Catalase overexpression attenuates angiotensinogen expression and apoptosis in diabetic mice.

Increased generation of reactive oxygen species (ROS) leads to oxidative stress in diabetes. Catalase is a highly conserved heme-containing protein that reduces hydrogen peroxide to water and oxygen and is an important factor decreasing cellular injury owing to oxidative stress. Hyperglycemic conditions increase oxidative stress and angiotensinogen gene expression. Angiotensinogen conversion to angiotensin II leads to a furtherance in oxidative stress through increased generation of reactive oxygen species. In this study, we utilized mice transgenically overexpressing rat catalase in a kidney-specific manner to determine the impact on ROS, angiotensinogen and apoptotic gene expression in proximal tubule cells of diabetic animals. Proximal tubules isolated from wild-type and transgenic animals without or with streptozotocin-induced diabetes were incubated in low glucose media in the absence or presence of angiotensin II or in a high-glucose media. Our results show that the overexpression of catalase prevents the stimulation of ROS and angiotensinogen mRNA in tubules owing to elevated glucose or angiotensin II in vitro. Additionally, overexpression of catalase attenuated ROS generation, angiotensinogen and proapoptotic gene expression and apoptosis in the kidneys of diabetic mice in vivo. Our studies point to an important role of ROS in the pathophysiology of diabetic nephropathy.

Transforming growth factor-beta 1 stimulates angiotensinogen gene expression in kidney proximal tubular cells.

The present study investigated whether transforming growth factor-beta 1 (TGF-beta1) exerts an autocrine positive effect on angiotensinogen (ANG) gene expression in rat kidney proximal tubular cells, and delineates its underlying mechanism(s) of action. Rat immortalized renal proximal tubular cells (IRPTCs) and freshly isolated mouse renal proximal tubules were incubated in the absence or presence of active human TGF-beta1. IRPTCs were also stably transfected with rat TGF-beta1 or p53 tumor suppressor protein (p53) cDNA in sense (S) and antisense (AS) orientations. ANG mRNA and p53 protein expression were assessed by reverse transcription-polymerase chain reaction and Western blotting, respectively. Reactive oxygen species (ROS) generation was quantified by lucigenin assay. Active TGF-beta1 evoked ROS generation and stimulated ANG mRNA and p53 protein expression, whereas a superoxide scavenger and inhibitors of nicotinamide adenine dinucleotide oxidase and p38 mitogen-activated protein kinase (p38 MAPK) abolished the TGF-beta1 effect. Stable transfer of p53 cDNA (S) enhanced and p53 cDNA (AS) abolished the stimulatory effect of TGF-beta1 on ANG mRNA expression in IRPTCs. Our results demonstrate that TGF-beta1 stimulates ANG gene expression and its action is mediated, at least in part, via ROS generation, p38 MAPK activation, and p53 expression, suggesting that angiotensin II and TGF-beta1 may form a positive feedback loop to enhance their respective gene expression, leading to renal injury.

RAS blockade decreases blood pressure and proteinuria in transgenic mice overexpressing rat angiotensinogen gene in the kidney.

Angiotensinogen (ANG) is the sole substrate of the renin-angiotensin system (RAS). Clinical studies have shown that RAS activation may lead to hypertension, a major cardiovascular and renal risk factor. To delineate the underlying mechanisms of hypertension-induced nephropathy, we generated transgenic mice that overexpress rat ANG (rANG) in the kidney to establish whether intrarenal RAS activation alone can evoke hypertension and kidney damage and whether RAS blockade can reverse these effects. Transgenic mice overexpressing renal rANG were generated by employing the kidney-specific, androgen-regulated protein promoter linked to rANG cDNA. This promoter targets rANG cDNA to renal proximal tubules and responds to androgen stimulation. Transgenic mice displayed kidney-specific expression of rANG, significantly increased blood pressure (BP) and albuminuria in comparison to non-transgenic littermates. Administration of losartan (an angiotensin II (type 1)-receptor antagonist) or perindopril (an angiotensin-converting enzyme inhibitor) reversed these abnormalities in transgenic animals. Renal injury was evident on examination of the kidneys in transgenic mice, and attenuated by losartan and perindopril treatment. We conclude that the overproduction of ANG alone in the kidney induces an increase in systemic BP, proteinuria, and renal injury. RAS blockers prevent these abnormalities. These data support the role of the intrarenal RAS in the development of hypertension and renal injury.

The angiotensin II type 1 receptor and receptor-associated proteins.

The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang II. Among the structures required for regulation and activation of the receptor, its carboxyl-terminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstream signaling pathway by G proteins and the Ang II signal transduction pathways leading to specific cellular responses are discussed. In addition, recent work on the identification and characterization of novel proteins associated with carboxyl-terminus of the AT1 receptor is presented. These novel proteins will advance our understanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors.

Increased gene expression of components of the renin-angiotensin system in glomeruli of genetically hypertensive rats.

OBJECTIVE: The renin-angiotensin system (RAS) is implicated in the development of hypertensive glomerulosclerosis. However, no experimental evidence exists that clearly demonstrates activation of glomerular RAS in hypertensive nephropathy. We used stroke-prone spontaneously hypertensive rats (SHRSP) to examine whether RAS components are increased in glomeruli of SHRSP and whether this increase leads to an increase in mRNA levels for transforming growth factor-beta1 (TGF-beta1). METHODS: We examined the sequential changes of urinary albumin excretion (UAE), morphology, and glomerular mRNA expression for TGF-beta1 and fibronectin (FN) in relation to glomerular mRNA expression for angiotensinogen (ATN), angiotensin converting enzyme (ACE), angiotensin II type 1a (AT1a), and type 1b (AT1b) receptors, and intervention with angiotensin II type 1 receptor antagonist candesartan and equihypotensive hydralazine. RESULTS: In SHRSP, UAE was normal at 9 weeks of age, but became higher, beginning at 12 weeks of age, than that in the age-matched Wistar-Kyoto (WKY) rats, while SHRSP showed no glomerulosclerosis until 14 weeks of age; it was marked at 24 weeks. Plasma renin activity and plasma angiotensin II level was equivalent in the 9- and 12-week-old SHRSP and the WKY rats; both parameters, however, were elevated in 24-week-old SHRSP as compared with age-matched control. RNase protection assays showed that glomerular levels of ATN, ACE, and AT1a and AT1b receptors mRNA were significantly increased in 9-, 12-, and 14-week-old, but not in 24-week-old SHRSP, compared with age-matched WKY rats. Northern blot analysis showed that glomerular levels of TGF-beta1 and FN mRNA were higher in SHRSP than in WKY rats at all time points. Candesartan reduced UAE to control levels, whereas hydralazine reduced UAE but not to control levels. Candesartan administration for 12 weeks virtually prevented the progression of glomerulosclerosis. While candesartan reduced mRNA levels for RAS components, TGF-beta1, and FN to control levels, hydralazine was not effective in this respect. Conclusion Results suggest that increases in glomerular RAS components that occur independently of circulating RAS alter glomerular permselectivity and increase the glomerular expression of TGF-beta1 and FN in young SHRSP. Findings in old SHRSP suggest that altered glomerular permselectivity and an increased glomerular expression of TGF-beta1 and FN may be associated with the activation of systemic RAS.

HCaRG, a novel calcium-regulated gene coding for a nuclear protein, is potentially involved in the regulation of cell proliferation.

Since a negative calcium balance is present in spontaneously hypertensive rats, we searched for the gene(s) involved in this dysregulation. A cDNA library was constructed from the spontaneously hypertensive rat parathyroid gland, which is a key regulator of serum-ionized calcium. From seven overlapping DNA fragments, a 1100-base pair novel cDNA containing an open reading frame of 224 codons was reconstituted. This novel gene, named HCaRG (hypertension-related, calcium-regulated gene), was negatively regulated by extracellular calcium concentration, and its basal mRNA levels were higher in hypertensive animals. The deduced protein showed no transmembrane domain, 67% alpha-helix content, a mutated calcium-binding site (EF-hand motif), four putative "leucine zipper" motifs, and a nuclear receptor-binding domain. At the subcellular level, HCaRG had a nuclear localization. We cloned the human homolog of this gene. Sequence comparison revealed 80% homology between rats and humans at the nucleotide and amino acid sequences. Tissue distribution showed a preponderance in the heart, stomach, jejunum, kidney (tubular fraction), liver, and adrenal gland (mainly in the medulla). HCaRG mRNA was significantly more expressed in adult than in fetal organs, and its levels were decreased in tumors and cancerous cell lines. We observed that after 60-min ischemia followed by reperfusion, HCaRG mRNA declined rapidly in contrast with an increase in c-myc mRNA. Its levels then rose steadily to exceed base line at 48 h of reperfusion. HEK293 cells stably transfected with HCaRG exhibited much lower proliferation, as shown by cell count and [(3)H]thymidine incorporation. Taken together, our results suggest that HCaRG is a nuclear protein potentially involved in the control of cell proliferation.

Inhibition of the expression of the gene for the angiotensin AT1 receptor by angiotensin II in the rat adrenal gland.

The expression of angiotensin AT1A and AT1B receptor mRNA after continuous angiotensin II administration was investigated in the rat adrenal gland. Angiotensin AT1 receptor mRNA detected by Northern blot analysis decreased to 52.7+/-16.1% of control after the administration of angiotensin II (20 microg/h) for 24 h, and to 70.8+/-8.0% after 1 week. A low dose of angiotensin II (0.2 microg/h) also decreased angiotensin AT1 receptor mRNA to 73.0+/-5.5% after 1 week. Competitive reverse transcription and polymerase chain reaction (RT-PCR) experiments revealed that both angiotensin AT1A and AT1B receptor mRNAs decreased after administration of angiotensin II (20 or 0.2 microg/h) for 1 week. Analysis of the angiotensin AT1A promoter by using luciferase-reporter system showed that angiotensin II (up to 1 microM) did not have any effects on the promoter activity (106+/-5.7% after 0.1 microM angiotensin II stimulation) in Y1 cells and cultured vascular smooth muscle cells, although phorbol myristate acetate (PMA) decreased the promoter activity by about 40% compared with control. These results suggest that angiotensin AT1 receptor gene expression in the rat adrenal gland is inhibited by angiotensin II and it may not be due to suppression of promoter activity. Other mechanisms such as destabilization of angiotensin AT1 receptor mRNA or angiotensin II-induced increased blood pressure may be involved in the inhibition.

Role of cytoplasmic tail of the type 1A angiotensin II receptor in agonist- and phorbol ester-induced desensitization.

To investigate mechanisms underlying the agonist-induced desensitization of the type 1A angiotensin II receptor (AT1A-R), we have stably expressed in Chinese hamster ovary (CHO) cells the wild-type receptor and truncated mutants lacking varying lengths of the cytoplasmic tail. Assay of inositol 1,4,5-trisphosphate (IP3) formation in response to agonist demonstrated that the truncated mutants T318, T328, and T348 lacking the last 42, 32, or 12 amino acid residues, respectively, couple with Gq protein with an efficiency similar to that of full-length receptors, whereas coupling of Gq protein was abolished in the T310 truncated mutant devoid of the carboxyl-terminal 50 amino acids. Exposure of CHO/AT1A-R cells expressing the wild-type AT1A-R to angiotensin II resulted in rapid and dose-dependent homologous desensitization of receptor-mediated IP3 formation, which was independent of the receptor internalization. Mastoparan, an activator of G protein-coupled receptor kinase (GRK), induced desensitization of the AT1A-R. The agonist-induced desensitization of the receptor was largely prevented by heparin, a potent inhibitor of GRK, whereas it was only partially attenuated by a protein kinase C (PKC)-specific inhibitor. The homologous or heterologous desensitization of the receptor was greatly impaired in the truncated mutants T318 and T328, lacking the Ser/Thr-rich (13 or 12 Ser/Thr residues) cytoplasmic tail of the AT1A-R. Deletion of the last two Ser residues, including one PKC consensus site in the receptor tail, prevented only phorbol 12-myristate 13-acetate-induced desensitization by 30%. Moreover, we found an agonist-induced translocation of a heparin-sensitive kinase activity. The angiotensin II-stimulated heparin-sensitive kinase could phosphorylate a thioredoxin fusion protein containing the entire AT1A-R cytoplasmic tail (N295 to E359), which lacks consensus phosphorylation sites for GRK1, GRK2, and GRK3. The heparin-sensitive kinase may not be GRK2, GRK3, or GRK6 expressed in CHO/AT1A-R cells, since angiotensin II did not induce translocation of these receptor kinases. Potential Ser/Thr phosphorylation sites located between S328 and S347 in the cytoplasmic tail of AT1A-R seem to play a critical role in the heterologous and homologous desensitization of the receptor. A heparin-sensitive kinase other than GRK2, GRK3, or GRK6 may be involved in the agonist-induced homologous desensitization of the AT1A-R.

Interaction of the adaptor protein Shc and the adhesion molecule cadherin.

In mitogenic signaling pathways, Shc participates in the growth factor activation of Ras by interacting with activated receptors and/or the Grb-2.Sos complex. Using several experimental approaches we demonstrate that Shc, through its SH2 domain, forms a complex with the cytoplasmic domain of cadherin, a transmembrane protein involved in the Ca2+-dependent regulation of cell-cell adhesion. This interaction is demonstrated in a yeast two-hybrid assay, by co-precipitation from mammalian cells, and by direct biochemical analysis in vitro. The Shc-cadherin association is phosphotyrosine-dependent and is abrogated by addition of epidermal growth factor to A-431 cells maintained in Ca2+-free medium, a condition that promotes changes in cell shape. Shc may therefore participate in the control of cell-cell adhesion as well as mitogenic signaling through Ras.

Candesartan prevents the progression of glomerulosclerosis in genetic hypertensive rats.

The renin-angiotensin system (RAS) has been implicated in the development of hypertensive glomerulosclerosis. However, there are no experimental findings clearly demonstrating activation of glomerular RAS in hypertensive nephropathy. Using the stroke-prone spontaneously hypertensive rat (SHRSP) as an animal model of hypertensive glomerulosclerosis, we examined the relationship between the sequential changes in urinary albumin excretion (UAE), renal morphology, and glomerular mRNA expression for transforming growth factor-beta (TGF-beta) and fibronectin (FN) and glomerular mRNA levels for RAS components, and determined the effects of the angiotensin II (Ang II) type 1 (AT-1) receptor antagonist (candesartan) and equihypotensive hydralazine on these parameters. In SHRSP, UAE was normal at nine weeks of age and increased by 12 weeks. Plasma renin activity, plasma Ang II concentration, and angiotensin converting enzyme (ACE) activity were not higher in 9- and 12-week-old SHRSP than in WKY. RNase protection assay revealed higher glomerular mRNA levels for angiotensinogen, ACE, and AT-1a and AT-1b receptors in 9-, 12-, and 14-week-old SHRSP than in WKY. The glomerular mRNA levels for TGF-beta and FN in SHRSP were increased from nine weeks of age. SHRSP had a greater glomerulosclerosis index (GSI) at 24 weeks of age than did WKY. Administration of candesartan for two weeks, but not of hydralazine, markedly reduced UAE and normalized mRNA levels for TGF-beta, FN, and RAS components. Candesartan administration for 12 weeks virtually prevented the progression of glomerulosclerosis in rats. We conclude that in SHRSP, RAS activation and increased sensitivity to Ang II in glomeruli play important roles in the progression of glomerulosclerosis.

Regulation of ANG II-receptor subtype and its gene expression in adrenal gland.

We have previously demonstrated that two isoforms (AT1A and AT1B) of the angiotensin II (ANG II) type 1 (AT1) receptor exist in the rat kidney and are differentially regulated by a low-sodium diet. The present experiment was designed to test the hypothesis that sodium deficiency upregulates AT1A and AT1B gene expression in the adrenal gland by activating the AT1 receptor. Wistar rats (7 wk old) were divided into four groups (n = 10 each) and fed normal sodium (0.5%; NS), NS plus 3 mg.kg-1.day-1 losartan (DUP-753; i.e., DUP), low sodium (0.07%; LS), and LS plus DUP. After 2 wks, body weight and mean arterial pressure were not different (P > 0.05). Northern blot analysis showed that the ratio of AT1A: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA in the adrenal gland was increased (P < 0.001) by 172% in LS but was unchanged in NS + DUP and LS + DUP vs. NS. The ratio of adrenal AT1B:GAPDH mRNA was increased (P < 0.001) by 245% in LS and unchanged in NS + DUP and LS + DUP vs. NS. Radioligand binding indicated that AT1 receptors (fmol/mg protein) in the adrenal gland were increased in LS (141 +/- 17; P < 0.001) vs. NS (54 +/- 3), NS + DUP (43 +/- 5), and LS + DUP (56 +/- 6). We conclude that sodium deficiency increases both AT1A and AT1B gene expression and elevates the AT1 receptor density in the adrenal gland. Blockade of the binding of ANG II to the AT1 receptor by losartan prevents the increases in AT1A and AT1B mRNA expression and the AT1 receptor density induced by sodium depletion, suggesting that these changes in the adrenal gland are mediated by activation of the AT1 receptor. These results will provide a basis for future experiments to further elucidate transcriptional regulation or functional activity of each of the receptor subtypes.

Identification of a cis-acting glucocorticoid responsive element in the rat angiotensin II type 1A promoter.

Enhanced vascular responsiveness to angiotensin II at the AT1 receptor has been considered one of the major contributing factors to vascular hypertrophy and high blood pressure. The transcription of the rat angiotensin II type 1A receptor gene is stimulated by glucocorticoids. To clarify the molecular mechanism for glucocorticoid action in rat vascular smooth muscle cells, we investigated the effects of dexamethasone on the promoter activity of the angiotensin II type 1A receptor by using promoter/luciferase reporter gene constructs and heterologous context constructs (containing the thymidine kinase promoter) in transfected vascular smooth muscle cells (< 12 passages). There are three putative glucocorticoid responsive elements (GREs) in the promoter. However, only one GRE was found to respond to dexamethasone (1 mumol/L) and was located at positions -756 to -770 bp upstream from the transcription initiation site. When compared with the consensus sequence of GRE, 9 of 12 bases were identical. RU38486, a glucocorticoid antagonist, completely blocked the induction by dexamethasone, suggesting that the GRE was functional through a specific glucocorticoid receptor. The response to dexamethasone was lost in vascular smooth muscle cells at higher passage numbers (> 8 passages) but was restored when the cells were transfected with a glucocorticoid-receptor expression construct. This finding provided additional support that the response to dexamethasone was mediated by the glucocorticoid receptor. The gel mobility supershift assay showed that the GRE binds in vitro-translated rat glucocorticoid receptors in a specific manner. Compared with the angiotensin II type 1A receptor promoter, no effect by dexamethasone was observed in vascular smooth muscle cells transfected with the angiotensin II type 1B receptor promoter/luciferase reporter gene constructs.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenesis and the molecular modeling of the rat angiotensin II receptor (AT1).

The molecular interaction involved in the ligand binding of the rat angiotensin II receptor (AT1A) was studied by site-directed mutagenesis and receptor model building. The three-dimensional structure of AT1A was constructed on the basis of a multiple amino acid sequence alignment of seven transmembrane domain receptors and angiotensin II receptors and after the beta 2 adrenergic receptor model built on the template of the bacteriorhodopsin structure. These data indicated that there are conserved residues that are actively involved in the receptor-ligand interaction. Eleven conserved residues in AT1, His166, Arg167, Glu173, His183, Glu185, Lys199, Trp253, His256, Phe259, Thr260, and Asp263, were targeted individually for site-directed mutation to Ala. Using COS-7 cells transiently expressing these mutated receptors, we found that the binding of angiotensin II was not affected in three of the mutations in the second extracellular loop, whereas the ligand binding affinity was greatly reduced in mutants Lys199-->Ala, Trp253-->Ala, Phe259-->Ala, Asp263-->Ala, and Arg167-->Ala. These amino acid residues appeared to provide binding sites for Ang II. The molecular modeling provided useful structural information for the peptide hormone receptor AT1A. Binding of EXP985, a nonpeptide angiotensin II antagonist, was found to be involved with Arg167 but not Lys199.

Steroid hormones upregulate rat angiotensin II type 1A receptor gene: role of glucocorticoid responsive elements in rat angiotensin II type 1A promoter.

The transcription of the rat angiotensin II type 1A receptor gene is stimulated by glucocorticoids. To clarify the molecular mechanism for glucocorticoid action in rat vascular smooth muscle cells, we investigated the effects of dexamethasone on the promoter activity of the angiotensin II type 1A receptor by using promoter/luciferase reporter gene constructs and heterologous context constructs (containing the thymidine kinase promoter) in transfected vascular smooth muscle cells. There are three putative glucocorticoid responsive elements in the promoter. However, only one glucocorticoid responsive element was found to respond to dexamethasone (1 microM). The region was located at positions, -756 to -770 bp upstream of the transcription initiation site. A glucocorticoid antagonist, RU38486, completely blocked the induction by dexamethasone, suggesting that the glucocorticoid responsive element was functional through a specific glucocorticoid receptor. Compared with the angiotensin II type 1A receptor promoter, no effect by dexamethasone was observed in vascular smooth muscle cells transfected with the angiotensin II type 1B receptor promoter/luciferase reporter gene constructs. We concluded that the dexamethasone-induced increase in the transcription of the angiotensin II type 1A receptor gene occurred through the binding to GRE up the glucocorticoid-specific receptor.

Localization of the genes encoding the three rat angiotensin II receptors, Agtr1a, Agtr1b, Agtr2, and the human AGTR2 receptor respectively to rat chromosomes 17q12, 2q24 and Xq34, and the human Xq22.

Using fluorescence in situ hybridization, we determined the regional localization of the 3 rat genes encoding angiotensin II receptors at 17q12 (Agtr1a), 2q24 (Agtr1b) and Xq34 (Agtr2). In parallel, we showed that the type 2 human gene, AGTR2, also maps on the X chromosome, at band Xq22.

Cloning, expression and regulation of angiotensin II receptors.

Complementary DNAs for angiotensin II type 1 receptor isoforms AT1A and AT1B were cloned by expression cloning from bovine adrenal and rat vascular smooth muscles. Human AT1 receptor was also cloned. Seven transmembrane structures emerged. The AT1 type receptor interacted with more than one type of G-proteins. The ligand binding site of AT1 involving Arg167, Lys199, and Asp263 has been identified by site directed mutagenesis. The regulation of the receptors occur at many stages. The isoform, AT2, was also expression cloned from rat pheochromocytoma cells. Although its ligand binding is not affected by stable GTP analogs, it is a seven transmembrane domain receptor. It mediates the modulations of phosphotyrosine phosphatase by angiotensin II and AT2 specific CGP42112A. The modulation was abolished by pertussis toxin. Thus, AT2 belongs to a new class of angiotensin receptors with unique signalling and regulatory mechanisms. AT1 mediates cellular growth. Interestingly, AT2 expression is inversely related to the mitogenic activity of cells.

Cloning, expression and regulation of angiotensin II receptors.

Angiotensin II isoform 1 (AT1) receptor cDNAs were cloned by expression cloning from bovine adrenal and rat vascular smooth muscles. Human AT1 receptor was also cloned. Seven transmembrane structures emerged. A single type of receptor seems to interact with more than one type of G-protein. AT1 consists of subtypes AT1A and AT1B, and the regulation of the receptors occurs at many stages. The isoform AT2 was also expression cloned from rat pheochromocytoma cells. Although its ligand binding is not affected by GTP analogs, it is a seven transmembrane domain receptor. It mediates the inhibition of phosphotyrosine phosphatase by angiotensin II and AT2 specific CGP42112A; the inhibition was abolished by pertussis toxin. Thus, AT2 belongs to a new class of angiotensin receptors with unique signalling and regulatory mechanisms.

Role of carboxyl tail of the rat angiotensin II type 1A receptor in agonist-induced internalization of the receptor.

Binding of angiotensin II (Ang II) to its receptor type 1A (AT1A) is known to trigger its internalization. We studied the role of cytosolic segments of AT1A in the internalization, and obtained results indicating a functional role of the cytosolic carboxyl terminal tail of AT1A in the internalization. Deletion of 50 amino acids from the carboxyl terminus abolished the receptor internalization. Deletion mutants lacking 13 and 32 amino acid residues in the carboxyl terminal cytosolic region were internalized to the same extent as wild type AT1A; however, internalization of a mutant lacking the last 42 residues was partially suppressed. Thus, residues 310 through 327 were shown to be essential for the internalization. We propose that a short domain in the cytoplasmic tail (residues 310 to 327) may play a dominant role in the agonist-induced receptor internalization of AT1A. Our results also suggest that the molecular determinants of the AT1A receptor involved in receptor internalization are distinct from those participating in the desensitization process.

Molecular biology of angiotensin II receptors: an overview.

ISOFORMS OF ANGIOTENSIN II RECEPTORS: So far, three isoforms of angiotensin II receptors have been identified by complementary DNA cloning, all with seven transmembrane domain structures. AT1A and AT1B are the most common isoforms. They are coupled to phospholipase C through Gq/G11 proteins and to a calcium channel, and negatively coupled to adenyl cyclase. AT2 is only remotely related to the AT1 family. KNOWN STRUCTURAL DETAILS OF ANGIOTENSIN II RECEPTORS: Ligand-binding domains are being defined in the space surrounded by transmembrane helices. Coupling to Gq seems to involve the second cytosolic loop. Receptor proteins undergo transition to a low-affinity form, which is desensitized and internalized. CHROMOSOME LOCATION: In the rat, AT1A, AT1B and AT2 are located on chromosomes 17, 2 and X, respectively. SIGNALING PATHWAY: Studies with receptors are revealing several different pathways of angiotensin signaling that modulate protein tyrosine phopsphorylation.