This project is focused on the receptors and signal transduction pathways that mediate the actions of the pressor octapeptide, angiotensin II, on its target cells. The diverse physiological actions of angiotensin II (Ang II) in cardiovascular, renal, neuronal, hepatic, and endocrine cells are mediated by the Gq protein-coupled AT1 receptor. The functions of the distantly related AT2 receptor are less well defined, and in general counteract the growth-related actions of the AT1 receptor. Studies on AT1 receptor expression revealed the importance of a juxtamembrane aromatic residue in the cytoplasmic tail in plasma-membrane insertion of the seven transmembrane-domain receptor. Investigation of the nature and role of glycosylation in AT1 receptor expression showed that all three of the asparagine residues within consensus sites for N-linked glycosylation are glycosylated in transfected COS-7 cells, and appear to be glycosylated in the endogenous AT1 receptor of bovine adrenal glomerulosa cells. Mutations at each glycosylation site, either alone or in combination, had little effect on ligand binding affinity. However, progressive reduction of glycosylation was associated with decreasing cell-surface expression, which was minimal for the non-glycosylated triple mutant receptor. The decrease in surface expression of mutant AT1 receptors was correlated with a progressive reduction in total cell receptor content estimated by immunoblotting with an AT1 receptor antibody. These findings suggest that glycosylation favors receptor expression, possibly by enhancing receptor stability by protecting the adjacent proteins from proteolytic degradation. In other studies on the structure-function properties of the AT1 receptor, an analysis of the roles of two adjacent aspargine residues (Asn294 and Asn295) in the seventh transmembrane domain of the receptor revealed that Asn295 is required for the integrity of the intramembrane binding pocket of the AT1 receptor, but is not essential for signal generation. It also demonstrated the importance of the seventh transmembrane helix in the formation of the binding site for non-peptide receptor agonists. On the other hand, mutation of Asn294 impaired G protein coupling and attenuated inositol phosphate signaling indicating the importance of this residue in receptor activation. Previous observations on the importance of an STL sequence in the C-terminal cytoplasmic domain of the AT1 receptor in the process of agonist-induced internalization of the receptor were extended by an analysis of the phosphorylation sites in this region of the receptor. All of the phosphorylated serine or threonine residues of the agonist-activated receptor were confined to an 11-residue sequence in the C-terminal tail of the receptor. This study also demonstrated a correlation between the degree of phosphorylation of the AT1 receptor and the extent of its agonist-induced internalization. Studies were also initiated on the phosphorylation of the angiotensin AT2 receptor, employing an epitope-tagged receptor expressed in COS-1 cells. The AT2 receptor is also phosphorylated on its cytoplasmic tail during ligand-induced activation, but less extensively than the AT1 receptor. This work is being extended to an analysis of the location and nature of the phosphorylated residues, and the relationship between AT2 receptor phosphorylation and its functions including inhibition of AT1-induced growth responses. Earlier studies on the pathways by which the AT1 receptor activates bovine glomerulosa cell proliferation revealed that Ang II stimulated the MAP kinase cascade through protein kinase C-dependent and independent pathways. An investigation of the mechanisms by which Ang II activates Raf-1, the upstream intermediate in MAP kinase phosphorylation, revealed that this process is dependent on Ras activation, and is mediated in part via Gi and PI-3-kinase. In addition, activation of Raf-1 kinase by Ang II was negatively modulated by increased calcium influx, and by a downstream negative signaling element. In contrast to the mechanisms of Raf-1 activation by Ang II in other cell types, its activation in adrenal glomerulosa cells was independent of PKC and elevated intracellular calcium, and was only partially dependent on tyrosine kinases.
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