Angiotensin II Type 1a Receptor Research Articles

Apparent loss-of-function mutant GPCRs revealed as constitutively desensitized receptors.

The DRY motif is a triplet amino acid sequence (aspartic acid, arginine, and tyrosine) that is highly conserved in G protein-coupled receptors (GPCRs). Recently, we have shown that a molecular determinant for nephrogenic diabetes insipidus, the vasopressin receptor with a substitution at the DRY motif arginine (V2R R137H), is a constitutively desensitized receptor that is unable to couple to G proteins due to its constitutive association with beta-arrestin [Barak, L. S. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 93-98]. Additionally, the mutant receptors are localized in endocytic vesicles, identical to wild-type receptors stimulated with agonist. In this study, we asked whether the constitutively desensitized phenotype observed in the V2R R137H represents a general paradigm that may be extended to other GPCRs. We show that arginine substitutions in the DRY motifs of the alpha(1B) adrenergic receptor (alpha(1B)-AR) and angiotensin II type 1A receptor (AT(1A)R) result in receptors that are uncoupled from G proteins, associated with beta-arrestins, and found localized in endocytic vesicles rather than at the plasma membrane in the absence of agonists. The localization of the alpha(1B)-ARs and AT(1A)Rs with arginine substitutions can be restored to the plasma membrane by either using selective antagonists or preventing the endocytosis of the beta-arrestin-receptor complexes. These results indicate that the arginine residue of the DRY motif is essential for preserving the localization of the inactive receptor complex. Furthermore, constitutive desensitization may underlie some loss-of-function receptor phenotypes and represent an unappreciated mechanism of hormonal resistance.

Beta-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking.

The two widely coexpressed isoforms of beta-arrestin (termed beta arrestin 1 and 2) are highly similar in amino acid sequence. The beta-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of beta-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the beta-arrestins (beta arr1-KO and beta arr2-KO) or both (beta arr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the beta(2)-adrenergic receptor (beta(2)-AR) and the angiotensin II type 1A receptor (AT(1A)-R). Both beta arr1-KO and beta arr2-KO cells showed similar impairment in agonist-stimulated beta(2)-AR and AT(1A)-R desensitization, when compared with their WT control cells, and the beta arr1/2-KO cells were even further impaired. Sequestration of the beta(2)-AR in the beta arr2-KO cells was compromised significantly (87% reduction), whereas in the beta arr1-KO cells it was not. Agonist-stimulated internalization of the AT(1A)-R was only slightly reduced in the beta arr1-KO but was unaffected in the beta arr2-KO cells. In the beta arr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two beta-arrestins to sequester the beta(2)-AR revealed beta-arrestin 2 to be 100-fold more potent than beta-arrestin 1. Down-regulation of the beta(2)-AR was also prevented in the beta arr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two beta-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.

Receptor/β-Arrestin Complex Formation and the Differential Trafficking and Resensitization ofβ2-Adrenergic and Angiotensin II Type 1A Receptors

Beta-arrestins target G protein-coupled receptors (GPCRs) for endocytosis via clathrin-coated vesicles. Beta-arrestins also become detectable on endocytic vesicles in response to angiotensin II type 1A receptor (AT1AR), but not beta2-adrenergic receptor (beta2AR), activation. The carboxyl-terminal tails of these receptors contribute directly to this phenotype, since a beta2AR bearing the AT1AR tail acquired the capacity to stimulate beta-arrestin redistribution to endosomes, whereas this property was lost for an AT1AR bearing the beta2AR tail. Using beta2AR/AT1AR chimeras, we tested whether the beta2AR and AT1AR carboxyl-terminal tails, in part via their association with beta-arrestins, might regulate differences in the intracellular trafficking and resensitization patterns of these receptors. In the present study, we find that beta-arrestin formed a stable complex with the AT1AR tail in endocytic vesicles and that the internalization of this complex was dynamin dependent. Internalization of the beta2AR chimera bearing the AT1AR tail was observed in the absence of agonist and was inhibited by a dominant-negative beta-arrestin1 mutant. Agonist-independent AT1AR internalization was also observed after beta-arrestin2 overexpression. After internalization, the beta2AR, but not the AT1AR, was dephosphorylated and recycled back to the cell surface. However, the AT1AR tail prevented beta2AR dephosphorylation and recycling. In contrast, although the beta2AR-tail promoted AT1AR recycling, the chimeric receptor remained both phosphorylated and desensitized, suggesting that receptor dephosphorylation is not a property common to all receptors. In summary, we show that the carboxyl-terminal tails of GPCRs not only contribute to regulating the patterns of receptor desensitization, but also modulate receptor intracellular trafficking and resensitization patterns.

Properties of Secretin Receptor Internalization Differ from Those of the β2-Adrenergic Receptor

The endocytic pathway of the secretin receptor, a class II GPCR, is unknown. Some class I G protein-coupled receptors (GPCRs), such as the beta(2)-adrenergic receptor (beta(2)-AR), internalize in clathrin-coated vesicles and this process is mediated by G protein-coupled receptor kinases (GRKs), beta-arrestin, and dynamin. However, other class I GPCRs, for example, the angiotensin II type 1A receptor (AT(1A)R), exhibit different internalization properties than the beta(2)-AR. The secretin receptor, a class II GPCR, is a GRK substrate, suggesting that like the beta(2)-AR, it may internalize via a beta-arrestin and dynamin directed process. In this paper we characterize the internalization of a wild-type and carboxyl-terminal (COOH-terminal) truncated secretin receptor using flow cytometry and fluorescence imaging, and compare the properties of secretin receptor internalization to that of the beta(2)-AR. In HEK 293 cells, sequestration of both the wild-type and COOH-terminal truncated secretin receptors was unaffected by GRK phosphorylation, whereas inhibition of cAMP-dependent protein kinase mediated phosphorylation markedly decreased sequestration. Addition of secretin to cells resulted in a rapid translocation of beta-arrestin to plasma membrane localized receptors; however, secretin receptor internalization was not reduced by expression of dominant negative beta-arrestin. Thus, like the AT(1A)R, secretin receptor internalization is not inhibited by reagents that interfere with clathrin-coated vesicle-mediated internalization and in accordance with these results, we show that secretin and AT(1A) receptors colocalize in endocytic vesicles. This study demonstrates that the ability of secretin receptor to undergo GRK phosphorylation and beta-arrestin binding is not sufficient to facilitate or mediate its internalization. These results suggest that other receptors may undergo endocytosis by mechanisms used by the secretin and AT(1A) receptors and that kinases other than GRKs may play a greater role in GPCR endocytosis than previously appreciated.

Cellular Trafficking of G Protein-coupled Receptor/β-Arrestin Endocytic Complexes

beta-Arrestins are multifunctional proteins identified on the basis of their ability to bind and uncouple G protein-coupled receptors (GPCR) from heterotrimeric G proteins. In addition, beta-arrestins play a central role in mediating GPCR endocytosis, a key regulatory step in receptor resensitization. In this study, we visualize the intracellular trafficking of beta-arrestin2 in response to activation of several distinct GPCRs including the beta2-adrenergic receptor (beta2AR), angiotensin II type 1A receptor (AT1AR), dopamine D1A receptor (D1AR), endothelin type A receptor (ETAR), and neurotensin receptor (NTR). Our results reveal that in response to beta2AR activation, beta-arrestin2 translocation to the plasma membrane shares the same pharmacological profile as described for receptor activation and sequestration, consistent with a role for beta-arrestin as the agonist-driven switch initiating receptor endocytosis. Whereas redistributed beta-arrestins are confined to the periphery of cells and do not traffic along with activated beta2AR, D1AR, and ETAR in endocytic vesicles, activation of AT1AR and NTR triggers a clear time-dependent redistribution of beta-arrestins to intracellular vesicular compartments where they colocalize with internalized receptors. Activation of a chimeric AT1AR with the beta2AR carboxyl-terminal tail results in a beta-arrestin membrane localization pattern similar to that observed in response to beta2AR activation. In contrast, the corresponding chimeric beta2AR with the AT1AR carboxyl-terminal tail gains the ability to translocate beta-arrestin to intracellular vesicles. These results demonstrate that the cellular trafficking of beta-arrestin proteins is differentially regulated by the activation of distinct GPCRs. Furthermore, they suggest that the carboxyl-tail of the receptors might be involved in determining the stability of receptor/betaarrestin complexes and cellular distribution of beta-arrestins.

Inhibition of adenosine-1 receptor-mediated preglomerular vasoconstriction in AT1A receptor-deficient mice.

The effect of the adenosine type 1 receptor agonist N6-cyclohexyladenosine (CHA) on glomerular vascular reactivity was studied in male angiotensin II type 1A (AT1A) receptor knockout mice (9). Vascular reactivity was assessed as the response of stop-flow pressure (PSF) to infusion of CHA into loops of Henle using micropuncture techniques. In AT1A +/+ mice at ambient arterial blood pressure (96.7 +/- 2.8 mmHg), the presence of CHA (10 (-5) M) in the perfusate increased PSF responses from 6.8 +/- 0.6 to 14.3 +/- 0.9 mmHg when the loop of Henle of the index nephron was perfused and from 0.7 +/- 0.3 to 12.3 +/- 1.0 mmHg when the loop of an adjacent nephron was perfused. At reduced arterial blood pressure (82.8 +/- 1. 3 mmHg), index nephron perfusion with CHA increased PSF responses from 4.5 +/- 0.3 to 9.4 +/- 0.4 mmHg. In AT1A -/- mice with a mean arterial blood pressure of 80 +/- 1.9 mmHg, CHA increased PSF responses only from 0.1 +/- 0.3 to 3.6 +/- 0.54 mmHg during index nephron perfusion and from 0.25 +/- 0.2 to 2.7 +/- 0.55 mmHg during adjacent nephron perfusion, significantly less than in wild-type animals (P < 0.001). Responses to CHA were intermediate in AT1A +/- mice. Thus AT1A receptor knockout mice show a markedly reduced constrictor response to CHA both in the presence and absence of simultaneous activation of the tubuloglomerular feedback system. These data support the notion of a functional interaction between adenosine and angiotensin II in the regulation of afferent arteriolar tone.

Angiotensin II maintains the structure and function of glomerular mesangium via type 1a receptor. What we have learned from null mutant mice minus the angiotensin II type 1a receptor gene.

The angiotensin II type 1a (AT1a) receptor is the major receptor effecting the multiple actions of angiotensin II on the cardiovascular system. It is expressed abundantly in the glomerular mesangial cells of the kidney. We investigated glomerular changes in null mutant mice minus the AT1a receptor gene to gain an understanding of the in vivo action of angiotensin II via AT1a on the mesangium. Morphological observations and morphometric analysis revealed that the glomerular volume was greatly increased owing to the expansion of the mesangial area, which contained fluid-filled spaces with a small amount of fibrillar components. The mesangial cells lost contact with each other and with the perimesangial area of the glomerular basement membrane (GBM), so that the glomerular capillary neck was greatly widened. These findings suggest a defect of the anchoring function of mesangial cells resulting from some abnormality in mesangial matrix formation. We conclude that angiotensin II has an important role in the structural and functional maintenance of the mesangium via the AT1a receptor, especially by reinforcing the connection between mesangial cells and GBM via the mesangial matrix.

Visualization of Dynamic Trafficking of a Protein Kinase C βII/Green Fluorescent Protein Conjugate Reveals Differences in G Protein-coupled Receptor Activation and Desensitization

Protein kinase C (PKC) links various extracellular signals to intracellular responses and is activated by diverse intracellular factors including diacylglycerol, Ca2+, and arachidonic acid. In this study, using a fully functional green fluorescent protein conjugated PKCbetaII (GFP-PKCbetaII), we demonstrate a novel approach to study the dynamic redistribution of PKC in live cells in response to G protein-coupled receptor activation. Agonist-induced PKC translocation was rapid, transient, and selectively mediated by the activation of Gqalpha- but not Gsalpha- or Gialpha-coupled receptors. Interestingly, although the stimuli were continuously present, only one brief peak of PKC membrane translocation was observed, consistent with rapid desensitization of the signaling pathway. Moreover, when GFP-PKCbetaII was used to examine cross-talk between two Gqalpha-coupled receptors, angiotensin II type 1A receptor (AT1AR) and endothelin A receptor (ETAR), activation of ETARs resulted in a subsequent loss of AT1AR responsiveness, whereas stimulation of AT1ARs did not cause desensitization of the ETAR signaling. The development of GFP-PKCbetaII has allowed not only the real time visualization of the dynamic PKC trafficking in live cells in response to physiological stimuli but has also provided a direct and sensitive means in the assessment of activation and desensitization of receptors implicated in the phospholipase C signaling pathway.

The C-terminal Third Intracellular Loop of the Rat AT1A Angiotensin Receptor Plays a Key Role in G Protein Coupling Specificity and Transduction of the Mitogenic Signal

To identify the role(s) of the third intracellular loop of the angiotensin II (AngII) type 1A (AT1A) receptor in G protein coupling specificity and receptor activation, several chimerae were constructed and characterized. The cDNA sequence encoding the C-terminal segment of the third intracellular loop of the AT1A receptor (residues 234-240) was replaced with the homologous regions of the alpha1B adrenergic (alpha1B-AR), the beta2 adrenergic (beta2-AR), and the AngII type 2 (AT2) receptors. These chimeric receptors were stably expressed in Chinese hamster ovary cells, and their pharmacological and functional properties were characterized, including AngII-induced inositol phosphate and cyclic AMP (cAMP) productions, [3H]thymidine incorporation into DNA, and internalization. The affinities of these chimeric receptors for [Sar1]AngII, [Sar1,Ile8]AngII, and losartan were essentially normal; however, the affinity of these mutants was increased by a factor of 10-40 for the AT2-specific ligand CGP42112A. The functional properties of the alpha1B-AR chimera were essentially identical to those of the wild type AT1A receptor. On the other hand, replacement with the beta2-AR segment produced a partial reduction of the inositol phosphate production, a measurable AngII-induced cAMP accumulation, a reduced internalization, and a total impairment to transduce the mitogenic effect of AngII. The AT2 chimera presented a normal internalization, but was inactive in all the other functional tests. In conclusion, the distal segment of the third intracellular loop of the rat AT1A receptor plays a pivotal role in coupling selectivity and receptor signaling via G protein(s) as well as in the activation of the specific signaling pathways involved in the mitogenic actions of AngII.

Angiotensin II type1a receptor gene expression in the heart: AP-1 and GATA-4 participate in the response to pressure overload.

Hypertrophy of mammalian cardiac muscle is mediated, in part, by angiotensin II through an angiotensin II type1a receptor (AT1aR)-dependent mechanism. To understand how the level of AT1aRs is altered in this pathological state, we studied the expression of an injected AT1aR promoter-luciferase reporter gene in adult rat hearts subjected to an acute pressure overload by aortic coarctation. This model was validated by demonstrating that coarctation increased expression of the alpha-skeletal actin promoter 1.7-fold whereas the alpha-myosin heavy chain promoter was unaffected. Pressure overload increased expression from the AT1aR promoter by 1. 6-fold compared with controls. Mutations introduced into consensus binding sites for AP-1 or GATA transcription factors abolished the pressure overload response but had no effect on AT1aR promoter activity in control animals. In extracts from coarcted hearts, but not from control hearts, a Fos-JunB-JunD complex and GATA-4 were detected in association with the AP-1 and GATA sites, respectively. These results establish that the AT1aR promoter is active in cardiac muscle and its expression is induced by pressure overload, and suggest that this response is mediated, in part, by a functional interaction between AP-1 and GATA-4 transcription factors.

Translational regulation of angiotensin II type 1A receptor. Role of upstream AUG triplets.

The cDNA sequence of rat angiotensin II type 1A receptor (AT1AR) shows that AT1AR transcripts have AUG triplets in the 5'-leader region that may begin a short open reading frame encoding an 11-amino acid peptide. In this study, the mutational inactivation of the start codon of the short open reading frame in AT1AR-chloramphenicol acetyltransferase (CAT) reporter gene constructs resulted in a 2.6-fold increase in CAT activity, whereas CAT transcript levels were not affected. Furthermore, experiments with rat AT1AR cDNA-transfected Cos-7 cells revealed that mutagenesis of the upstream AUG increased the AT1AR protein up to 2.5-fold, although AT1AR transcript levels showed no changes. The synthetic peptide corresponding to the sequence of the short open reading frame significantly suppressed the amount of AT1AR product in the in vitro translation system. The inhibiting effect of the short open reading frame appears to operate at least in part at the level of translation initiation, because polysome analysis with transfected Cos-7 cells showed that mutagenesis of the upstream AUG resulted in a shift of AT1AR mRNA distribution from a smaller to larger fraction of polysomes. Taken together, these results show that the upstream AUG inhibits translational regulation, suggesting that the short open reading frame in the 5'-leader region of AT1AR transcripts has a certain role in the translation of AT1AR protein.

Identification of a Negative Cis-regulatory Element and Trans-acting Protein That Inhibit Transcription of the Angiotensin II Type 1a Receptor Gene

The rat angiotensin II type 1a receptor (AT1a-R) gene is expressed in a cell-specific manner. We demonstrated that the negative regulatory element (NRE) between -489 and -331 is active in PC12 cells (Murasawa, S., Matsubara, H., Urakami, M., and Inada, M. (1993) J. Biol. Chem. 268, 26996-27003). Gel retardation assays confirmed that PC12 cells have a trans-acting factor bound to the NRE. By means of a DNase I footprint assay we identified the core of the NRE as an (A+T)-rich sequence (TAATCTTTTATTTTA) located at nucleotides -456 to -442. Oligonucleotides corresponding to the NRE core sequence bound to nuclear protein. Site-directed mutagenesis at nucleotides -451 to -448 eliminated the specific protein/DNA binding and restored expression of the AT1a-R in transient transfection assays (2.7-fold increase). The NRE did not negatively affect the thymidine kinase promoter. No homology was found with known NREs, suggesting that this is a novel NRE. Southwestern blotting revealed a 53-kDa, specific binding protein in PC12 cells and the rat brain, but not in the liver, spleen, adrenal gland, and kidney. These findings demonstrate that the NRE of the rat AT1a-R is an (A+T)-rich sequence located at nucleotides -456 to -442 and the 53-kDa protein is a specific binding protein, and suggest that this protein may be a trans-acting factor which determines the neuron-specific down-regulation of the AT1a-R gene.

Regulatory elements that mediate expression of the gene for the angiotensin II type 1a receptor for the rat.

To analyze the mechanism of the cell type-specific expression of rat angiotensin II type 1a receptor (AT1a-R) gene, we isolated the 5'-portion of the gene and identified multiple positive and negative regulatory sequences that regulate its transcription. Primer extension and S1 mapping identified a transcriptional initiation site at 33 (position +1) base pairs (bp) downstream of TATA sequence. The transcriptional activities of various 5'-deletion mutants of the AT1a-R gene upstream region, fused to the chloramphenicol acetyltransferase (CAT) gene, were examined using rat vascular smooth muscle cells (A10) and glial cells expressing AT1a-R mRNA predominantly or PC12 cells expressing AT2-R and very small amounts of AT1a-R mRNA. A 980-bp 5'-flanking sequence contained at least three positive elements, P1 (-560 to -489), P2 (-331 to -201), and P3 (-201 to -61). P1 and P3 were active in the tested three cells, and P2 was functional only in glial and PC12 cells. In addition to these positive elements, there was negative element, N1 (-489 to -331), which was active only in PC12 cells. These elements, when cotransfected with the AT1a-CAT fusion gene, had competitive effects against its promoter activity. The present study suggests the presence of multiple trans-acting factors that act on these positive and negative cis-acting elements and regulate the cell type-specific expression of the rat AT1a-R gene.