This project addresses the signaling mechanisms and functions of angiotensin II (AngII) via the AT1 receptor (AT1R), which mediates the physiological actions of AngII on blood pressure, aldosterone secretion, and electrolyte balance. In functional studies on the AT1 receptor, a molecular mechanism for the constitutive activity of mutants of the AT1R at position 111 was suggested by molecular modeling. This involves a cascade of conformational changes in spatial positions of side chains along transmembrane helix (TM3) from L112 to Y113 to F117, which in turn, results in conformational changes in TM4 (residues I152 and M155) leading to the movement of TM4 as a whole. The mechanism is consistent with the available data of site-directed mutagenesis, as well as with correct predictions of constitutive activity of mutants L112F and L112C. It was also predicted that the double mutant N111G/L112A might possess basal constitutive activity comparable with that of the N111G mutant, whereas the double mutants N111G/Y113A, N111G/F117A, and N111G/I152A would have lower levels of basal activity. Experimental studies of the above double mutants showed significant constitutive activity of N111G/L112A and N111G/F117A. The basal activity of N111G/I152A was higher than expected, and that of N111G/Y113A was not determined due to poor expression of the mutant. The proposed mechanism of constitutive activity of the AT1R reveals a novel nonsimplistic view on the general problem of constitutive activity, and demonstrates the inherent complexity of the process of GPCR activation.? ? The regulation of adrenal function, including aldosterone production from adrenal glomerulosa cells, is dependent on a variety of GPCRs and receptor tyrosine kinases (RTKs). In many cell types, GPCR-induced MAPK 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 cells express receptors for lysophosphatidic acid (LPA) and EGF. In cultured bovine adrenal glomerulosa cells, LPA, which is predominantly coupled to Gi and partially to Gq/PKCs alpha 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. 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 heparin-binding EGF. LPA-induced ERK1/2 activation occurs predominantly through EGF-R transactivation by Gi/Src and partly through activation of protein kinase C, which acts downstream of EGF-R and Ras. In contrast, LPA-induced phosphorylation of Shc and ERK1/2 in C9 hepatocytes 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 an 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 responses that differ from those of native cells and tissues.? ? Our recent review on the RAS describes Ang II-induced AT1R stimulation of phospholipases A2, C, and D, and activates InsP3/Ca2+ signaling, protein kinase C isoforms, and MAPK via Gq/11, as well as several tyrosine kinases (Pyk2, Src, Tyk2, FAK), scaffold proteins (arrestins, GPCR kinase-interacting protein 1, p130Cas, paxillin, vinculin), RTKs, and the NF-kappaB pathway. The AT1R also signals via Gi/o and G11/12 and stimulates G protein-independent signaling systems, such as beta-arrestin-mediated MAPK activation and the Jak/STAT pathway. In addition to their roles in the physiological control of blood pressure, thirst, and sodium balance, these responses also exert diverse pathological actions in cardiovascular, renal, and other cell types. It is noteworthy that locally generated Ang II, rather than the circulating octapeptide, often initiates such deleterious effects of AT1R activation. In heart failure the harmful effects of aldosterone exceed those of the RAS. AT1R-mediated overproduction of reactive oxygen species exerts potent growth-promoting, proinflammatory, and profibrotic actions by exerting positive feedback effects that amplify its signaling in cardiovascular cells, leukocytes, and monocytes. Agonist-induced activation of the AT1R also promotes the development of metabolic diseases and can increase tumor progression and metastasis through its growth-promoting and proangiogenic activities. The recognition of Ang II's pathogenic actions is broadening the clinical applications of angiotensin-converting enzyme (ACE) inhibitors and AT1R antagonists, in addition to their established therapeutic actions in essential hypertension. Furthermore, recent reports suggest that intracellular Ang II can exert actions on cell growth that are independent of the AT1 receptor. This implies that certain disorders secondary to angiotensin action could be responsive to ACE inhibitors but not to AT1R antagonists.? ? In addition to the many examples of GPCR-mediated activation of the EGF-R and other RTKs, there is increasing evidence for the converse effect of agonist-activated RTKs upon specific GPCRs. We have observed that the agonist-activated EGF-R caused phosphorylation of the AT1R that was mediated by the EGFR, PKC and PI 3-kinase, and decreased both membrane-associated AT1Rs and inositol phosphate responses to Ang II. These effects were dependent on caveolin-1, which was endogenously phosphorylated and distributed in plasma membrane patches that underwent redistribution during Ang II stimulation. This involved the agonist-induced phosphorylation and association of caveolin 1 with the AT1R, suggesting a scaffolding role of caveolin during transactivation of the EGF-R by Ang II. These studies demonstrated that agonist-stimulation promotes the formation of a caveolin-dependent signalplex that includes GPCRs, RTKs and probably other signaling proteins. This complex could facilitate protein phosphorylation that mediates Ang II-induced transactivation of the epidermal growth factor receptor and activation of ERKs, and epidermal growth factor-induced InsP3 accumulation and phosphorylation/desensitization of the AT1R. This work has extended current insights into the nature and complexity of signaling via GPCRs and receptor tyrosine kinases, and of the importance of caveolin and plasma membrane compartmentalization in signal transduction.
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