β-Arrestin 1 and 2 stabilize the angiotensin II type I receptor in distinct high-affinity conformations (original) (raw)
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Differential β-arrestin binding of AT1and AT2angiotensin receptors
FEBS Letters, 2005
Agonist stimulation of G protein-coupled receptors causes receptor activation, phosphorylation, b-arrestin binding and receptor internalization. Angiotensin II (AngII) causes rapid internalization of the AT 1 receptors, whereas AngII-bound AT 2 receptors do not internalize. Although the activation of the rat AT 1A receptor with AngII causes translocation of b-arrestin2 to the receptor, no association of this molecule with the AT 2 receptor can be detected after AngII treatment with confocal microscopy or bioluminescence resonance energy transfer. These data demonstrate that the two subtypes of angiotensin receptors have different mechanisms of regulation.
Regulatory Peptides, 2004
h-Arrestins play a role in AT 1 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 h-arrestin to the activated receptor are poorly defined. In this study, we used CHO-K1 and HEK 293 cells expressing wild-type or mutant AT 1 to investigate two possibilities: activated AT 1 induces global relocation of h-arrestins to the plasma membrane or the phosphorylated C-terminus acts as bait to attract h-arrestins. Results obtained using high osmolarity and dominant-negative h-arrestin confirmed that internalization of AT 1 in both CHO-K1 and HEK 293 cells is predominately via clathrin-mediated endocytosis involving h-arrestin, and substitution of T332, S335, T336 and S338 with alanine to preclude phosphorylation markedly attenuated AT 1 internalization. Confocal microscopy revealed that wild-type AT 1 induced a timedependent translocation of GFP-tagged h-arrestins 1 and 2 to the cell surface. In contrast, the TSTS/A mutant did not traffic h-arrestin 1 at all, and only trafficked h-arrestin 2 weakly. Results of rescue-type experiments were consistent with the idea that both h-arrestins are able to interact with the non-phosphorylated receptor, albeit with much lower affinity and h-arrestin 1 less so than h-arrestin 2. In conclusion, this study shows that the high affinity binding of h-arrestins to the phosphorylated C-terminus is the predominant mechanism of agonist-induced h-arrestin recruitment to the cell surface and AT 1 receptor. D
Journal of Biological Chemistry, 2011
The angiotensin II peptide analog [Sar1,Ile4,Ile8]AngII (SII) is a biased AT1A receptor agonist that stimulates receptor phosphorylation, β-arrestin recruitment, receptor internalization, and β-arrestin-dependent ERK1/2 activation without activating heterotrimeric G-proteins. To determine the scope of G-protein-independent AT1A receptor signaling, we performed a gel-based phosphoproteomic analysis of AngII and SII-induced signaling in HEK cells stably expressing AT1A receptors. A total of 34 differentially phosphorylated proteins were detected, of which 16 were unique to SII and eight to AngII stimulation. MALDI-TOF/TOF mass fingerprinting was employed to identify 24 SII-sensitive phosphoprotein spots, of which three (two peptide inhibitors of protein phosphatase 2A (I1PP2A and I2PP2A) and prostaglandin E synthase 3 (PGES3)) were selected for validation and further study. We found that phosphorylation of I2PP2A was associated with rapid and transient inhibition of a β-arrestin 2-ass...
Biased Signaling of the Angiotensin II Type 1 Receptor Can Be Mediated through Distinct Mechanisms
2010
Background: Seven transmembrane receptors (7TMRs) can adopt different active conformations facilitating a selective activation of either G protein or b-arrestin-dependent signaling pathways. This represents an opportunity for development of novel therapeutics targeting selective biological effects of a given receptor. Several studies on pathway separation have been performed, many of these on the Angiotensin II type 1 receptor (AT1R). It has been shown that certain ligands or mutations facilitate internalization and/or recruitment of b-arrestins without activation of G proteins. However, the underlying molecular mechanisms remain largely unresolved. For instance, it is unclear whether such selective G proteinuncoupling is caused by a lack of ability to interact with G proteins or rather by an increased ability of the receptor to recruit b-arrestins. Since uncoupling of G proteins by increased ability to recruit b-arrestins could lead to different cellular or in vivo outcomes than lack of ability to interact with G proteins, it is essential to distinguish between these two mechanisms.
Science Signaling, 2012
The angiotensin type 1 receptor (AT1R) and its octapeptide ligand, angiotensin II (AngII) engages multiple downstream signaling pathways. Ligand-dependent AT1R functional selectivity has unveiled G protein-and βarrestin-dependent signaling. Here, we examine AT1R-mediated Gα q and βarrestin signaling using multiple AngII analogs bearing substitutions at the critical position Phe 8. Using assays that discriminate between ligand-promoted recruitment of βarrestin to the AT1R and its resulting conformational rearrangement and downstream signaling events, we extend the concept of biased signaling to include the analogs propensity to differentially promote βarrestin conformational changes. The relative contributions of GRK2 and GRK6 were also found to vary among analogs indicating a role for the kinases in these βarrestin responses. We demonstrate that the efficacy of AngII analogs at activating ERK1/2 correlated with the stability of the complexes between βarrestin and AT1R in endosomes, and not with the extent of βarrestin recruitment to the receptor, supporting the importance of the nature of this complex towards selective signaling modes. In vascular smooth muscle cells, we establish a link between βarrestin's conformational change promoted by ligands and their effect on βarrestin-dependent migration or proliferation responses. Our data reveal the existence of novel modalities of biased signaling at the AT1R and βarrestin level, and suggest that different AngII analogs selectively engage distinct βarrestin conformations leading to specific signaling events and cell responses.
Journal of Biological Chemistry, 2013
Background: Hemodynamic regulation involves extensive cross-talk between the renin-angiotensin and kallikrein-kinin systems. Results: In vascular smooth muscle, "biased" AT 1 agonists inhibit both AT 1 and B 2 signaling by internalizing AT 1-B 2 heterodimers. Conclusion: AT 1 antagonists and arrestin-selective biased AT 1 agonists have opposing effects on B 2 signaling. Significance: Negative allosteric modulation of B 2 signaling by biased AT 1 agonists may impact their clinical utility. The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT 1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B 1 and B 2 bradykinin receptors. The reninangiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT 1-B 2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT 1 agonist, [Sar 1 ,Ile 4 ,Ile 8 ]-An-gII, but not the neutral AT 1 antagonist, losartan, inhibits endogenous B 2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar 1 ,Ile 4 , Ile 8 ]-AngII require the AT 1 receptor and result from arrestin-dependent co-internalization of AT 1-B 2 heterodimers. BRET 50 measurements indicate that AT 1 and B 2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar 1 ,Ile 4 ,Ile 8 ]-AngII blunts B 2 receptor activation of G q/11-dependent intracellular calcium influx and G i/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar 1 ,Ile 4 ,Ile 8 ]-AngII has no effect on B 2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar 1 ,Ile 4 ,Ile 8 ]-AngII promotes internalization of AT 1-B 2 heterodimers. Thus, [Sar 1 ,Ile 4 ,Ile 8 ]-AngII exerts lateral allosteric modulation of B 2 receptor signaling by binding to the orthosteric ligand binding site of the AT 1 receptor and promoting co-sequestration of AT 1-B 2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT 1 receptor ligands may translate into different pharmacologic actions. The renin-angiotensin system (RAS) 2 is a critical regulator of vascular tone and volume homeostasis and an important therapeutic target in hypertension and congestive heart failure (1). In response to a drop in systemic blood pressure, renin, a protease produced in the juxtaglomerular apparatus of the kidney, converts circulating angiotensinogen to angiotensin I, which is in turn cleaved by angiotensin converting enzyme (ACE) to generate the biologically active peptide, Ang[1-8] or angiotensin II (AngII). Most of the physiological effects of AngII are mediated by G protein-coupled type 1 angiotensin II (AT 1) receptors (2-4). AT 1 receptors couple primarily to G q/11-phospholipase C-protein kinase C (PKC) and G 12/13-Rho-GEF (guanine nucleotide exchange factor) signaling pathways. In vascular smooth muscle cells (VSMC), AT 1 receptors increase vascular tone by triggering phospholipase C-inositol-trisphosphate-dependent intracellular calcium release and calcium-dependent contraction. In the adrenal cortex, AngII stimulates production of aldosterone, a potent mineralocorticoid that promotes renal sodium retention to expand blood volume. Clinically, prolonged activation of the AT 1 receptor has been asso-* This work was supported, in whole or in part, by National Institutes of Health Grants R01 HL087986 and HL077192 (to A. A. J.), R01 DK55524 (to L. M. L.), and F30 DK083208 (to P. C. W.
Heteromerization of angiotensin receptors changes trafficking and arrestin recruitment profiles
Cellular Signalling, 2011
The cardiovascular hormone angiotensin II (AngII) exerts its actions via two G protein-coupled receptor (GPCR) subtypes, AT 1 and AT 2 , which often display antagonistic functions. Methodological constraints have so far precluded detailed analyses of the ligand-dependency, cellular localization, and functional relevance of AngII receptor interactions in live cells. In this study, we utilize a protein-fragment complementation assay (PCA) and GPCR-Heteromer Identification Technology (GPCR-HIT) to provide the first detailed investigation of the liganddependency and cellular localization of AngII receptor interactions in human embryonic kidney 293 cells. Fluorescent-tagged receptor constructs for PCA and GPCR-HIT displayed normal affinity and selectivity for AngII (AT 1 : IC 50 =1.0-1.6 nM; AT 2 : IC 50 =2.0-3.0 nM). Well-characterized angiotensin receptor interactions were used as positive and negative controls to demonstrate the sensitivity and specificity of these fluorescence-based assays. We report that AT 1 -AT 2 receptor heteromers form constitutively, are localized to the plasma membrane and perinuclear compartments, and do not internalize following AngII stimulation despite arrestin being recruited specifically to the heteromer. Our findings using novel fluorescence-based technologies reveal a previously unrecognized mechanism of angiotensin receptor cross-talk involving cross-inhibition of AT 1 receptor internalization through heteromerization with the AT 2 receptor subtype.
Decoding Angiotensin II Type 1 Receptor Allosteric Communication to Gq and β-arrestin
2021
The allosteric communication between the agonist binding site and the G protein or β-arrestin coupling sites in G protein-coupled receptors (GPCRs) play an important role in determining ligand efficacy towards these two signaling pathways and hence the ligand bias. Knowledge of the amino acid residue networks involved in the allosteric communication will aid understanding GPCR signaling and the design of biased ligands. Angiotensin II type I receptor (AT1R) is an ideal model GPCR to study the molecular basis of ligand bias as it has multiple β-arrestin2 and Gq protein biased agonists as well as three-dimensional structures. Using Molecular Dynamics simulations, dynamic allostery analysis, and functional BRET assays, we identified a network of residues involved in allosteric communication from the angiotensin II binding site to the putative Gq coupling sites and another network to the β-arrestin2 coupling sites, with 6 residues common to both pathways located in TM3, TM5 and TM6. Our...