The Interaction of a Constitutively Active Arrestin with the Arrestin-Insensitive 5-HT2A Receptor Induces Agonist-Independent Internalization (original) (raw)
Related papers
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.
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.
Agonist-Receptor-Arrestin, an Alternative Ternary Complex with High Agonist Affinity
Journal of Biological Chemistry, 1997
The rapid decrease of a response to a persistent stimulus, often termed desensitization, is a widespread biological phenomenon. Signal transduction by numerous G protein-coupled receptors appears to be terminated by a strikingly uniform two-step mechanism, most extensively characterized for the  2-adrenergic receptor ( 2 AR), m2 muscarinic cholinergic receptor (m2 mAChR), and rhodopsin. The model predicts that activated receptor is initially phosphorylated and then tightly binds an arrestin protein that effectively blocks further G protein interaction. Here we report that complexes of  2 AR-arrestin and m2 mAChR-arrestin have a higher affinity for agonists (but not antagonists) than do receptors not complexed with arrestin. The percentage of phosphorylated  2 AR in this high affinity state in the presence of full agonists varied with different arrestins and was enhanced by selective mutations in arrestins. The percentage of high affinity sites also was proportional to the intrinsic activity of an agonist, and the coefficient of proportionality varies for different arrestin proteins. Certain mutant arrestins can form these high affinity complexes with unphosphorylated receptors. Mutations that enhance formation of the agonistreceptor-arrestin complexes should provide useful tools for manipulating both the efficiency of signaling and rate and specificity of receptor internalization.
Cellular signalling, 2017
Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M2 muscarinic receptor, so that agonist activation of the M2 did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M2, whereas its interactions with other receptors, including the β2-adrenergic receptor and the D1 and D2 dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-...
Journal of Biological Chemistry, 2008
Homologous desensitization of  2 -adrenergic and other G-protein-coupled receptors is a two-step process. After phosphorylation of agonist-occupied receptors by G-protein-coupled receptor kinases, they bind -arrestins, which triggers desensitization and internalization of the receptors. Because it is not known which regions of the receptor are recognized by -arrestins, we have investigated -arrestin interaction and internalization of a set of mutants of the human  2 -adrenergic receptor. Mutation of the four serine/threonine residues between residues 355 and 364 led to the loss of agonist-induced receptor--arrestin2 interaction as revealed by fluorescence resonance energy transfer (FRET), translocation of -arrestin2 to the plasma membrane, and receptor internalization. Mutation of all seven serine/threonine residues distal to residue 381 did not affect agonist-induced receptor internalization and -arrestin2 translocation. A  2 -adrenergic receptor truncated distal to residue 381 interacted normally with -arrestin2, whereas its ability to internalize in an agonist-dependent manner was compromised. A similar impairment of internalization was observed when only the last eight residues of the C terminus were deleted. Our experiments show that the C terminus distal to residue 381 does not affect the initial interaction between receptor and -arrestin, but its last eight amino acids facilitate receptor internalization in concert with -arrestin2.
Journal of Neurochemistry, 2008
Understanding the precise structure and function of the intracellular domains of G protein-coupled receptors is essential for understanding how receptors are regulated, and how they transduce their signals from the extracellular milieu to intracellular sites. To understand better the structure and function of the intracellular domain of the 5-hydroxytryptamine 2A (5-HT 2A ) receptor, a model G ␣q -coupled receptor, we overexpressed and purified to homogeneity the entire third intracellular loop (i3) of the 5-HT 2A receptor, a region previously implicated in G-protein coupling. Circular dichroism spectroscopy of the purified i3 protein was consistent with ␣-helical and -loop, -turn, and -sheet structure. Using random peptide phage libraries, we identified several arrestin-like sequences as i3-interacting peptides. We subsequently found that all three known arrestins (-arrestin, arrestin-3, and visual arrestin) bound specifically to fusion proteins encoding the i3 loop of the 5-HT 2A receptor. Competition binding studies with synthetic and recombinant peptides showed that the middle portion of the i3 loop, and not the extreme N and C termini, was likely to be involved in i3-arrestin interactions. Dual-label immunofluorescence confocal microscopic studies of rat cortex indicated that many cortical pyramidal neurons coexpressed arrestins (-arrestin or arrestin-3) and 5-HT 2A receptors, particularly in intracellular vesicles. Our results demonstrate (a) that the i3 loop of the 5-HT 2A receptor represents a structurally ordered domain composed of ␣-helical and -loop, -turn, and -sheet regions, (b) that this loop interacts with arrestins in vitro, and is hence active, and (c) that arrestins are colocalized with 5-HT 2A receptors in vivo. Key Words: -Arrestin-Structure-function-5-HT 2A receptor-Receptor-effector coupling.
Journal of Biological Chemistry, 2012
Background: WT non-visual arrestins are promiscuous, binding numerous GPCRs. Results: Mutations of very few receptor discriminator residues greatly increase receptor specificity of arrestin-3. Conclusion: Targeted manipulation of key residues that determine receptor preference is a viable approach to the construction of arrestins with high specificity for particular GPCR subtypes. Significance: Non-visual arrestins with high receptor specificity make therapeutic use of signaling-biased arrestin mutants feasible. Based on the identification of residues that determine receptor selectivity of arrestins and the analysis of the evolution in the arrestin family, we introduced 10 mutations of "receptor discriminator" residues in arrestin-3. The recruitment of these mutants to M2 muscarinic (M2R), D1 (D1R) and D2 (D2R) dopamine, and  2-adrenergic receptors ( 2 AR) was assessed using bioluminescence resonance energy transfer-based assays in cells. Seven of 10 mutations differentially affected arrestin-3 binding to individual receptors. D260K and Q262P reduced the binding to  2 AR, much more than to other receptors. The combination D260K/Q262P virtually eliminated  2 AR binding while preserving the interactions with M2R, D1R, and D2R. Conversely, Y239T enhanced arrestin-3 binding to  2 AR and reduced the binding to M2R, D1R, and D2R, whereas Q256Y selectively reduced recruitment to D2R. The Y239T/Q256Y combination virtually eliminated the binding to D2R and reduced the binding to  2 AR and M2R, yielding a mutant with high selectivity for D1R. Eleven of 12 mutations significantly changed the binding to light-activated phosphorhodopsin. Thus, manipulation of key residues on the receptor-binding surface modifies receptor preference, enabling the construction of non-visual arrestins specific for particular receptor subtypes. These findings pave the way to the construction of signalingbiased arrestins targeting the receptor of choice for research or therapeutic purposes. G protein-coupled receptors (GPCRs) 2 are the largest and the most functionally and structurally diverse family of signal-ing proteins in mammals (1, 2). Different species have from 800 to Ͼ3,400 GPCR subtypes encoded by 3-10% of their genes (SEVENS database, available on the Computational Biology Research Center Web site). Different GPCRs respond to a wide variety of stimuli, from light, small molecules, and extracellular calcium to peptide and protein hormones and extracellular protease activity. Active receptors sequentially activate multiple G protein molecules, amplifying the signal. This process is stopped when G protein-coupled receptor kinases selectively phosphorylate activated receptors (3) and arrestins bind to active phosphoreceptor (4), blocking further G protein coupling by steric exclusion (5, 6). Mammals have only seven G protein-coupled receptor kinases that serve hundreds of different GPCRs (7, 8), whereas the complement of arrestins is even smaller, only four subtypes (9). Arrestin-1 3 and-4 are specifically expressed in photoreceptor cells and quench light-induced signaling by rhodopsin and cone opsins (10, 11). In contrast, arrestin-2 and-3 are expressed in virtually every cell in the body and regulate the great majority of GPCRs (10). In most cells, including mature neurons that express the highest levels of non-visual arrestins, arrestin-2 outnumbers arrestin-3 by ϳ10-20:1 (12, 13). Thus, evolution produced arrestins with high receptor specificity, such as arrestin-1 with high preference for rhodopsin (14-16), along with fairly promiscuous non-visual arrestins (16, 17). Several genetic disorders are associated with mutations in GPCRs (18). In the case of loss-of-function mutations, gene replacement therapy introducing a functional version of the affected protein is the most logical therapeutic approach. However, the treatment of patients with gain-of-function mutations requires a different strategy; these mutations are dominant, which means that the other perfectly normal allele does not help. Excessive GPCR activity can be dampened by enhanced arrestins with preactivating mutations in vitro (19), in Xenopus
Receptor-Arrestin Interactions: The GPCR Perspective
Biomolecules
Arrestins are a small family of four proteins in most vertebrates that bind hundreds of different G protein-coupled receptors (GPCRs). Arrestin binding to a GPCR has at least three functions: precluding further receptor coupling to G proteins, facilitating receptor internalization, and initiating distinct arrestin-mediated signaling. The molecular mechanism of arrestin–GPCR interactions has been extensively studied and discussed from the “arrestin perspective”, focusing on the roles of arrestin elements in receptor binding. Here, we discuss this phenomenon from the “receptor perspective”, focusing on the receptor elements involved in arrestin binding and emphasizing existing gaps in our knowledge that need to be filled. It is vitally important to understand the role of receptor elements in arrestin activation and how the interaction of each of these elements with arrestin contributes to the latter’s transition to the high-affinity binding state. A more precise knowledge of the molec...