(Verbatim from the application): The renin-angiotensin system is important in normal physiology and the diseases hypertension and atherosclerosis. Angiotensin II (Ang II), interacts with, among others, the Ang II type 1A receptor (AT, AR) of vascular smooth muscle cells (VSMCs), leading to activation of several phospholipases (PL). The second or tonic phase of signaling requires endocytosis or sequestration of the AT1 4R to an undefined discrete signaling domain, a major focus of this proposal. Additionally, Ang II increases intracellular calcium, and activates protein kinase C, Src family kinases and Jak kinases, as well as mitogen activated protein kinases that mediate VSMC hypertrophy and growth. Importantly, Mg II induces robust generation of reactive oxygen species (ROS) that are major modifiers of signal generation. Calcium-dependent transactivation of the epidermal growth factor receptor (EGF R) serves as a """"""""scaffold"""""""" for the assembly of a signaling complex including cSrc, Pyk2, and downstream adaptors (Shc, Grb2, Sos), leading to ERK1/2 and protein kinase B/Akt activation. Little is known of the spatial-temporal organization of AT,A R signaling in the VSMC membrane and how specificity is achieved. Evidence suggests that receptors and associated signaling molecules are not randomly distributed but are localized in specialized domains. Functionally distinct microdomains with characteristic lipid composition, """"""""lipid rafts,"""""""" have been identified in plasma membranes. """"""""Rafts"""""""" localize signaling molecules including receptors, G-protein subunits, adaptor molecules, and a host of signal generating proteins. They serve as scaffolds to facilitate interactions among pathways and signaling proteins. Caveolae are cell membrane invaginations that contain the major structural protein caveolin and are specialized forms of rafts. We have shown that Ang II stimulation of VSMCs causes movement of the AT, A R into caveolae-enriched membrane fractions. Its original, basal location is undefined. Another major signaling domain is the focal adhesion or complex, which contains integrin receptors and many associated signaling molecules as well as caveolin. The EGF R has been associated with both caveolae and focal adhesions. Our preliminary data indicate that the Mg Il-induced transmodulation of the EGF-R and certain downstream pathways are dependent upon ROS and suggest involvement of both caveolin and focal adhesion associated molecules. We hypothesize that the full repertoire of Ang 11-induced signals in VSMC is dependent upon several major """"""""scaffolds"""""""" and discrete signaling domains. To gain insight into these issues we propose three Specific Aims involving: 1) the basal location and mechanisms by which AT1ARS move to caveolae-enriched fractions; 2) determination of the functional consequences of this movement or sequestration; and 3) determination of the role of ROS in affecting the related signaling pathways. Insights gained in understanding of the mechanisms of Ang II signaling will lead to increased understanding of the pathogenesis and potential treatment of hypertension and atherosclerosis.
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