Small-molecule WNK inhibition regulates cardiovascular and renal function.

The With-No-Lysine (K) (WNK) kinases play a critical role in blood pressure regulation and body fluid and electrolyte homeostasis. Herein, we introduce the first orally bioavailable pan-WNK-kinase inhibitor, WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.

A stress response pathway regulates DNA damage through ß2-adrenoreceptors and ß-arrestin-1.

The human mind and body respond to stress, a state of perceived threat to homeostasis, by activating the sympathetic nervous system and secreting the catecholamines adrenaline and noradrenaline in the 'fight-or-flight' response. The stress response is generally transient because its accompanying effects (for example, immunosuppression, growth inhibition and enhanced catabolism) can be harmful in the long term. When chronic, the stress response can be associated with disease symptoms such as peptic ulcers or cardiovascular disorders, and epidemiological studies strongly indicate that chronic stress leads to DNA damage. This stress-induced DNA damage may promote ageing, tumorigenesis, neuropsychiatric conditions and miscarriages. However, the mechanisms by which these DNA-damage events occur in response to stress are unknown. The stress hormone adrenaline stimulates ß(2)-adrenoreceptors that are expressed throughout the body, including in germline cells and zygotic embryos. Activated ß(2)-adrenoreceptors promote Gs-protein-dependent activation of protein kinase A (PKA), followed by the recruitment of ß-arrestins, which desensitize G-protein signalling and function as signal transducers in their own right. Here we elucidate a molecular mechanism by which ß-adrenergic catecholamines, acting through both Gs-PKA and ß-arrestin-mediated signalling pathways, trigger DNA damage and suppress p53 levels respectively, thus synergistically leading to the accumulation of DNA damage. In mice and in human cell lines, ß-arrestin-1 (ARRB1), activated via ß(2)-adrenoreceptors, facilitates AKT-mediated activation of MDM2 and also promotes MDM2 binding to, and degradation of, p53, by acting as a molecular scaffold. Catecholamine-induced DNA damage is abrogated in Arrb1-knockout (Arrb1(-/-)) mice, which show preserved p53 levels in both the thymus, an organ that responds prominently to acute or chronic stress, and in the testes, in which paternal stress may affect the offspring's genome. Our results highlight the emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and reveal how DNA damage may accumulate in response to chronic stress.

S-nitrosylation of beta-arrestin regulates beta-adrenergic receptor trafficking.

Signal transduction through G protein-coupled receptors (GPCRs) is regulated by receptor desensitization and internalization that follow agonist stimulation. Nitric oxide (NO) can influence these processes, but the cellular source of NO bioactivity and the effects of NO on GPCR-mediated signal transduction are incompletely understood. Here, we show in cells and mice that beta-arrestin 2, a central element in GPCR trafficking, interacts with and is S-nitrosylated at a single cysteine by endothelial NO synthase (eNOS), and that S-nitrosylation of beta-arrestin 2 is promoted by endogenous S-nitrosogluthathione. S-nitrosylation after agonist stimulation of the beta-adrenergic receptor, a prototypical GPCR, dissociates eNOS from beta-arrestin 2 and promotes binding of beta-arrestin 2 to clathrin heavy chain/beta-adaptin, thereby accelerating receptor internalization. The agonist- and NO-dependent shift in the affiliations of beta-arrestin 2 is followed by denitrosylation. Thus, beta-arrestin subserves the functional coupling of eNOS and GPCRs, and dynamic S-nitrosylation/denitrosylation of beta-arrestin 2 regulates stimulus-induced GPCR trafficking.

Erythrocyte plasma membrane-bound ERK1/2 activation promotes ICAM-4-mediated sickle red cell adhesion to endothelium.

The core pathology of sickle cell disease (SCD) starts with the erythrocyte (RBC). Aberration in MAPK/ERK1/2 signaling, which can regulate cell adhesion, occurs in diverse pathologies. Because RBCs contain abundant ERK1/2, we predicted that ERK1/2 is functional in sickle (SS) RBCs and promotes adherence, a hallmark of SCD. ERK1/2 remained active in SS but not normal RBCs. ß(2)-adrenergic receptor stimulation by epinephrine can enhance ERK1/2 activity only in SS RBCs via PKA- and tyrosine kinase p72(syk)-dependent pathways. ERK signaling is implicated in RBC ICAM-4 phosphorylation, promoting SS RBC adhesion to the endothelium. SS RBC adhesion and phosphorylation of both ERK and ICAM-4 all decreased with continued cell exposure to epinephrine, implying that activation of ICAM-4-mediated SS RBC adhesion is temporally associated with ERK1/2 activation. Furthermore, recombinant ERK2 phosphorylated α- and ß-adducins and dematin at the ERK consensus motif. Cytoskeletal protein 4.1 also showed dynamic phosphorylation but not at the ERK consensus motif. These results demonstrate that ERK activation induces phosphorylation of cytoskeletal proteins and the adhesion molecule ICAM-4, promoting SS RBC adhesion to the endothelium. Thus, blocking RBC ERK1/2 activation, such as that promoted by catecholamine stress hormones, could ameliorate SCD pathophysiology.

Hemodynamic responses elicited by systemic injections of isotonic and hypertonic saline in hemorrhaged rats.

PURPOSE: The objectives of this study were (i) to characterize the hemodynamic responses caused by controlled hemorrhage (HEM) in pentobarbital-anesthetized rats, and (ii) to determine the responses elicited by systemic bolus injections of isotonic saline (0.15M) or hypertonic saline (3M) given 5min after completion of HEM. RESULTS: Controlled HEM (4.3±0.2ml/rat at 1.5ml/min) resulted in a pronounced and sustained fall in mean arterial blood pressure (MAP) to about 40mmHg. The fall in MAP was associated with a reduction in hindquarter vascular resistance (HQR) but no changes in renal (RR) or mesenteric (MR) vascular resistances. Systemic injections of isotonic saline (96-212µmol/kg i.v., in 250-550µl) did not produce immediate responses but promoted the recovery of MAP to levels below pre-HEM values. Systemic injections of hypertonic saline (750-3000µmol/kg, i.v., in 250-550µl) produced immediate and pronounced falls in MAP, RR, MR and especially HQR of 30-120s in duration. However, hypertonic saline prompted a full recovery of MAP, HQR and RR to pre-HEM levels and an increase in MR to levels above pre-HEM values. CONCLUSIONS: This study demonstrates that (i) HEM induced a pronounced fall in MAP which likely involved a fall in cardiac output and HQR, (ii) isotonic saline did not fully normalize MAP, and (iii) hypertonic saline produced dramatic initial responses, and promoted normalization of MAP probably by restoring blood volume and cardiac output through sequestration of fluid from intracellular compartments.

Selective engagement of G protein coupled receptor kinases (GRKs) encodes distinct functions of biased ligands.

CCL19 and CCL21 are endogenous agonists for the seven-transmembrane receptor CCR7. They are equally active in promoting G protein stimulation and chemotaxis. Yet, we find that they result in striking differences in activation of the G protein-coupled receptor kinase (GRK)/ss-arrestin system. CCL19 leads to robust CCR7 phosphorylation and beta-arrestin2 recruitment catalyzed by both GRK3 and GRK6 whereas CCL21 activates GRK6 alone. This differential GRK activation leads to distinct functional consequences. Although each ligand leads to beta-arrestin2 recruitment, only CCL19 leads to redistribution of beta-arrestin2-GFP into endocytic vesicles and classical receptor desensitization. In contrast, these agonists are both capable of signaling through GRK6 and beta-arrestin2 to ERK kinases. Thus, this mechanism for "ligand bias" whereby endogenous agonists activate different GRK isoforms leads to functionally distinct pools of beta-arrestin.

beta-arrestins: traffic cops of cell signaling.

Once thought to function only in the desensitization of seven membrane spanning receptors (7MSRs), the ubiquitous beta-arrestin molecules are increasingly appreciated to play important roles in the endocytosis and signaling of these receptors. These functions reflect the ability of the beta-arrestins to bind an ever-growing list of signaling and endocytic elements, often in an agonist-dependent fashion. One heavily studied system is that leading to MAP kinase activation via beta-arrestin-mediated scaffolding of these pathways in a receptor-dependent fashion. The beta-arrestins are also found to be involved in the regulation of novel receptor systems, such as Frizzled and TGFbeta receptors.

Beta-blockers alprenolol and carvedilol stimulate beta-arrestin-mediated EGFR transactivation.

Recent evidence suggests that binding of agonist to its cognate receptor initiates not only classical G protein-mediated signaling, but also beta-arrestin-dependent signaling. One such beta-arrestin-mediated pathway uses the beta(1)-adrenergic receptor (beta(1)AR) to transactivate the EGFR. To determine whether beta-adrenergic ligands that do not activate G protein signaling (i.e., beta-blockers) can stabilize the beta(1)AR in a signaling conformation, we screened 20 beta-blockers for their ability to stimulate beta-arrestin-mediated EGFR transactivation. Here we show that only alprenolol (Alp) and carvedilol (Car) induce beta(1)AR-mediated transactivation of the EGFR and downstream ERK activation. By using mutants of the beta(1)AR lacking G protein-coupled receptor kinase phosphorylation sites and siRNA directed against beta-arrestin, we show that Alp- and Car-stimulated EGFR transactivation requires beta(1)AR phosphorylation at consensus G protein-coupled receptor kinase sites and beta-arrestin recruitment to the ligand-occupied receptor. Moreover, pharmacological inhibition of Src and EGFR blocked Alp- and Car-stimulated EGFR transactivation. Our findings demonstrate that Alp and Car are ligands that not only act as classical receptor antagonists, but can also stimulate signaling pathways in a G protein-independent, beta-arrestin-dependent fashion.

Distinct conformational changes in beta-arrestin report biased agonism at seven-transmembrane receptors.

Beta-arrestins critically regulate G protein-coupled receptors (GPCRs), also known as seven-transmembrane receptors (7TMRs), both by inhibiting classical G protein signaling and by initiating distinct beta-arrestin-mediated signaling. The recent discovery of beta-arrestin-biased ligands and receptor mutants has allowed characterization of these independent "G protein-mediated" and "beta-arrestin-mediated" signaling mechanisms of 7TMRs. However, the molecular mechanisms underlying the dual functions of beta-arrestins remain unclear. Here, using an intramolecular BRET (bioluminescence resonance energy transfer)-based biosensor of beta-arrestin 2 and a combination of biased ligands and/or biased mutants of three different 7TMRs, we provide evidence that beta-arrestin can adopt multiple "active" conformations. Surprisingly, phosphorylation-deficient mutants of the receptors are also capable of directing similar conformational changes in beta-arrestin as is the wild-type receptor. This indicates that distinct receptor conformations induced and/or stabilized by different ligands can promote distinct and functionally specific conformations in beta-arrestin even in the absence of receptor phosphorylation. Our data thus highlight another interesting aspect of 7TMR signaling--i.e., functionally specific receptor conformations can be translated to downstream effectors such as beta-arrestins, thereby governing their functional specificity.

Selectively engaging ß-arrestins at the angiotensin II type 1 receptor reduces blood pressure and increases cardiac performance.

Biased G protein-coupled receptor ligands engage subsets of the receptor signals normally stimulated by unbiased agonists. However, it is unclear whether ligand bias can elicit differentiated pharmacology in vivo. Here, we describe the discovery of a potent, selective ß-arrestin biased ligand of the angiotensin II type 1 receptor. TRV120027 (Sar-Arg-Val-Tyr-Ile-His-Pro-D-Ala-OH) competitively antagonizes angiotensin II-stimulated G protein signaling, but stimulates ß-arrestin recruitment and activates several kinase pathways, including p42/44 mitogen-activated protein kinase, Src, and endothelial nitric-oxide synthase phosphorylation via ß-arrestin coupling. Consistent with ß-arrestin efficacy, and unlike unbiased antagonists, TRV120027 increased cardiomyocyte contractility in vitro. In rats, TRV120027 reduced mean arterial pressure, as did the unbiased antagonists losartan and telmisartan. However, unlike the unbiased antagonists, which decreased cardiac performance, TRV120027 increased cardiac performance and preserved cardiac stroke volume. These striking differences in vivo between unbiased and ß-arrestin biased ligands validate the use of biased ligands to selectively target specific receptor functions in drug discovery.

The central fibroblast growth factor receptor/beta klotho system: Comprehensive mapping in Mus musculus and comparisons to nonhuman primate and human samples using an automated in situ hybridization platform.

Central activation of fibroblast growth factor (FGF) receptors regulates peripheral glucose homeostasis and reduces food intake in preclinical models of obesity and diabetes. The current work was undertaken to advance our understanding of the receptor expression, as sites of ligand action by FGF19, FGF21, and FGF1 in the mammalian brain remains unresolved. Recent advances in automated RNAscope in situ hybridization and droplet digital PCR (ddPCR) technology allowed us to interrogate central FGFR/beta klotho (Klb) system at the cellular level in the mouse, with relevant comparisons to nonhuman primate and human brain. FGFR1-3 gene expression was broadly distributed throughout the CNS in Mus musculus, with FGFR1 exhibiting the greatest heterogeneity. FGFR4 expression localized only in the medial habenula and subcommissural organ of mice. Likewise, Klb mRNA was restricted to the suprachiasmatic nucleus (SCh) and select midbrain and hindbrain nuclei. ddPCR in the rodent hypothalamus confirmed that, although expression levels are indeed low for Klb, there is nonetheless a bonafide subpopulation of Klb+ cells in the hypothalamus. In NHP and human midbrain and hindbrain, Klb + cells are quite rare, as is expression of FGFR4. Collectively, these data provide the most robust central map of the FGFR/Klb system to date and highlight central regions that may be of critical importance to assess central ligand effects with pharmacological dosing, such as the putative interactions between the endocrine FGFs and FGFR1/Klb, or FGF19 with FGFR4.

Downregulation of propranolol-sensitive beta-adrenoceptor signaling after inhibition of nitric oxide synthesis.

The beta-adrenoceptor agonist, isoprenaline, elicits vasodilation and tachycardia in anesthetized rats via activation of propranolol-sensitive beta1- and beta2-adrenoceptors and also by propranolol-insensitive beta1- and beta3-adrenoceptors. The aim of this study was to determine whether the relative contribution of propranolol-sensitive and -insensitive beta-adrenoceptors to the changes in heart rate (HR) and vascular resistances elicited by isoprenaline is altered after blockade of nitric oxide (NO) synthase, in pentobarbital-anesthetized rats. The hemodynamic responses elicited by isoprenaline (0.1 and 0.5 microg kg(-1), i.v.) were determined before and after injection of saline or the NO synthase inhibitor, N(G)-nitro-L-arginine methylester (L-NAME, 50 micromol kg(-1), i.v.), and again after injection of the beta1- and beta2-adrenoceptor antagonist, propranolol (1 mg kg(-1), i.v.). The responses elicited by the above doses of isoprenaline were also determined before and during infusion of the alpha1-adrenoceptor agonist, phenylephrine (3 microg kg(-1) min(-1), i.v.), and again 15-20 min after injection of propranolol (1.0 mg kg(-1), i.v.). Both doses of isoprenaline elicited tachycardia and reductions in vascular resistances. Propranolol eliminated the responses elicited by the lower dose of isoprenaline and substantially diminished the responses elicited by the higher dose of the beta1-, beta2- and beta3-adrenoceptor agonist. The maximal vasodilator responses elicited by both doses of isoprenaline were not diminished whereas the maximal increases in HR were higher after injection of L-NAME. The ability of propranolol to diminish the hemodynamic actions of isoprenaline was substantially diminished in L-NAME-treated rats, whereas propranolol retained its potency in rats that received an equi-pressor infusion of the alpha1-adrenoceptor agonist, phenylephrine. The finding that the maximal vasodilator responses elicited by isoprenaline were not diminished by L-NAME suggests that the vasodilation elicited by this drug was due to direct activation of beta-adrenoceptors on vascular smooth muscle and that the full compliment of isoprenaline-sensitive receptors was not changed after inhibition of NO synthesis. However, these results suggest that the activities of propranolol-sensitive beta-adrenoceptors are downregulated, whereas propranolol-insensitive beta-adrenoceptors are upregulated upon the loss of exposure to endothelial nitrosyl factors.

Intracellular cGMP may promote Ca2+-dependent and Ca2+-independent release of catecholamines from sympathetic nerve terminals.

OBJECTIVE: This study examined the hypothesis that intracellular cGMP stimulates the release of catecholamines from sympathetic nerve terminals (SNTs) in conscious rats. METHODS: Conscious rats were prepared to determine the effects of intravenously-administered agents on heart rate (HR) and mean arterial blood pressure (MAP). RESULTS: Bolus intravenous injections of the membrane-permeable cGMP analogue, 8-(4-chlorophenylthio)-cGMP (8-CPT-cGMP), elicited immediate and pronounced increases in HR before any changes in MAP were observed. In contrast, injections of cGMP did not elicit changes in HR or MAP. The 8-CPT-cGMP-induced tachycardia was markedly diminished by (1) the beta(1,2)-adrenoceptor antagonist, propranolol, (2) the ganglion blocking agent, chlorisondamine, and (3) bretylium, which blocks Ca2+-dependent mobilization of vesicular stores of catecholamines from SNTs. 8-CPT-cGMP also elicited minor falls in MAP in propranolol-treated rats but elicited pronounced falls in MAP in rats treated with chlorisondamine, bretylium, or combined administration of bretylium and the muscarinic receptor antagonist, methyl-atropine. CONCLUSIONS: These findings suggest that (1) intracellular cGMP elicits the release of Ca2+-sensitive and Ca2+-insensitive stores of catecholamines from SNTs in conscious rats, and (2) cGMP-mediated release of catecholamines from SNTs antagonizes cGMP-mediated relaxation of vascular smooth muscle in resistance arteries. Taken together, these findings support the concept that increases in intracellular cGMP levels by atrial natriuretic peptide and endothelium- and cardiac-derived nitric oxide regulate sympathetic control of the heart and the microvasculature of conscious rats via cGMP-dependent release of catecholamines.

PKA-mediated phosphorylation of the beta1-adrenergic receptor promotes Gs/Gi switching.

Recently, it has been shown that PKA-mediated phosphorylation of the beta(2)-adrenergic receptor (beta(2)-AR) by the cyclic AMP-dependent protein kinase (PKA) reduces its affinity for G(s) and increases its affinity for G(i). Here we demonstrate that, like the beta(2)-AR, the beta(1)-AR is also capable of "switching" its coupling from G(s) to G(i) in a PKA-dependent manner. The beta(1)-AR is capable of activating adenylate cyclase via G(s), and can also activate the extracellular-regulated kinases, p44 and p42 (ERK1/2). In transfected CHO cells, the observed beta(1)-AR-mediated activation of ERK is both sensitive to pertussis toxin (PTX), indicating involvement of G(i)/G(o), and to the PKA inhibitor, H-89. beta(1)-ARs with PKA phosphorylation sites mutated to alanines are unable to activate ERK. Mutating these same residues to aspartic acid, mimicking PKA phosphorylation, leads to a decrease in G(s)-stimulated cAMP accumulation and an increase in PTX-sensitive ERK activation. These results strongly support the hypothesis that the beta(1)-AR, like the beta(2)-AR, can undergo PKA-dependent "G(s)/G(i) switching".

Therapeutic potential of ß-arrestin- and G protein-biased agonists.

Members of the seven-transmembrane receptor (7TMR), or G protein-coupled receptor (GPCR), superfamily represent some of the most successful targets of modern drug therapy, with proven efficacy in the treatment of a broad range of human conditions and disease processes. It is now appreciated that ß-arrestins, once viewed simply as negative regulators of traditional 7TMR-stimulated G protein signaling, act as multifunctional adapter proteins that regulate 7TMR desensitization and trafficking and promote distinct intracellular signals in their own right. Moreover, several 7TMR biased agonists, which selectively activate these divergent signaling pathways, have been identified. Here we highlight the diversity of G protein- and ß-arrestin-mediated functions and the therapeutic potential of selective targeting of these in disease states.

Cardiorenal actions of TRV120027, a novel ß-arrestin-biased ligand at the angiotensin II type I receptor, in healthy and heart failure canines: a novel therapeutic strategy for acute heart failure.

BACKGROUND: The angiotensin II type 1 receptor (AT1R) plays a key role in regulating cardiorenal function. Classic "unbiased" AT1R antagonists block receptor coupling to both G(αq) and ß-arrestin-mediated signals, which desensitize G-protein signaling as well as transduce G-protein-independent signals. TRV120027 is a novel ß-arrestin-biased AT1R ligand, which engages ß-arrestins while blocking G-protein signaling. At the AT1R, TRV120027 can inhibit angiotensin II-mediated vasoconstriction, whereas, through ß-arrestin coupling, increase cardiomyocyte contractility. We defined for the first time the acute cardiorenal actions of TRV120027 in healthy and heart failure (HF) canines. METHODS AND RESULTS: Healthy and HF canines (induced by tachypacing) were anesthetized. After instrumentation and equilibration, a 30-minute baseline clearance was performed, followed by further clearance with escalating doses of intravenous TRV120027 (0.01, 0.1, 1, 10, and 100 µg/kg per minute) and a 30-minute washout. In healthy canines, TRV120027 decreased pulmonary capillary wedge pressure and systemic and renal vascular resistances, while increasing cardiac output, renal blood flow, glomerular filtration rate, and urinary sodium excretion. In HF canines, TRV120027 decreased mean arterial pressure, right atrial pressure, and pulmonary capillary wedge pressure, systemic and renal vascular resistances and increased cardiac output and renal blood flow. Glomerular filtration rate and urinary sodium excretion were maintained. CONCLUSIONS: We report for the first time the cardiorenal actions of the novel ß-arrestin-biased AT1R ligand TRV120027. In both normal and HF canines, TRV120027 demonstrated cardiac unloading actions while preserving renal function. With this beneficial pharmacological profile, TRV120027 represents a novel strategy for the treatment of HF.

Oxygen-regulated beta(2)-adrenergic receptor hydroxylation by EGLN3 and ubiquitylation by pVHL.

Agonist-induced ubiquitylation and degradation of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) play an essential role in surface receptor homeostasis, thereby tuning many physiological processes. Although beta-arrestin and affiliated E3 ligases mediate agonist-stimulated lysosomal degradation of the beta(2)-adrenergic receptor (beta(2)AR), a prototypic GPCR, the molecular cues that mark receptors for ubiquitylation and the regulation of receptor degradation by the proteasome remain poorly understood. We show that the von Hippel-Lindau tumor suppressor protein (pVHL)-E3 ligase complex, known for its regulation of hypoxia-inducible factor (HIF) proteins, interacts with and ubiquitylates the beta(2)AR, thereby decreasing receptor abundance. We further show that the interaction of pVHL with beta(2)AR is dependent on proline hydroxylation (proline-382 and -395) and that the dioxygenase EGLN3 interacts directly with the beta(2)AR to serve as an endogenous beta(2)AR prolyl hydroxylase. Under hypoxic conditions, receptor hydroxylation and subsequent ubiquitylation decrease dramatically, thus attenuating receptor degradation and down-regulation. Notably, in both cells and tissue, the abundance of endogenous beta(2)AR is shown to reflect constitutive turnover by EGLN3 and pVHL. Our findings provide insight into GPCR regulation, broaden the functional scope of prolyl hydroxylation, and expand our understanding of the cellular response to hypoxia.

beta-Arrestin1 mediates nicotinic acid-induced flushing, but not its antilipolytic effect, in mice.

Nicotinic acid is one of the most effective agents for both lowering triglycerides and raising HDL. However, the side effect of cutaneous flushing severely limits patient compliance. As nicotinic acid stimulates the GPCR GPR109A and Gi/Go proteins, here we dissected the roles of G proteins and the adaptor proteins, beta-arrestins, in nicotinic acid-induced signaling and physiological responses. In a human cell line-based signaling assay, nicotinic acid stimulation led to pertussis toxin-sensitive lowering of cAMP, recruitment of beta-arrestins to the cell membrane, an activating conformational change in beta-arrestin, and beta-arrestin-dependent signaling to ERK MAPK. In addition, we found that nicotinic acid promoted the binding of beta-arrestin1 to activated cytosolic phospholipase A2 as well as beta-arrestin1-dependent activation of cytosolic phospholipase A2 and release of arachidonate, the precursor of prostaglandin D2 and the vasodilator responsible for the flushing response. Moreover, beta-arrestin1-null mice displayed reduced cutaneous flushing in response to nicotinic acid, although the improvement in serum free fatty acid levels was similar to that observed in wild-type mice. These data suggest that the adverse side effect of cutaneous flushing is mediated by beta-arrestin1, but lowering of serum free fatty acid levels is not. Furthermore, G protein-biased ligands that activate GPR109A in a beta-arrestin-independent fashion may represent an improved therapeutic option for the treatment of dyslipidemia.

beta-arrestin-biased agonism at the beta2-adrenergic receptor.

Classically, the beta 2-adrenergic receptor (beta 2AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including beta 2AR, can signal via beta-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among beta 2AR agonists there exist ligands that disproportionately signal via G proteins or beta-arrestins and are hence "biased." Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known beta 2AR agonists exhibit relative efficacies for beta-arrestin-associated activities (beta-arrestin membrane translocation and beta 2AR internalization) identical to the irrelative efficacies for G protein-dependent signaling (cyclic AMP generation). However, for three betaAR ligands there is a marked bias toward beta-arrestin signaling; these ligands stimulate beta-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these biased ligands reveals that all three are catecholamines containing an ethyl substitution on the alpha-carbon, a motif absent on all of the other, unbiased ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR ligands with a distinct bias for beta-arrestin-mediated signaling.

Beta-arrestin scaffolding of phosphatidylinositol 4-phosphate 5-kinase Ialpha promotes agonist-stimulated sequestration of the beta2-adrenergic receptor.

Members of the seven-transmembrane receptor (7TMR) superfamily are sequestered from the plasma membrane following stimulation both to limit cellular responses as well as to initiate novel G protein-independent signaling pathways. The best studied mechanism for 7TMR internalization is via clathrin-coated pits, where clathrin and adaptor protein complex 2 nucleate and polymerize upon encountering the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) to form the outer layer of the clathrin-coated vesicle. Activated receptors are recruited to clathrin-coated pits by beta-arrestins, scaffolding proteins that interact with agonist-occupied 7TMRs as well as adaptor protein complex 2 and clathrin. We report here that following stimulation of the beta2-adrenergic receptor (beta2-AR), a prototypical 7TMR, beta-arrestins bind phosphatidylinositol 4-phosphate 5-kinase (PIP5K) Ialpha, a PIP(2)-producing enzyme. Furthermore, beta-arrestin2 is required to form a complex with PIP5K Ialpha and agonist-occupied beta2-AR, and beta-arrestins synergize with the kinase to produce PIP(2) in response to isoproterenol stimulation. Interestingly, beta-arrestins themselves bind PIP(2), and a beta-arrestin mutant deficient in PIP(2) binding no longer internalizes 7TMRs, fails to interact with PIP5K Ialpha, and is not associated with PIP kinase activity assayed in vitro. However, a chimeric protein in which the core kinase domain of PIP5K Ialpha has been fused to the same beta-arrestin mutant rescues internalization of beta2-ARs. Collectively, these data support a model in which beta-arrestins direct the localization of PIP5K Ialpha and PIP(2) production to agonist-activated 7TMRs, thereby regulating receptor internalization.