application) Angiotensin II (angII) is a pleiotropic hormone with an extraordinary repertoire of signaling responses associated with activation of its receptors. The prototype ang II receptor is the AT1AR of vascular smooth muscle (VSMC). AT1AR activation in VSMC is associated with the sequential activation of phospholipase Cs (PLC) and phospholipase D (PLD). The initial PLC activation rapidly desensitizes and is followed immediately by PLD protein coupled AT1AR also activates a variety of receptor tyrosine kinase. The PIs propose that these various signals are organized by being associated with specific temporal and spatial domains. The major general object of this proposal is to understand the signaling repertoire by providing insights into the coupling protein linking the AT1AR to effector molecules and into the mechanisms controlling receptor trafficking into signaling or recycling domains. Preliminary evidence suggests that this domain is the caveola. A major underlying hypothesis is that the receptor trafficking controls the pattern of signal generation. An important recent accomplishment of the program is the development and refinement of the technique of electroportation of antibodies against specific signaling components into VSMC. This approach, for example, has permitted assignment of a pivotal role for the Gg subunit in transducing AT1AR signaling. The PIs have developed a general model that guides development and testing of specific mechanistic hypotheses concerning angII signaling events in VSMC. To test the general model we propose the following specific aims: 1) Define the specific membrane domains to which the activated AT1AR is mobilized after activation; 2) Determine the role of caveolae as the site of activation of the tonic component of the biphasic signaling response; 3) Determine the mechanism of activation of tyrosine kinase by the AT1AR in caveolae; 4) Examine the role of caveolae in oxidant- sensitive AT1AR-mediated signal transduction; 5) Using the baculovirus expression system and information from cell studies, determine which components of the AT1AR signaling pathways physically interact. These studies should provide new insights into the mechanisms by which ang II fulfills its pivotal physiologic and pathophysiologic roles.
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