Agonist-dependent internalization of the angiotensin II type one receptor (AT1): role of C-terminus phosphorylation in recruitment of β-arrestins (original) (raw)
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British Journal of Pharmacology, 2010
The angiotensin II type 1 (AT1) receptor belongs to family A of 7 transmembrane (7TM) receptors. The receptor has important roles in the cardiovascular system and is commonly used as a drug target in cardiovascular diseases. Interaction of 7TM receptors with G proteins or b-arrestins often induces higher binding affinity for agonists. Here, we examined interactions between AT1A receptors and b-arrestins to look for differences between the AT1A receptor interaction with b-arrestin1 and b-arrestin2.
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.
β-Arrestin- and Dynamin-Dependent Endocytosis of the AT1Angiotensin Receptor
Molecular Pharmacology, 2001
The major mechanism of agonist-induced internalization of G protein-coupled receptors (GPCRs) is -arrestinand dynamindependent endocytosis via clathrin-coated vesicles. However, recent reports have suggested that some GPCRs, exemplified by the AT 1 angiotensin receptor expressed in human embryonic kidney (HEK) 293 cells, are internalized by a -arrestinand dynamin-independent mechanism, and possibly via a clathrin-independent pathway. In this study, agonist-induced endocytosis of the rat AT 1A receptor expressed in Chinese hamster ovary (CHO) cells was abolished by clathrin depletion during treatment with hyperosmotic sucrose and was unaffected by inhibition of endocytosis via caveolae with filipin. In addition, internalized fluorescein-conjugated angiotensin II appeared in endosomes, as demonstrated by colocalization with transferrin. Overexpression of -arrestin1(V53D) and -arres-tin1(1-349) exerted dominant negative inhibitory effects on the endocytosis of radioiodinated angiotensin II in CHO cells. GTPase-deficient (K44A) mutant forms of dynamin-1 and dynamin-2, and a pleckstrin homology domain-mutant (K535A) dynamin-2 with impaired phosphoinositide binding, also inhibited the endocytosis of AT 1 receptors in CHO cells. Similar results were obtained in COS-7 and HEK 293 cells. Confocal microscopy using fluorescein-conjugated angiotensin II showed that overexpression of dynamin-1(K44A) and dynamin-2(K44A) isoforms likewise inhibited agonist-induced AT 1 receptor endocytosis in CHO cells. Studies on the angiotensin II concentration-dependence of AT 1 receptor endocytosis showed that at higher agonist concentrations its rate constant was reduced and the inhibitory effects of dominant negative dynamin constructs were abolished. These data demonstrate the importance of -arrestinand dynamin-dependent endocytosis of the AT 1 receptor via clathrin-coated vesicles at physiological angiotensin II concentrations. The pressor octapeptide hormone, angiotensin II (Ang II), exerts the majority of its physiological effects on cardiovascular regulation and saltwater balance by activating the G q-coupled AT 1 angiotensin receptor (De Gasparo et al., 2000). The AT 1 receptor also activates intracellular signaling pathways that stimulate cell growth including activation of tyrosine kinases and small GTP-binding proteins (Berk, 1999; De Gasparo et al., 2000), and is rapidly internalized after Ang II binding (Thomas, 1999; Hunyady et al., 2000). Agonist-induced endocytosis of G protein-coupled receptors (GPCRs) initiates a process by which desensitized receptors are resensitized and recycled to the plasma membrane (Krupnick and Benovic, 1998). Sequestration of the  2-adrenergic receptor has been shown to require the binding of -arrestin proteins to its cytoplasmic tail after agonist-induced activation and phosphorylation by G protein-coupled receptor kinases (Zhang et al., 1996; Ferguson et al., 1997; Krupnick and Benovic, 1998). -arrestins direct the phosphorylated receptors to clathrin-coated pits and induce the formation of clathrin-coated vesicles (Goodman et al., 1996). The role of -arrestins in receptor internalization has been demonstrated for several GPCRs (Bü nemann et al., 1999). Although -arrestins translocate to the plasma membrane upon agonist stimulation of many GPCRs (Zhang et al., 1999), it has been reported that the internalization of some of
Journal of Biological Chemistry, 2003
The vast majority of G protein-coupled receptors are desensitized by a uniform two-step mechanism: phosphorylation of an active receptor followed by arrestin binding. The arrestin⅐receptor complex is then internalized. Internalized receptor can be recycled back to the plasma membrane (resensitization) or targeted to lysosomes for degradation (down-regulation). The intracellular compartment where this choice is made and the molecular mechanisms involved are largely unknown. Here we used two arrestin2 mutants that bind with high affinity to phosphorylated and unphosphorylated agonist-activated 2-adrenergic receptor to manipulate the receptor-arrestin interface. We found that mutants support rapid internalization of 2-adrenergic receptor similar to wild type arrestin2. At the same time, phosphorylation-independent arrestin2 mutants facilitate receptor recycling and sharply reduce the rate of receptor loss, effectively protecting 2-adrenergic receptor from down-regulation even after very long (up to 24 h) agonist exposure. Phosphorylation-independent arres-tin2 mutants dramatically reduce receptor phosphorylation in response to an agonist both in vitro and in cells. Interestingly, co-expression of high levels of -adrenergic receptor kinase restores receptor down-regulation in the presence of mutants to the levels observed with wild type arrestin2. Our data suggest that unphosphorylated receptor internalized in complex with mutant arrestins recycles faster than phosphoreceptor and is less likely to get degraded. Thus, targeted manipulation of the characteristics of an arrestin protein that binds to a G protein-coupled receptors can dramatically change receptor trafficking and its ultimate fate in a cell.
Molecular Pharmacology, 2003
5-HT 2A serotonin receptors are unusual among G-protein coupled receptors in that they can be internalized and desensitized, in some cell types, in an arrestin-independent manner. The molecular basis of the arrestin-insensitivity of 5-HT 2A receptors is unknown but is probably caused, in part, by the apparent lack of agonist-induced 5-HT 2A receptor phosphorylation. Because the arrestin-insensitivity of 5-HT 2A receptors is cell-type selective, we used a "constitutively active" arrestin mutant that can interact with agonist-activated but nonphosphorylated receptors. We show here that this "constitutively active" arrestin mutant (Arr2-R169E) can force 5-HT 2A receptors to be regulated by arrestins. Cotransfection of 5-HT 2A receptors with Arr2-R169E induced agonist-independent 5-HT 2A receptor internalization, and a constitutive translocation of the Arr2-R169E mutant to the plasma membrane, whereas wild-type Arrestin-2 had no effect. Additionally, Arr2-R169E, unlike wild-type arrestin-2, induced a significant decrease in efficacy of agonist-induced phosphoinositide hydrolysis with an unexpected increase in agonist potency. Radioligand binding assays demonstrated that the fraction of receptors in the high-affinity agonist binding-state increased with expression of Arr2-R169E, indicating that Arr2-R169E stabilizes the agonisthigh affinity state of the 5-HT 2A receptor (R*). Intriguingly, the agonist-independent interaction of Arr2-R169E with 5-HT 2A receptors was inhibited by inverse agonist treatment and is thus probably caused by the high level of 5-HT 2A receptor constitutive activity. This is the first demonstration that a constitutively active arrestin mutant can both induce agonist-independent internalization and stabilize the agonist-high affinity state of an arrestin-insensitive G protein coupled receptor.
Role of Arrestins in Endocytosis and Signaling of alpha 2-Adrenergic Receptor Subtypes
Journal of Biological Chemistry, 1999
We investigated the role of arrestins in the trafficking of human ␣ 2 -adrenergic receptors (␣ 2 -ARs) and the effect of receptor trafficking on p42/p44 MAP kinase activation. ␣ 2 -ARs expressed in COS-1 cells demonstrated a modest level of agonist-mediated internalization, with ␣ 2c > ␣ 2b > ␣ 2a . However, upon coexpression of arrestin-2 (-arrestin-1) or arrestin-3 (-arrestin-2), internalization of the ␣ 2b AR was dramatically enhanced and redistribution of receptors to clathrin coated vesicles and endosomes was observed. Internalization of the ␣ 2c AR was selectively promoted by coexpression of arrestin-3, while ␣ 2a AR internalization was only slightly stimulated by coexpression of either arrestin. Coexpression of GRK2 had no effect on the internalization of any ␣ 2 -AR subtype, either in the presence or absence of arrestins. Internalization of the ␣ 2b and ␣ 2c ARs was inhibited by coexpression of dominant negative dynamin-K44A. However, ␣ 2 -AR-mediated activation of either endogenous or cotransfected p42/p44 mitogen-activated protein (MAP) kinase was not affected by either dynamin-K44A or arrestin-3. Moreover, activation of p42/ p44 MAP kinase by endogenous epidermal growth factor, lysophosphatidic acid, and  2 -adrenergic receptors was also unaltered by dynamin-K44A. In summary, our data suggest that internalization of the ␣ 2b , ␣ 2c , and to a lesser extent ␣ 2a ARs, is both arrestin-and dynamin-dependent. However, endocytosis does not appear to be required for ␣ 2 -adrenergic, epidermal growth factor, lysophosphatidic acid, or  2 -adrenergic receptor-mediated p42/p44 MAP kinase activation in COS-1 cells.
The Role of Arrestin α-Helix I in Receptor Binding
Journal of Molecular Biology, 2010
Arrestins rapidly bind phosphorylated activated forms of their cognate G protein-coupled receptors (GPCRs), thereby preventing G protein coupling and often switching the signaling to other pathways. Amphipathic α-helix I (residues 100-111) has been implicated in receptor binding, but the mechanism of its action is not yet determined. Here we show that several mutations in the helix itself and adjacent hydrophobic residues in the body of the N-domain reduce arrestin1 binding to phosphorylated light-activated rhodopsin (PRh*). On the background of phosphorylationindependent mutants that bind with high affinity to both P-Rh* and light-activated unphosphorylated rhodopsin (Rh*), these mutations reduce the stability of the arrestin complex with P-Rh*, but not with Rh*. Using site-directed spin labeling we found that the local structure around α helix I changes upon binding to rhodopsin. However, the intra-molecular distances between α-helix I and adjacent α-strand I, or the rest of the N-domain, measured using double electron-electron resonance, do not change, ruling out relocation of the helix due to receptor binding. Collectively, these data demonstrate that α-helix I plays an indirect role in receptor binding, likely keeping α-strand I, carrying several phosphate-binding residues, in a position favorable for its interaction with receptor-attached phosphates.
Molecular Endocrinology, 2000
-Arrestins target G protein-coupled receptors (GPCRs) for endocytosis via clathrin-coated vesicles. -Arrestins also become detectable on endocytic vesicles in response to angiotensin II type 1A receptor (AT 1A R), but not  2-adrenergic receptor ( 2 AR), activation. The carboxyl-terminal tails of these receptors contribute directly to this phenotype, since a  2 AR bearing the AT 1A R tail acquired the capacity to stimulate -arrestin redistribution to endosomes, whereas this property was lost for an AT 1A R bearing the  2 AR tail. Using  2 AR/AT 1A R chimeras, we tested whether the  2 AR and AT 1A R carboxyl-terminal tails, in part via their association with -arrestins, might regulate differences in the intracellular trafficking and resensitization patterns of these receptors. In the present study, we find that -arrestin formed a stable complex with the AT 1A R tail in endocytic vesicles and that the internalization of this complex was dynamin dependent. Internalization of the  2 AR chimera bearing the AT 1A R tail was observed in the absence of agonist and was inhibited by a dominant-negative -arrestin1 mutant. Agonist-independent AT 1A R internalization was also observed after -arrestin2 overexpression. After internalization, the  2 AR, but not the AT 1A R, was dephosphorylated and recycled back to the cell surface. However, the AT 1A R tail prevented  2 AR dephosphorylation and recycling. In contrast, although the  2 AR-tail promoted AT 1A R 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.
Journal of Biological Chemistry, 2002
Nonvisual arrestins (arrestin-2 and-3) serve as adaptors to link agonist-activated G protein-coupled receptors to the endocytic machinery. Although many G protein-coupled receptors bind arrestins, the molecular determinants involved in binding remain largely unknown. Because arrestins selectively promote the internalization of the ␣ 2b-and ␣ 2c-adrenergic receptors (ARs) while having no effect on the ␣ 2a AR, here we used ␣ 2 ARs to identify molecular determinants involved in arrestin binding. Initially, we assessed the ability of purified arrestins to bind glutathione S-transferase fusions containing the third intracellular loops of the ␣ 2a AR, ␣ 2b AR, or ␣ 2c AR. These studies revealed that arrestin-3 directly binds to the ␣ 2b AR and ␣ 2c AR but not the ␣ 2a AR, whereas arrestin-2 only binds to the ␣ 2b AR. Truncation mutagenesis of the ␣ 2b AR identified two arrestin-3 binding domains in the third intracellular loop, one at the N-terminal end (residues 194-214) and the other at the C-terminal end (residues 344-368). Site-directed mutagenesis further revealed a critical role for several basic residues in arrestin-3 binding to the ␣ 2b AR third intracellular loop. Mutation of these residues in the holo-␣ 2b AR and subsequent expression in HEK 293 cells revealed that the mutations had no effect on the ability of the receptor to activate ERK1/2. However, agonist-promoted internalization of the mutant ␣ 2b AR was significantly attenuated as compared with wild type receptor. These results demonstrate that arrestin-3 binds to two discrete regions within the ␣ 2b AR third intracellular loop and that disruption of arrestin binding selectively abrogates agonist-promoted receptor internalization.