This project is focused on the function and signaling of G protein-coupled receptors, in particular the AT1 receptor (AT1R) that mediates the physiological actions of angiotensin II (Ang II) on blood pressure, aldosterone secretion, and sodium balance. Ang II and the AT1R have also been implicated in the development of atheroma, cardiac hypertrophy and failure, and renal disease. 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. In C9 hepatic cells, which express endogenous AT1R, Ang II-induced ERK phosphorylation is largely dependent on transactivation of the EGFR by a PKCdelta-dependent mechanism, and is mediated by the Src/Pyk2 complex via transactivation of the EGFR. 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. Agonist-induced MMP activation in C9 cells led to shedding of HB-EGF and stimulation of ERK1/2 phosphorylation. Also, blockade of HB-EGF action by neutralizing antibody or its selective inhibitor, CRM197, attenuated ERK1/2 activation by Ang II. Consistent with its agonist action, 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. The Tyr319 residue of the AT1R has been proposed to be an essential regulator of EGFR transactivation. However, stimulation of both wild type and mutant (Y319F) AT1R expressed in COS-7 cells caused EGF-R transactivation and subsequent ERK1/2 phosphorylation through release of HB-EGF in a Src-dependent manner. In contrast, the noninvolvement of MMPs in HEK 293 cells, which may reflect the absence of Src activation by Ang II, was associated with lack of transactivation of the EGFR. These findings indicate 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. In previous studies on the function of a highly conserved DRY domain in the second intracellular loop of the AT1R, the D125A mutant AT1R exhibited moderate constitutive activity as indicated by increased basal ERK activation and enhanced inositol phosphate responses to partial agonists. Agonist-induced stimulation of the Elk1 promoter showed parallel impairment with inositol phosphate signal generation after mutations in this region of the AT1R. These data are consistent with the role of the conserved DRY sequence in AT1R activation, and of Asp125 in constraining the receptor in its inactive conformation. It is likely that an apolar surface in the cytoplasmic extension of the third transmembrane helix has a direct role in G protein coupling and signal generation. While the actions of Ang II in adrenal, renal, cardiovascular, and neural cells are primarily mediated by the AT1R and Gq-mediated signaling, its binding to the AT2 angiotensin receptor activates phosphatases that inhibit phosphotyrosine signaling by AT1 and growth factor receptors. The differential properties of the AT1 and AT2 receptors were analyzed in chimeric receptors created by exchanging the second (IL2) and third (IL3) intracellular loops and/or the cytoplasmic tail (CT) between these receptors. In transfected COS-7 cells, the chimeric receptors retained their ligand binding properties. Measurements of Ang II-induced receptor endocytosis and inositol phosphate responses showed that the CT is required for normal AT1R internalization, and that IL2 is a determinant of G protein activation, while the amino-terminal portion of IL3 is required for both receptor functions. However, only substitution of IL2 impaired Ang II-induced ERK activation, suggesting that alternative mechanisms are responsible for ERK activation in signaling-deficient mutant AT1 receptors. Substitution of IL2, IL3, or CT of the AT1 receptor into the AT2 receptor sequence did not enable it to internalize or to mediate inositol phosphate signaling responses. These data suggest that the lack of receptor internalization and inositol phosphate signal generation by the AT2 receptor is a consequence of its specific activation mechanism, rather than the inability of its cytoplasmic domains to couple to intracellular effectors. In related studies, ubiquitylation of the cytoplasmic serine/threonine-rich region of the AT1A receptor on lysine residues was not required for its agonist-induced internalization, suggesting that endocytosis of mammalian GPCRs occurs by a different mechanism than that of yeast GPCRs. Corticotropin-releasing hormone (CRH) regulates pituitary ACTH secretion and mediates behavioral and autonomic responses to stress through activation of CRHR1 receptors located in pituitary corticotrophs and the brain. Although CRHR1 sites are essential for ACTH responses to stress, their number in the pituitary gland does not correlate with corticotroph responsiveness, suggesting that activation of a small number of receptors is sufficient for maximum ACTH production. Previous studies on CRH binding and hybridization studies in groups of adrenalectomized, glucocorticoid-treated, and stressed rats revealed divergent changes in CRH receptors and CRHR1 mRNA in the pituitary gland, with a reduction in receptor binding but normal or elevated CRHR1 mRNA levels. Analysis of CRHR1 expression in pituitary glands of adrenalectomized rats showed unchanged receptor mRNA levels and increased receptor protein despite the impaired agonist binding, suggesting that the latter results from homologous desensitization rather than reduced receptor synthesis. In contrast, the fall in CRH binding following glucocorticoid administration is associated with decreased CRHR1 protein, suggesting inhibition of CRHR1 mRNA translation. This suggests that post-transcriptional regulatory mechanisms that permit rapid changes in CRH receptor activity are important for adaptation of corticotroph responsiveness to continuous changes in physiological demands. In studies on human retinoblastoma Y79 cells, CRH stimulation caused ~30-fold increases in intracellular cAMP accumulation without changing inositol phosphate levels, consistent with coupling of the CRHR1 to Gs but not Gq, and predominant signaling via the PKA cascade. Direct activation of PKC by PMA or DOG desensitized CRHR1 receptors in Y79 cells, reducing the maximum CRF-stimulated cAMP accumulation by 56% and 40%, respectively. Inhibition of PKC with BIM markedly reduced PMA's desensitizing action on CRH-stimulated cAMP production, but did not affect homologous CRHR1 desensitization. Retinoblastoma cells express PKCalpha, betaI, betaII, delta, and lambda isoforms, and down-regulation of PKC alpha and beta by 48-h PMA exposure prevented subsequent PMA-induced CRHR1 receptor desensitization. In transfected COS-7 cells, CRHR1 receptor phosphorylation after 5-min exposure to PMA was increased 2.3-fold, and was abolished by pretreatment with BIM. These studies indicate that activation of specific protein kinase C isoforms is an important factor in the heterologous phosphorylation and desensitization of the CRHR1 receptor.
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