Design of Super-arrestins for Gene Therapy of Diseases Associated with Excessive Signaling of G Protein-Coupled Receptors
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Numerous congenital disorders, ranging from night blindness to cancer, are associated with signaling by overactive GPCR mutants. Excessive GPCR signaling also underlies certain acquired pathological conditions. The coupling of most GPCRs to their cognate G proteins is stopped by the natural desensitization mechanism, which includes receptor phosphorylation by GRKs followed by arrestin binding to active phosphoreceptor. Elucidation of the molecular mechanisms of arrestin binding to GPCRs enabled the construction of enhanced arrestins that bind active receptors with higher affinity and even interact with unphosphorylated GPCRs. These mutants have therapeutic potential due to their ability to compensate, reducing excessive activity of mutant and normal GPCRs. The feasibility of this compensational approach was demonstrated in rod photoreceptors predominantly expressing only one receptor, rhodopsin, and visual arrestin-1. Mammals have two nonvisual arrestins, which are fairly promiscuous, interacting with hundreds of GPCR subtypes. Identification of arrestin elements responsible for their receptor preference enables the construction of nonvisual arrestins specifically targeting particular receptors. This allows the construction of enhanced receptor-specific mutants for targeted suppression of signaling by one GPCR, but not others expressed by the same cell. Modification of elements responsible for the interactions with non-receptor partners creates signaling-biased arrestins that direct the signaling to the pathways of choice, further expanding potential therapeutic uses of "designer" arrestins.Keywords:
Arrestin
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G protein-coupled receptors (GPCRs) can be activated by multiple ligands and exhibit the capacity to couple to numerous intracellular signal transduction pathways. This property allows GPCRs to be modulated by biased agonists that selectively activate specific subsets of GPCR-regulated cellular signaling proteins. The angiotensin II type 1 receptor (AT1R) is a GPCR that endogenously binds to the peptide ligand angiotensin II. More recently it has been demonstrated that a modified peptide, [Sar1I-le4-Ile8]-angiotensin II (SII) acts as a biased agonist towards the AT1R. SII binds to the AT1R without promoting heterotrimeric G protein-coupling, but serves to link the receptor to the beta-arrestin-dependent activation of the mitogen activated protein kinase pathway. The present mini-review summarizes current knowledge regarding the role of biased agonists in stimulating biased AT1R signaling. Keywords: Angiotensin II, SII, biased agonism, GPCR, G-protein, phosphorylation, GRK6-dependent, Cardiovascular Disease, Mechanical Stretch, phosphoinositol-4
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Rhodopsin-like receptors
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Heptahelical, G protein-coupled or seven transmembrane-spanning receptors, such as the β-adrenergic and the angiotensin II type 1 receptors, are the most diverse and therapeutically important family of receptors in the human genome, playing major roles in the physiology of various organs/tissues including the heart and blood vessels. Ligand binding activates heterotrimeric G proteins that transmit intracellular signals by regulating effector enzymes or ion channels. G protein signaling is terminated, in large part, by phosphorylation of the agonist-bound receptor by the G-protein coupled receptor kinases (GRKs), followed by βarrestin binding, which uncouples the phosphorylated receptor from the G protein and subsequently targets the receptor for internalization. As the receptor-βarrestin complex enters the cell, βarrestin-1 and -2, the two mammalian βarrestin isoforms, serve as ligand-regulated scaffolds that recruit a host of intracellular proteins and signal transducers, thus promoting their own wave of signal transduction independently of G-proteins. A constantly increasing number of studies over the past several years have begun to uncover specific roles played by these ubiquitously expressed receptor adapter proteins in signal transduction of several important heptahelical receptors regulating the physiology of various organs/ systems, including the cardiovascular (CV) system. Thus, βarrestin-dependent signaling has increasingly been implicated in CV physiology and pathology, presenting several exciting opportunities for therapeutic intervention in the treatment of CV disorders. Additionally, the discovery of this novel mode of heptahelical receptor signaling via βarrestins has prompted a revision of classical pharmacological concepts such as receptor agonism/antagonism, as well as introduction of new terms such as "biased signaling", which refers to ligand-specific activation of selective signal transduction pathways by the very same receptor. The present review gives an overview of the current knowledge in the field of βarrestin-dependent signaling, with a specific focus on CV heptahelical receptor βarrestin-mediated signaling and on "biased" CV heptahelical receptor ligands that promote or inhibit it. Exciting new possibilities for cardiovascular therapeutics arising from the delineation of this βarrestin-dependent signaling are also discussed.
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Rhodopsin-like receptors
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Numerous congenital disorders, ranging from night blindness to cancer, are associated with signaling by overactive GPCR mutants. Excessive GPCR signaling also underlies certain acquired pathological conditions. The coupling of most GPCRs to their cognate G proteins is stopped by the natural desensitization mechanism, which includes receptor phosphorylation by GRKs followed by arrestin binding to active phosphoreceptor. Elucidation of the molecular mechanisms of arrestin binding to GPCRs enabled the construction of enhanced arrestins that bind active receptors with higher affinity and even interact with unphosphorylated GPCRs. These mutants have therapeutic potential due to their ability to compensate, reducing excessive activity of mutant and normal GPCRs. The feasibility of this compensational approach was demonstrated in rod photoreceptors predominantly expressing only one receptor, rhodopsin, and visual arrestin-1. Mammals have two nonvisual arrestins, which are fairly promiscuous, interacting with hundreds of GPCR subtypes. Identification of arrestin elements responsible for their receptor preference enables the construction of nonvisual arrestins specifically targeting particular receptors. This allows the construction of enhanced receptor-specific mutants for targeted suppression of signaling by one GPCR, but not others expressed by the same cell. Modification of elements responsible for the interactions with non-receptor partners creates signaling-biased arrestins that direct the signaling to the pathways of choice, further expanding potential therapeutic uses of "designer" arrestins.
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The β1 and β2 adrenergic receptors (βARs) are the predominant G-protein-coupled receptors (GPCRs) subtypes expressed in the heart. It is now appreciated that ligands can induce multiple distinct “active” receptor conformations with unique downstream functional signaling profiles or efficacies. Our current understanding of GPCR signaling is that ligands can be biased toward activating either a G protein or a β-arrestin-signaling pathway, a concept known as biased ligand signaling. To identify novel biased signaling pathways mediated by β1- and β2ARs, we performed a proteomic interactome study of β1AR and β2AR using stable isotope labeling by amino acids in cell culture (SILAC). We identified several hundred proteins that distinctly bind to the β1AR or β2AR following stimulation with the unbiased full agonist, isoproterenol, or the β-arrestin-biased ligand carvedilol. We found that stimulation by the β-arrestin-biased agonist carvedilol of only β1ARs resulted in the recruitment of Gαi. No of the other ligand tested promoted Gαi recruitment, suggesting that carvedilol may be unique in its ability to activate Gαi-biased signaling. The Gαi inhibitor pertussis toxin blocked β-arrestin-dependent extracellular signal-regulated kinase (ERK) activation and epidermal growth factor receptor (EGFR) transactivation stimulated by carvedilol, suggesting the involvement of Gαi in carvedilol-induced β-arrestin biased signaling of β1ARs. Using β1AR/β2AR chimeric receptors we show that the C-terminal tail of the β1AR is required for carvedilol stimulated Gαi recruitment, but is unable to rescue the lack of Gαi recruitment by the β2AR. Since our current conceptual framework for biased signaling is based on the “bar code hypothesis”, ongoing phosphoproteomic experiments are testing whether recruitment of Gαi by carvedilol induces a distinct phosphorylation pattern on the c-tail of the β1AR. Our study shows that activation of β-arrestin-biased signaling requires G proteins for signaling and provides a new mechanistic understanding of how biased β-blockers can activate signaling.
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Abstract The β 1 adrenergic receptor (β 1 AR) is recognized as a classical Gα s -coupled receptor. Agonist binding not only initiates G protein-mediated signaling but also signaling through the multifunctional adapter protein β-arrestin. Some βAR ligands, such as carvedilol, stimulate βAR signaling preferentially through β-arrestin, a concept known as β-arrestin-biased agonism. Here, we identify a signaling mechanism, unlike that previously known for any Gα s -coupled receptor, whereby carvedilol induces the transition of the β 1 AR from a classical Gα s -coupled receptor to a Gα i -coupled receptor stabilizing a distinct receptor conformation to initiate β-arrestin-mediated signaling. Recruitment of Gα i is not induced by any other βAR ligand screened, nor is it required for β-arrestin-bias activated by the β 2 AR subtype of the βAR family. Our findings demonstrate a previously unrecognized role for Gα i in β 1 AR signaling and suggest that the concept of β-arrestin-bias may need to be refined to incorporate the selective bias of receptors towards distinct G protein subtypes.
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