The octapeptide hormone angiotensin II (Ang II) plays an important role in blood pressure regulation and the vascular smooth muscle cells (VSMCs) proliferative response. Two isoforms of Ang II receptors, AT1 and AT2 that belong to the superfamily of G protein coupled receptors (GPCR) mediate these responses. Surprisingly, in addition to G protein coupled responses, the AT1 receptor activates intracellular signal transduction pathways that are usually activated by cytokine and growth factor receptors. The AT2 receptor is a mediator of apoptosis. The applicant's studies show marked differences between the mode of action of the AT1 and AT2 receptor activation will provide a new paradigm for the study of small peptide hormone receptors. Site-directed mutagenesis combined with group-specific modifications of Ang II will be used to identify contact sites between Ang II and the AT1 and AT2 receptors. Systematic modifications of critical receptor residues and functional groups of Ang II necessary for agonism will reveal bonding interactions that are required for hormone-dependent receptor activation. Identification of those residues that upon mutation stabilize the activated-state of the receptor (i.e. those that generate a constitutively active mutant) and are the dock sites for agonist specific sidechains of Ang II will allow a molecular definition of receptor activation. Difference in binding pocket structure between the wild-type [R*] will then be identified by substituted cysteine accessibility mapping (SCAM); these studies will allow a prediction of the mechanical movements associated with receptor activation. Recent studies show that Ang II, via an action on the VSMCs AT1 receptor, causes an activation of Janus kinases (JAKs). Does the AT1 receptor activate JAKs directly or its activation subsequent to the activation of Gq (a G protein family that links GPCRs to phospholipase C)? Dominant negative mutants of Gq and AT1 receptor mutants that bind but do not activate Gq will be used to determine if JAK activation is direct or via Gq. Activated JAKs do not always lead to an activation of latent STATs (signal transducers and activators of transcription) and stimulation of c-fos gene expression. Using gel mobility shift of a CIS-inducible element and a c-fos -luciferase reporter gene the applicant will examine if STATs and c-fos gene are activated, respectively. These studies are likely to be important in defining the cellular signaling mechanisms involved in VSMCs proliferation, a step considered to be important in the natural progression of vascular disease.
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