1 has a scaffolding role in the process of MAPK activation. Furthermore, Cav1 phosphorylation by Ang II and EGF was regulated by calcium and Src. Phosphorylation of Cav1 and the EGFR by Ang II, but not ERK1/2 activation, are both dependent on calcium. The PI 3-kinase inhibitors, LY294002 and wortmannin, differentially modulated both Cav1 and EGF receptor activation by Ang II through calcium. These findings further demonstrate the importance of Cav1 in conjunction with receptor-mediated signaling pathways involved in cell proliferation and survival. It is clear that differential signaling pathways are operative in Ang II- and EGF- stimulated C9 cells, and that cholesterol-enriched microdomains are essential components in cellular signaling processes that are dependent on specific agonists and/or cell types. Relatively little is known about the protein-protein interactions that regulate the trafficking of the AT1R through the biosynthetic pathway. The membrane-proximal region of the cytoplasmic tail of the AT1R has been defined by site-directed mutagenesis studies as a site required for normal AT1R folding and surface expression. Based on yeast two-hybrid screening of a human embryonic kidney cDNA library with the AT1R carboxyl-terminal tail as bait, the Invariant chain (Ii) was identified as a novel receptor-interacting protein. This association was confirmed by co-immunoprecipitation and co-localization studies and the binding site for Ii on the AT1R carboxyl-terminal tail was localized to a region that has been identified as important for exit of the AT1R from the endoplasmic reticulum (ER), and is conserved in many G protein-coupled receptors. Transient co-expression of Ii with the AT1R in CHO cells consistently reduced the AT1R density at the cell surface. Furthermore, the interaction of Ii with the carboxyl-terminal tail of the AT1R promotes its retention in the ER and promotes its proteasomal degradation. These observations indicate that Ii and the AT1R become associated in the early biosynthetic pathway, and demonstrate that the Ii protein is a negative regulator of AT1R expression. In previous studies the molecular mechanism of the constitutive activity of AT1R mutants at position 111 was evaluated by molecular modeling. This involved a cascade of conformational changes in spatial positions of side chains along transmembrane helix 3 (TM3) from L112 to Y113 to F117, which in turn causes conformational changes in TM4 (residues I152 and M155) leading to its movement as a whole. This mechanism is consistent with the available data of site-directed mutagenesis, and with correct predictions of constitutive activity of mutants L112F and L112C. More recently, the network of inter-residue interactions within the transmembrane region of the AT1R was investigated by site-directed mutagenesis and molecular modeling studies. Mutagenesis was focused on residues Tyr292, Asn294, and Asn298 in transmembrane helix 7, and the conserved Asp74 in helix 2 and other polar residues. Functional interactions between pairs of residues were evaluated by determining the effects of single and double-reciprocal mutations on agonist-induced AT1R activation. Reciprocal mutations of Asp74/Asn294, as well as Ser115/Asn294, Ser252/Asn294, and Asn298/Ser115 caused additive impairment of function, suggesting that these pairs of residues make independent contributions to AT1R activation. In contrast, mutations of the Asp74/Tyr298 pair revealed that the D74N/N298D reciprocal mutation substantially increased the impaired inositol phosphate responses of the D74N and N298D receptors. Extensive molecular modeling yielded 3D models of the transmembrane region of the AT1R and the mutants as well as of their complexes with Ang II, which were used to identify possible mechanisms of impaired function of specific mutants. These data indicate that Asp74 and Asn298 are not optimally positioned for direct and strong interaction in the resting conformation of the AT1R. However, the balance of interactions between residues in helix 2 (such as D74) and helix 7 (such as N294, N295 and N298) of the AT1R is a crucial factor in determining their functional activity and levels of expression. Ang II promotes cell growth and proliferation, and has been implicated in several forms of tumorigenesis. The role of Ang II in prostate cancer was investigated in collaborative studies performed in the laboratory of Dr. Simon Louis in Melbourne, Australia. Ang II is present in the basal cell layer of the normal prostate gland and in benign prostatic hyperplasia (BPH), and stimulates prostate cell growth via the AT1R. Furthermore, AT1R blockers have been shown to reduce prostate-specific antigen and to inhibit prostate cancer cell growth. An analysis of Ang II expression in BPH and prostate cancer, including high grade prostatic intraepithelial neoplasia (HGPIN), showed its presence in basal epithelial cells in BPH and also in proliferating malignant cells in prostate cancer (Gleason grades 2-5), and in the cytoplasm of LNCaP, DU145, and PC3 prostate cancer cell lines. These data demonstrated Ang II staining in malignant cells in all grades of prostate cancer, and indicate that Ang II expression in non-basal epithelial cells is an early index of pre-malignant and malignant changes. In view of its mitogenic activity, it is probable that Ang II contributes to the growth and infiltration of malignant epithelial cells in the prostate. Furthermore, based on the observation by Baker et al. (2006) that elevated levels of cytoplasmic Ang II can increase cell proliferation via a non-AT1R mechanism, it is possible that angiotensin converting enzyme (ACE) inhibitors would also be of value in the treatment of prostate cancer by reducing intracellular Ang II formation. In collaboration with Dr. Sue Goo Rhee, formerly of the NIH, the mechanism responsible for the Ang II-induced production of reactive oxygen species in non-phagocytic cells was investigated in HEK293 and CHO cells reconstituted with the AT1R, NADPH oxidase 1 (Nox1), Nox organizer 1 (Noxo1), and Nox activator 1 (Noxa1). Stimulation of the reconstituted cells with Ang II caused a substantial increase in superoxide production relative to the constitutive level mediated by the complex of Nox1, Noxo1, and Noxa1. This demonstrated that Nox1 is activated by cell-surface receptor-mediated signaling, and that the AT1R is coupled to Nox1. Expression of AT1R mutants showed that interaction of the receptor with G proteins, but not that with beta-arrestin or proteins (Jak2, phospholipase C-gamma1, SHP2) that bind to the carboxy-terminal region of the AT1R, was necessary for Ang II-induced superoxide production. Evaluation of the effects of constitutively active alpha subunits of G proteins and of various pharmacological agents suggested that signaling by a pathway comprising the AT1R, Galphaq/11, phospholipase C-beta, and protein kinase C was largely, but not exclusively, responsible for Ang II-induced activation of the Nox1-Noxo1-Noxa1 complex in the reconstituted cells. Contributions of Galpha12/13, phospholipase D,and PI3-kinase to Ang II-induced superoxide generation were also suggested, whereas the small GTPase, Rac1, and the EGF receptor do not appear to participate in this action of Ang II.

Caveolin1（Cav1）是与某些细胞类型中与AT1和EGF受体相关的血浆膜微域（例如小窝和脂质筏）的重要组成部分。在ANG II和EGF刺激的肝C9细胞中对CAV1与其他介导AT1R功能的其他信号分子之间的相互作用进行了分析。这项研究表明，富含胆固醇的结构域介导了早期的上游信号分子（例如SRC和细胞内钙）在ANG II刺激的细胞中的作用，但不受EGF的刺激，而CAV1在MAPK激活过程中具有脚手架作用。此外，ANG II和EGF的CAV1磷酸化受钙和SRC调节。 ANG II（而不是ERK1/2激活）对CAV1和EGFR的磷酸化均取决于钙。 Pi 3-激酶抑制剂LY294002和沃特曼宁差异通过ANG II通过钙调节CAV1和EGF受体的激活。这些发现进一步证明了CAV1与受体介导的信号通路的重要性，涉及细胞增殖和存活。显然，在ANG II和EGF刺激的C9细胞中，差异信号通路是可操作的，并且富含胆固醇的微区域是依赖于特定的激动剂和/或细胞类型的细胞信号传导过程中必不可少的组成部分。 关于通过生物合成途径调节AT1R运输的蛋白质 - 蛋白质相互作用的知之甚少。 AT1R的细胞质尾部的膜斑区域已通过位置定向的诱变研究定义为正常AT1R折叠和表面表达所需的位点。基于用AT1R羧基末端尾巴作为诱饵的人类胚胎肾cDNA库的酵母两杂化筛选，不变链（II）被确定为一种新型的受体相互作用蛋白。通过共免疫沉淀和共定位研究证实了这一关联，并且在AT1R羧基末端尾部的II的结合位点定位于该区域，该区域已被鉴定为对内质网（ER）AT1R的重要性，并在许多G蛋白质耦合的受体中保留。与CHO细胞中AT1R的II的瞬态共表达始终降低细胞表面的AT1R密度。此外，II与AT1R的羧基末端的相互作用促进了其在ER中的保留率并促进其蛋白酶体降解。这些观察结果表明，II和AT1R在早期生物合成途径中相关，并证明II蛋白是AT1R表达的负调节剂。 在先前的研究中，通过分子建模评估了位置111处AT1R突变体的组成型活性的分子机制。这涉及沿跨膜螺旋3（TM3）从L112到Y113到F117的侧链空间位置的一系列构象变化，进而导致TM4（残基I152和M155）的构象变化导致其整个运动。该机制与位置定向诱变的可用数据一致，并正确预测了突变体L112F和L112C的组成型活性。 最近，通过位置定向的诱变和分子建模研究，研究了AT1R跨膜区域内的跨膜间相互作用的网络。诱变集中在跨膜螺旋7中的Tyr292，ASN294和ASN298上，以及Helix 2和其他极性残基中保守的ASP74。 通过确定单个和双向重点突变对激动剂诱导的AT1R激活的影响，评估了残基对之间的功能相互作用。 ASP74/ASN294以及Ser115/ASN294，Ser252/ASN294和ASN298/Ser115的相互突变引起了功能添加障碍，这表明这些残基对AT1R激活产生了独立的贡献。 相反，ASP74/Tyr298对的突变表明D74N/N298D相互突变显着增加了D74N和N298D受体的受损肌醇磷酸盐反应。 广泛的分子建模产生了AT1R和突变体的跨膜区域的3D模型，以及与Ang II的复合物的复合物，这些模型用于鉴定特定突变体功能受损功能的可能机制。这些数据表明，ASP74和ASN298在AT1R的静息构象中的直接相互作用和强相互作用并未最佳地定位。 但是，AT1R的螺旋2（例如D74）和螺旋7（例如N294，N295和N298）中残基之间的相互作用平衡是确定其功能活性和表达水平的关键因素。 ANG II促进细胞生长和增殖，并与多种形式的肿瘤发生有关。在澳大利亚墨尔本的西蒙·路易斯（Simon Louis）博士实验室进行的合作研究中，研究了ANG II在前列腺癌中的作用。 ANG II存在于正常前列腺的基底细胞层中，在前列腺增生（BPH）中存在，并通过AT1R刺激前列腺细胞的生长。此外，已显示AT1R阻滞剂可减少前列腺特异性抗原并抑制前列腺癌细胞的生长。对BPH和前列腺癌中ANG II表达的分析，包括高级前列腺上皮内肿瘤（HGPIN），在BPH的基础上皮细胞中以及前列腺癌中的恶性细胞（Gleason 2-5级）（2-5级）以及LNCAP，DU145和PC3 PROCTACTAPCAP CALLICATCAP，PC3 PROSTACE cALLECTAPLAST癌症中表明其存在于BPH的基础上皮细胞中。这些数据表明，在所有级别的前列腺癌中，ANG II染色，表明非基质上皮细胞中的ANG II表达是恶性和恶性变化的早期指数。 鉴于其有丝分裂活性，ANG II很可能有助于前列腺中恶性上皮细胞的生长和浸润。此外，根据Baker等人的观察。 （2006年），升高的细胞质ANG II水平可以通过非AT1R机制增加细胞增殖，血管紧张素转化酶（ACE）抑制剂也可能通过减少细胞内ANG II的形成来治疗前列腺癌。 在HEK293中，研究了与ANG II诱导的非噬菌细胞中活性氧物种产生的机制，并与AT1R，NADPH氧化酶1（NOX1），NOX Organizer 1（nox Organizer 1（noxo1）和Noxa Acticator 1（Nox Acticator 1（noxa）1（noxaive）1（noxaive1）1（noxox Aircan1），NOX Organizer 1（noxo1）和NoxActotor 1（noxaive1 noxa），研究了NIH的Sue Goo Rhee博士，该机制是针对非噬菌体细胞中反应性氧的产生的机制。用ANG II刺激重建的细胞导致超氧化物的产生相对于由NOX1，NOXO1和NOXA1介导的组成型水平的大幅增加。这表明NOX1被细胞表面受体介导的信号传导激活，并且AT1R耦合到NOX1。 AT1R突变体的表达表明，受体与G蛋白的相互作用，但与ANG II诱导的超级氧化物产生所必需的与AT1R的羧基末端区域结合的β-甲蛋白或蛋白（JAK2，磷脂酶C-GAMMA1，SHP2，SHP2）的相互作用。 Evaluation of the effects of constitutively active alpha subunits of G proteins and of various pharmacological agents suggested that signaling by a pathway comprising the AT1R, Galphaq/11, phospholipase C-beta, and protein kinase C was largely, but not exclusively, responsible for Ang II-induced activation of the Nox1-Noxo1-Noxa1 complex in the reconstituted cells.还提出了Galpha12/13，磷脂酶D和PI3-激酶对ANG II诱导的超氧化物产生的贡献，而小的GTPase，RAC1和EGF受体似乎并未参与ANG II的这一动作。