Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0004135 (ATM)
 13,001 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of N-glycosylation in the pharmacological properties and cell surface expression of AT1 receptor was evaluated. Using site-directed mutagenesis, we substituted both separately and simultaneously the asparagine residues in all three putative N-linked glycosylation consensus sequences (N-X-S/T) of AT1 receptor (positions 4, 176, and 188) with aspartic acid. Expression of these mutant receptors in COS-7 cells followed by photolabeling with [125I]-[p-benzoyl-Phe8]AngII and SDS-PAGE revealed ligand-receptor complexes of four different molecular sizes, indicating that the three N-glycosylation sites are actually occupied by oligosaccharides. Binding studies showed that the affinity of each mutant receptor for [Sar1,Ile8]Ang II was not significantly different from that of wild-type AT1 receptor. Moreover, the functional properties of all mutant receptors were unaffected as evaluated by inositol phosphate production. However, the expression levels of the aglycosylated mutant were 5-fold lower than that of the wild-type AT1 receptor. Use of green fluorescent protein-AT1 receptor fusion proteins in studying the cellular location of the aglycosylated mutant demonstrated that it was distributed at a much higher density to the ER-Golgi complex than to the plasma membrane in HEK 293 cells. Together, these results suggest an important role of N-glycosylation in the proper trafficking of AT1 receptor to the plasma membrane.
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PMID:Role of N-glycosylation in the expression and functional properties of human AT1 receptor. 1039 37

Agonist-induced endocytosis and processing of the G protein-coupled AT1 angiotensin II (Ang II) receptor (AT1R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)- or hemagglutinin epitope-tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5-GFP and Rab4-GFP in early endosomes, and subsequently with Rab11-GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand-receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa(R)-transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine-Ang II was also segregated into small internal vesicles. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase-dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.
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PMID:Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor. 1207 Jan 29

Beta-arrestins play a role in AT1 endocytosis by binding the cytoplasmic, C-terminus region T332-S338, the major site of angiotensin II (Ang II)-induced phosphorylation. However, the processes responsible for recruiting beta-arrestin to the activated receptor are poorly defined. In this study, we used CHO-K1 and HEK 293 cells expressing wild-type or mutant AT1 to investigate two possibilities: activated AT1 induces global relocation of beta-arrestins to the plasma membrane or the phosphorylated C-terminus acts as bait to attract beta-arrestins. Results obtained using high osmolarity and dominant-negative beta-arrestin confirmed that internalization of AT1 in both CHO-K1 and HEK 293 cells is predominately via clathrin-mediated endocytosis involving beta-arrestin, and substitution of T332, S335, T336 and S338 with alanine to preclude phosphorylation markedly attenuated AT1 internalization. Confocal microscopy revealed that wild-type AT1 induced a time-dependent translocation of GFP-tagged beta-arrestins 1 and 2 to the cell surface. In contrast, the TSTS/A mutant did not traffic beta-arrestin 1 at all, and only trafficked beta-arrestin 2 weakly. Results of rescue-type experiments were consistent with the idea that both beta-arrestins are able to interact with the non-phosphorylated receptor, albeit with much lower affinity and beta-arrestin 1 less so than beta-arrestin 2. In conclusion, this study shows that the high affinity binding of beta-arrestins to the phosphorylated C-terminus is the predominant mechanism of agonist-induced beta-arrestin recruitment to the cell surface and AT1 receptor.
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PMID:Agonist-dependent internalization of the angiotensin II type one receptor (AT1): role of C-terminus phosphorylation in recruitment of beta-arrestins. 1517 32

The expression of a constitutively active G protein-coupled receptor is expected to trigger diverse cellular changes ranging from normal to adaptive responses. We report that confluent HEK-293 cells stably expressing the constitutively active mutant N111G-AT1 receptor for angiotensin II spontaneously exhibited dramatic morphological changes and cytoskeletal reorganization. Phase-contrast microscopy revealed that these cells formed a dense monolayer, whereas cells expressing the WT-AT1 receptor displayed large intercellular spaces and numerous filopodia. Confocal microscopy revealed an elaborate web of polymerized actin at the apical and basolateral surfaces of cells expressing the N111G-AT1 receptor. Interestingly, these phenotypic changes were prevented by culturing the cells in the presence of the inverse agonist EXP3174. Similar morphologic rearrangements and de novo polymerized actin structures were found in Ang II-stimulated cells expressing the WT-AT1 receptor. We further showed that AT1 receptor-induced cell-cell contact formation did not require an increase in intracellular Ca2+ concentration or the activity of protein kinase C. However, pretreatment with Y-27632 revealed that Rho-kinase activity was required for cell-cell contact formation upon AT1 receptor activation. These observations demonstrate that the expression of the constitutively active mutant N111G-AT1 receptor had a significant impact on the morphology and cytoskeletal organization of HEK-293 cells, possibly via a mechanism involving the activity of Rho-kinase.
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PMID:The constitutively active N111G-AT1 receptor for angiotensin II modifies the morphology and cytoskeletal organization of HEK-293 cells. 1589 77

Many G protein coupled receptors (GPCRs) cause phosphorylation of MAP kinases through transactivation of the epidermal growth factor receptor (EGF-R), leading to increased cell survival and growth, motility, and migration. Phosphoinositide 3-kinase (PI3K) is one of the important cell survival signaling molecules activated by EGF-R stimulation. However, the extent to which EGF-R transactivation is essential for GPCR agonist-stimulated PI3K activation is not known. Here we examined the mechanism of PI3K activation that elicits GPCR-mediated ERK1/2 activation by pathways dependent and/or independent of EGF-R transactivation in specific cell types. Immortalized hypothalamic neurons (GT1-7 cells) express endogenous gonadotropin-releasing hormone receptors (GnRH-R) and their stimulation causes marked phosphorylation of ERK1/2 and Akt (Ser 473) through transactivation of the EGF-R and recruitment of PI3K. In C9 hepatocytes, agonist activation of AT1 angiotensin II (AT1-R), lysophosphatidic acid (LPA), and EGF receptors caused phosphorylation of Akt through activation of the EGF-R in a PI3K-dependent manner. However, ERK1/2 activation by these agonists in these cells was independent of PI3K activation. In contrast, agonist stimulation of HEK 293 cells stably expressing AT1-R caused ERK1/2 phosphorylation that was independent of EGF-R transactivation but required PI3K activation. LPA signaling in these cells showed partial and complete dependence on EGF-R and PI3K, respectively. These data indicate that GPCR-induced ERK1/2 phosphorylation is dependent or independent of PI3K in specific cell types, and that the involvement of PI3K during ERK1/2 activation is not dependent solely on agonist-induced transactivation of the EGF-R.
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PMID:Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs. 1592 Jul 62

1. Nitric oxide (NO) is known to affect the properties of various proteins via the S-nitrosylation of cysteine residues. This study evaluated the direct effects of the NO donor sodium nitroprusside (SNP) on the pharmacological properties of the AT1 receptor for angiotensin II expressed in HEK-293 cells. 2. SNP dose-dependently decreased the binding affinity of the AT1 receptor without affecting its total binding capacity. This modulatory effect was reversed within 5 min of removing SNP. 3. The effect of SNP was not modified in the presence of the G protein uncoupling agent GTPgammaS or the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. 4. The binding properties of a mutant AT1 receptor in which all five cysteine residues within the transmembrane domains had been replaced by serine was not affected by SNP. Systematic analysis of mutant AT1 receptors revealed that cysteine 289 conferred the sensitivity to SNP. 5. These results suggest that NO decreased the binding affinity of the AT1 receptor by S-nitrosylation of cysteine 289. This modulatory mechanism may be particularly relevant in pathophysiological situations where the beneficial effects of NO oppose the deleterious effects of angiotensin II.
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PMID:S-nitrosylation of cysteine 289 of the AT1 receptor decreases its binding affinity for angiotensin II. 1656 29

Expression of spermidine/spermine N(1)-acetyltransferase (SSAT) increases in kidneys subjected to ischemia-reperfusion injury (IRI). Increased expression of SSAT in vitro leads to alterations in cellular polyamine content, depletion of cofactors and precursors of polyamine synthesis, and reduced cell proliferation. In our model system, a >28-fold increase in SSAT levels in HEK-293 cells leads to depletion of polyamines and elevation in the enzymatic activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, suggestive of a compensatory reaction to increased polyamine catabolism. Increased expression of SSAT also led to DNA damage and G(2) arrest. The increased DNA damage was primarily due to the depletion of polyamines. Other factors such as increased production of H(2)O(2) due to polyamine oxidase activity may play a secondary role in the induction of DNA lesions. In response to DNA damage the ATM/ATR --> Chk1/2 DNA repair and cell cycle checkpoint pathways were activated, mediating the G(2) arrest in SSAT-expressing cells. In addition, the activation of ERK1 and ERK2, which play integral roles in the G(2)/M transition, is impaired in cells expressing SSAT. These results indicate that the disruption of polyamine homeostasis due to enhanced SSAT activity leads to DNA damage and reduced cell proliferation via activation of DNA repair and cell cycle checkpoint and disruption of Raf --> MEK --> ERK pathways. We propose that in kidneys subjected to IRI, one mechanism through which increased expression of SSAT may cause cellular injury and organ damage is through induction of DNA damage and the disruption of cell cycle.
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PMID:Spermidine/spermine N1-acetyltransferase overexpression in kidney epithelial cells disrupts polyamine homeostasis, leads to DNA damage, and causes G2 arrest. 1706 2

Peroxisome proliferator-activated receptor gamma (PPAR gamma) agonists are commonly used to treat cardiovascular diseases, and are reported to have several effects on cardiovascular function that may be due to PPAR gamma-independent signaling events. Select angiotensin receptor blockers (ARBs) interact with and modulate PPAR gamma activity, thus we hypothesized that a PPAR gamma agonist may exert physiologic effects via the angiotensin II type 1(A) receptor (AT1(A)R). In AT1(A)R-overexpressing HEK 293 cells, both angiotensin II (Ang II) and the PPAR gamma agonist troglitazone (Trog) enhanced AT1(A)R internalization and recruitment of endogenous beta-arrestin 1/2 (beta arr1/2) to the AT1(A)R. A fluorescence assay to measure diacylglycerol (DAG) accumulation showed that although Ang II induced AT1(A)R-G(q) protein-mediated DAG accumulation, Trog had no impact on DAG generation. Trog-mediated recruitment of beta arr1/2 was selective to AT1(A)R as the response was prevented by an ARB- and Trog-mediated beta arr1/2 recruitment to beta1-adrenergic receptor (beta 1AR) was not observed. In isolated mouse cardiomyocytes, Trog increased both % and rate of cell shortening to a similar extent as Ang II, effects which were blocked with an ARB. Additionally, these effects were found to be beta arr2-dependent, as cardiomyocytes isolated from beta arr2-KO mice showed blunted contractile responses to Trog. These findings show for the first time that the PPAR gamma agonist Trog acts at the AT1(A)R to simultaneously block G(q) protein activation and induce the recruitment of beta arr1/2, which leads to an increase in cardiomyocyte contractility.
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PMID:Troglitazone stimulates beta-arrestin-dependent cardiomyocyte contractility via the angiotensin II type 1A receptor. 2046 Jan 6

A cell-penetrating peptide consisting of the second intracellular loop (IC2) of the angiotensin II (AngII) type-I receptor (AT1) linked to the HIV-transactivating regulatory protein (TAT) domain was used to identify the role of this motif In intracellular signal transduction. HEK-293 cells stably transfected with AT1R cDNA and primary cultures of human pulmonary artery smooth muscle cells expressing endogenous AT1 receptor were exposed to the cell-penetrating peptide construct, and the effect on angiotensin II signaling was determined. The AT1 IC2 peptide effectively inhibited AngII-stimulated phosphatidylinositol turnover and calcium influx. It also limited the activation of Akt/PKB as determined by an inhibition of phosphorylation of Akt at Ser473, and completely abolished the AngII-dependent activation of the transcriptional factor NFkappaB. In contrast, the AT1 IC2 peptide had no effect on AngII/AT1 receptor activation of ERK. These results illustrate the potential of using cell-penetrating peptides to both delineate receptor-mediated signal transduction and to selectively regulate G protein-coupled receptor signaling.
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PMID:Limiting angiotensin II signaling with a cell-penetrating peptide mimicking the second intracellular loop of the angiotensin II type-I receptor. 2049 49

Signaling through both angiotensin AT1 receptors (AT1R) and dopamine D1 receptors (D1R) modulates renal sodium excretion and arterial BP. AT1R and D1R form heterodimers, but whether treatment with AT1R antagonists functionally modifies D1R via allosterism is unknown. In this study, the AT1R antagonist losartan strengthened the interaction between AT1R and D1R and increased expression of D1R on the plasma membrane in vitro. In rat proximal tubule cells that express endogenous AT1R and D1R, losartan increased cAMP generation. Losartan increased cAMP in HEK 293a cells transfected with both AT1R and D1R, but it did not increase cAMP in cells transfected with either receptor alone, suggesting that losartan induces D1R activation. Furthermore, losartan did not increase cAMP in HEK 293a cells expressing AT1R and mutant S397/S398A D1R, which disrupts the physical interaction between AT1R and D1R. In vivo, administration of a D1R antagonist significantly attenuated the antihypertensive effect of losartan in rats with renal hypertension. Taken together, these data imply that losartan might exert its antihypertensive effect both by inhibiting AT1R signaling and by enhancing D1R signaling.
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PMID:Binding of losartan to angiotensin AT1 receptors increases dopamine D1 receptor activation. 2219 84


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