This project is focused on the function and signaling of angiotensin II (Ang II) receptors, in particular the AT1 receptor (AT1R), which mediates the physiological actions of Ang II on blood pressure, aldosterone secretion, and sodium balance. This G protein-coupled receptor has also been implicated in hypertension, and the development of atheroma, cardiac hypertrophy and failure, renal disease, and diabetes. Agonist binding to the AT1R activates Gq/11 proteins, phosphoinositide-calcium signaling and specific PKC isoforms, leading to ERK1/2 phosphorylation via EGFR transactivation-dependent or -independent pathways. Many of the growth-related actions of Ang II are mediated by transactivation of the EGFR and initiation of ras-dependent phosphorylation of ERK1/2, leading to increased expression of genes regulating cell growth, differentiation and function. Studies in C9 hepatic cells, as well as transfected COS and HEK 293 cells, have indicated that the individual actions of Ang II on EGFR transactivation pathways in specific cell types are related to the differential involvement of MMP-dependent HB-EGF release in this process. Thus, further analysis of the mechanisms involved in agonist-induced EGF-R transactivation and subsequent ERK1/2 phosphorylation in C9 hepatocytes and cell lines transfected with the AT1R showed that Ang II-induced ERK1/2 activation was attenuated by inhibition of Src kinase and matrix metalloproteinases (MMPs) in C9 and COS-7 cells, but not in HEK 293 cells. In C9 cells, agonist-induced MMP activation causes shedding of HB-EGF and stimulation of ERK1/2 phosphorylation, and this is attenuated by blockade of HB-EGF action with a neutralizing antibody or its selective inhibitor, CRM197. HB-EGF stimulation of both cell types caused marked phosphorylation of the EGFR and its adapter molecule, Shc, as well as ERK1/2 and its dependent protein, RSK1, in a manner similar to that elicited by Ang II or EGF. In hepatic C9 cells, Ang II-induced activation of AT1-Rs stimulates ERK1/2 phosphorylation via transactivation of the endogenous EGF-R by a protein kinase Cdelta/Src/Pyk2-dependent pathway, leading to phosphorylation of the EGF-R as well as its subsequent internalization. On the other hand, EGF-induced activation of the EGF-R in C9 cells was found to cause phosphorylation of the AT1-R. This was prevented by selective inhibition of the intrinsic tyrosine kinase activity of the EGF-R by AG1478 and was reduced by inhibition of PKC and phosphoinositide 3-kinase. EGF-induced AT1-R phosphorylation was associated with a decrease in membrane-associated AT1-Rs and a reduced inositol phosphate response to Ang II. Agonist activation of endogenous AT1-Rs and EGF-Rs induced the formation of a multireceptor complex containing both the AT1-R and the transactivated EGF-R. The dependence of these responses on caveolin was indicated by the finding that cholesterol depletion of C9 cells abolished Ang II-induced inositol phosphate production, activation of Akt/PKB and ERK1/2, and AT1-R internalization. Confocal microscopy demonstrated that caveolin-1 is endogenously phosphorylated and was distributed on the plasma membrane in patches that undergo redistribution during Ang II stimulation. Agonist-induced phosphorylation and association of caveolin 1 with the AT1-R was observed, consistent with a scaffolding role of caveolin during transactivation of the EGF-R by Ang II. The EGF-induced AT1-R/caveolin association was abolished by AG1478, indicating that activation of the EGF-R promotes the association of caveolin and the AT1-R. This work has revealed further details of the complex mechanisms of signaling between GPCRs and receptor tyrosine kinases, and of the significant roles of membrane interactions and compartmentalization in ligand-induced signal transduction and cell responses. The regulation of adrenal function, including aldosterone production from adrenal glomerulosa cells, is dependent on a variety of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). In many cell types, GPCR-mediated MAP kinase activation is mediated through transactivation of RTKs, in particular the EGF-R. However, the extent to which this cross-communication between GPCRs and RTKs is operative in the adrenal glomerulosa has not been defined. Bovine adrenal glomerulosa (BAG) cells express receptors for Ang II, as well as lysophosphatidic acid (LPA) and EGF. In cultured BAG cells, LPA, which is predominantly coupled to Gi and partially to Gq/PKCalpha and epsilon, caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2) at Tyr402, EGF-R, protein kinase B/Akt, ERK1/2, and their dependent protein, p90 ribosomal S6 kinase (RSK-1). Overexpression of dominant negative mutants of Ras or EGF-R, and selective inhibition of EGF-R kinase with AG1478, significantly reduced LPA-induced ERK1/2 phosphorylation. However, this was not impaired by inhibition of matrix metalloproteinase (MMP) and HB-EGF. LPA-induced ERK1/2 activation occurs predominantly through EGF-R transactivation by Gi/Src and partly through activation of PKC, which acts downstream of EGF-R and Ras. In contrast, LPA-induced phosphorylation of Shc and ERK1/2 in C9 cells was primarily mediated through MMP-dependent transactivation of the EGF-R. These observations in adrenal glomerulosa and hepatic cells demonstrate that LPA phosphorylates ERK1/2 through EGF-R transactivation in a MMP-dependent or independent manner in individual target cells. This reflects the ability of GPCRs expressed in cell lines and neoplastic cells to utilize distinct signaling pathways that can elicit altered responses compared with those of native tissues. Protein kinase C (PKC) isoforms are important transducers of signals from GPCRs to diverse cellular targets, including ERK1/2. C9 cells express receptors for angiotensin II (AT1R), lysophosphatidic acid (LPA), and epidermal growth factor (EGF), and their stimulation causes transient ERK1/2 phosphorylation through transactivation of the epidermal growth factor receptor (EGF-R). Inhibition of PKC by Go6983 or PKC depletion by prolonged phorbol 12-myristate 13-acetate (PMA) treatment, attenuated ERK1/2 activation by Ang II and PMA, but not by LPA and EGF. In contrast, another PKC inhibitor, Go6976, enhanced basal and agonist-stimulated phosphorylation of ERK1/2, which was not caused by alteration in receptor binding and internalization, stimulation of inositol phosphate production, or activation of Pyk2 and Src tyrosine kinases. However, Go6976 enhanced agonist-induced tyrosine phosphorylation of the EGF receptor, possibly through inhibition of protein tyrosine phosphatase (PTP), because the PTP inhibitor sodium orthovanadate mimicked the effects of Go6976. Selective blockade of EGF-R kinase by AG1478 abolished the ERK1/2 activation induced by Go6976. Similar experiments were conducted in human embryonic kidney 293 cells, which express receptors for LPA and EGF but exhibit no significant cross-communication between them. Although Go6976 caused a significant increase in EGF-induced tyrosine phosphorylation of the EGF-R and subsequent ERK1/2 activation, it had no such effects on LPA-induced responses. In Chinese hamster ovary cells, which express receptors for LPA but not for EGF, Go6976 also had no significant effect on LPA-induced ERK1/2 activation. These data have shown that Go6976 potentiates agonist-induced ERK1/2 activation through stimulation of tyrosine phosphorylation of the EGF-R.