Novel mechanisms of G-protein-coupled receptors functions: AT1 angiotensin receptor acts as a signaling hub and focal point of receptor cross-talk

AT1 angiotensin receptor (AT1R), a prototypical G protein-coupled receptor (GPCR), is the main receptor, which mediates the effects of the renin-angiotensin system (RAS). AT1R plays a crucial role in the regulation of blood pressure and salt-water homeostasis, and in the development of pathological conditions, such as hypertension, heart failure, cardiovascular remodeling, renal fibrosis, inflammation, and metabolic disorders. Stimulation of AT1R leads to pleiotropic signal transduction pathways generating arrays of complex cellular responses. Growing amount of evidence shows that AT1R is a versatile GPCR, which has multiple unique faces with distinct conformations and signaling properties providing new opportunities for functionally selective pharmacological targeting of the receptor. Biased ligands of AT1R have been developed to selectively activate the β-arrestin pathway, which may have therapeutic benefits compared to the conventional angiotensin converting enzyme inhibitors and angiotensin receptor blockers. In this review, we provide a summary about the most recent findings and novel aspects of the AT1R function, signaling, regulation, dimerization or oligomerization and its cross-talk with other receptors, including epidermal growth factor (EGF) receptor, adrenergic receptors and CB1 cannabinoid receptor. Better understanding of the mechanisms and structural aspects of AT1R activation and cross-talk can lead to the development of novel type of drugs for the treatment of cardiovascular and other diseases. © 201

Differential β-arrestin2 requirements for constitutive and agonist-induced internalization of the CB1 cannabinoid receptor

CB1 cannabinoid receptor (CB1R) undergoes both constitutive and agonist-induced internalization, but the underlying mechanisms of these processes and the role of beta-arrestins in the regulation of CB1R function are not completely understood. In this study, we followed CB1R internalization using confocal microscopy and bioluminescence resonance energy transfer measurements in HeLa and Neuro-2a cells. We found that upon activation CB1R binds beta-arrestin2 (beta-arr2), but not beta-arrestin1. Furthermore, both the expression of dominant-negative beta-arr2 (beta-arr2-V54D) and siRNA-mediated knock-down of beta-arr2 impaired the agonist-induced internalization of CB1R. In contrast, neither beta-arr2-V54D nor beta-arr2-specific siRNA had a significant effect on the constitutive internalization of CB1R. However, both constitutive and agonist-induced internalization of CB1R were impaired by siRNA-mediated depletion of clathrin heavy chain. We conclude that although clathrin is required for both constitutive and agonist-stimulated internalization of CB1R, beta-arr2 binding is only required for agonist-induced internalization of the receptor suggesting that the molecular mechanisms underlying constitutive and agonist-induced internalization of CB1R are different

Új mechanizmusok az angiotenzin II élettani hatásainak szabályozásában = Novel mechanisms in the regulation of the physiological actions of angiotensin II

Az AT1 angiotenzin receptor (AT1R) egy Gq-fehérjéhez kapcsolt receptor, mely angiotenzin II (AngII) hatására a sejtekben Ca2+-jelet hoz létre. Kísérleteinkben kimutattuk, hogy az AT1R és más Gq-kapcsolt receptorok jelátviteli folyamata során keletkező diacil-glicerinből a sejtekben 2-arachidonoil-glicerin (2-AG) keletkezik, mely CB1 kannabinoid receptorok parakrin aktivációját hozhatja létre. Kimutattuk e mechanizmus jelentőségét az AngII vazokonstriktor hatásának szabályozásában, és felvetettük szerepét az AngII centrális vérnyomásemelő és gyomorvédő hatásának létrehozásában. Leírtuk, hogy az AngII mellékvesekéreg glomerulosa sejtekben fokozza a neurotrofinok családjába tartozó agyi eredetű növekedési faktor (BDNF) expresszióját. BRET módszeren alapuló bioszenzorok segítségével kimutattuk, hogy AngII hatására az endoplazmatikus retikulumban és a transz-Golgi-hálózatban is létrejöhet a Ras kis G-fehérje aktiválódása; valamint azt, hogy AngII hatására megváltozik az AT1R membrán mikrodoménekben való elhelyezkedése. Kimutattuk, hogy Ca2+-mobilizáló agonistával serkentett HEK293T sejtekben a Ca2+-válasszal párhuzamosan Mg2+-jel jön létre. Leírtuk e Mg2+-jel, valamint a p38 MAP-kináz és egy új típusú (novel) protein-kináz C enzim szerepét a mitokondriális Ca2+-felvétel és a mellékvesekéreg aldoszteron-termelésének szabályozásában. Eredményeink hozzájárulnak a magasvérnyomás és más keringési betegségek terápiájában fontos célpontot jelentő AT1R működésének jobb megértéséhez. | Angiotensin II (AngII) exerts its main biological effects via the AT1 angiotensin receptor (AT1R), which is coupled to Gq protein, and produces Ca2+ signal generation after stimulation. We have demonstrated that activation of AT1R and other Gq-coupled receptors leads to the generation of 2-arachidonoylglycerol (2-AG) from diacylglcycerol, formed during the signaling process, and 2-AG can cause paracrine activation of CB1 cannabinoid receptors. Our data show that this mechanism modulates the vasoconstrictor action of AngII, and suggest its role in the central hypertensive and gastroprotective effects of AngII. We have reported that AngII can stimulate the expression of brain-dervied neurtropic factor (BDNF), a major neurotrophin, in adrenal glomerulosa cells. We have demonstrated using BRET-based biosensors that AngII induces the activation of Ras small G protein in the endoplasmic reticulum and the trans-Golgi network, and alters the localization of AT1R in membrane microdomains. We have described that in HEK293T cells Ca2+-mobilizing agonists produce Mg2+ signal, parallel with the Ca2+ signal generation; and we have demonstrated the roles of the Mg2+ signal, p38 MAP kinase and a novel protein kinase C in the regulation of the mitochondrial Ca2+ uptake and aldosterone production of adrenal glomerulosa cells. Our results facilitate better understanding of the function of AT1R, which is a main target in the therapy of hypertension and other cardiovascular diseases

Mutations in the 'DRY' motif of the CB1 cannabinoid receptor result in biased receptor variants.

The role of the highly-conserved 'DRY' motif in the signaling of the CB1 cannabinoid receptor (CB1R) was investigated by introducing single, double and triple alanine mutations into this site of the receptor. We found that the CB1R-R3.50A mutant displays a partial decrease in its ability to activate heterotrimeric Go proteins (~85% of wild-type CB1R (CB1R-WT)). Moreover, this mutant showed impaired beta-arrestin binding in response to agonist stimulus, although its basal beta-arrestin binding was enhanced. More strikingly, the double mutant CB1R-D3.49A/R3.50A was biased toward beta-arrestins, as it gained a robustly increased beta-arrestin1 and beta-arrestin2 binding ability compared to the wild-type receptor, while its G protein activation was decreased. In contrast, the double mutant CB1R-R3.50A/Y3.51A proved to be G protein-biased, as it was practically unable to recruit beta-arrestin2 in response to agonist stimulus, while still activating G proteins, although at a reduced level (~75% of CB1R-WT). Agonist-induced ERK1/2 activation of the CB1R mutants showed good correlation with their beta-arrestin binding ability but not with their G protein activation or inhibition of cAMP accumulation. Our results suggest that G protein-activation and beta-arrestin-binding of the CB1R are mediated by distinct receptor conformations and the conserved 'DRY' motif plays different roles in the stabilization of these conformations, thus mediating both G protein- and beta-arrestin2-mediated functions of CB1R

Proteomic Changes of Osteoclast Differentiation in Rheumatoid and Psoriatic Arthritis Reveal Functional Differences

BACKGROUND: Osteoclasts play a crucial role in the maintenance, repair, and remodeling of bones of the adult vertebral skeleton due to their bone resorption capability. Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are associated with increased activity of osteoclasts. OBJECTIVES: Our study aimed to investigate the dynamic proteomic changes during osteoclast differentiation in healthy donors, in RA, and PsA. METHODS: Blood samples of healthy donors, RA, and PsA patients were collected, and monocytes were isolated and differentiated into osteoclasts in vitro using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANK-L). Mass spectrometry-based proteomics was used to analyze proteins from cell lysates. The expression changes were analyzed with Gene Set Enrichment Analysis (GSEA). RESULTS: The analysis of the proteomic changes revealed that during the differentiation of the human osteoclasts, expression of the proteins involved in metabolic activity, secretory function, and cell polarity is increased; by contrast, signaling pathways involved in the immune functions are downregulated. Interestingly, the differences between cells of healthy donors and RA/PsA patients are most pronounced after the final steps of differentiation to osteoclasts. In addition, both in RA and PsA the differentiation is characterized by decreased metabolic activity, associated with various immune pathway activities; furthermore by accelerated cytokine production in RA. CONCLUSIONS: Our results shed light on the characteristic proteomic changes during human osteoclast differentiation and expression differences in RA and PsA, which reveal important pathophysiological insights in both diseases

Angiotensin type 1A receptor regulates β-arrestin binding of the β2-adrenergic receptor via heterodimerization

Heterodimerization between angiotensin type 1A receptor (AT1R) and β2-adrenergic receptor (β2AR) has been shown to modulate G protein-mediated effects of these receptors. Activation of G protein-coupled receptors (GPCRs) leads to β-arrestin binding, desensitization, internalization and G protein-independent signaling of GPCRs. Our aim was to study the effect of heterodimerization on β-arrestin coupling. We found that β-arrestin binding of β2AR is affected by activation of AT1Rs. Costimulation with angiotensin II and isoproterenol markedly enhanced the interaction between β2AR and β-arrestins, by prolonging the lifespan of β2AR-induced β-arrestin2 clusters at the plasma membrane. While candesartan, a conventional AT1R antagonist, had no effect on the β-arrestin2 binding to β2AR, TRV120023, a β-arrestin biased agonist, enhanced the interaction. These findings reveal a new crosstalk mechanism between AT1R and β2AR, and suggest that enhanced β-arrestin2 binding to β2AR can contribute to the pharmacological effects of biased AT1R agonists. © 201

The Orexigenic Effect of Ghrelin Is Mediated through Central Activation of the Endogenous Cannabinoid System

INTRODUCTION Ghrelin and cannabinoids stimulate appetite, this effect possibly being mediated by the activation of hypothalamic AMP-activated protein kinase (AMPK), a key enzyme in appetite and metabolism regulation. The cannabinoid receptor type 1 (CB1) antagonist rimonabant can block the orexigenic effect of ghrelin. In this study, we have elucidated the mechanism of the putative ghrelin-cannabinoid interaction. METHODS The effects of ghrelin and CB1 antagonist rimonabant in wild-type mice, and the effect of ghrelin in CB1-knockout animals, were studied on food intake, hypothalamic AMPK activity and endogenous cannabinoid content. In patch-clamp electrophysiology experiments the effect of ghrelin was assessed on the synaptic inputs in parvocellular neurons of the hypothalamic paraventricular nucleus, with or without the pre-administration of a CB1 antagonist or of cannabinoid synthesis inhibitors. RESULTS AND CONCLUSIONS Ghrelin did not induce an orexigenic effect in CB1-knockout mice. Correspondingly, both the genetic lack of CB1 and the pharmacological blockade of CB1 inhibited the effect of ghrelin on AMPK activity. Ghrelin increased the endocannabinoid content of the hypothalamus in wild-type mice and this effect was abolished by rimonabant pre-treatment, while no effect was observed in CB1-KO animals. Electrophysiology studies showed that ghrelin can inhibit the excitatory inputs on the parvocellular neurons of the paraventricular nucleus, and that this effect is abolished by administration of a CB1 antagonist or an inhibitor of the DAG lipase, the enzyme responsible for 2-AG synthesis. The effect is also lost in the presence of BAPTA, an intracellular calcium chelator, which inhibits endocannabinoid synthesis in the recorded parvocellular neuron and therefore blocks the retrograde signaling exerted by endocannabinoids. In summary, an intact cannabinoid signaling pathway is necessary for the stimulatory effects of ghrelin on AMPK activity and food intake, and for the inhibitory effect of ghrelin on paraventricular neurons

Paracrine Transactivation of the CB1 Cannabinoid Receptor by AT1 Angiotensin and Other Gq/11 Protein-coupled Receptors*

Intracellular signaling systems of G protein-coupled receptors are well established, but their role in paracrine regulation of adjacent cells is generally considered as a tissue-specific mechanism. We have shown previously that AT1 receptor (AT1R) stimulation leads to diacylglycerol lipase-mediated transactivation of co-expressed CB1Rs in Chinese hamster ovary cells. In the present study we detected a paracrine effect of the endocannabinoid release from Chinese hamster ovary, COS7, and HEK293 cells during the stimulation of AT1 angiotensin receptors by determining CB1 cannabinoid receptor activity with bioluminescence resonance energy transfer-based sensors of G protein activation expressed in separate cells. The angiotensin II-induced, paracrine activation of CB1 receptors was visualized by detecting translocation of green fluorescent protein-tagged β-arrestin2. Mass spectrometry analyses have demonstrated angiotensin II-induced stimulation of 2-arachidonoylglycerol production, whereas no increase of anandamide levels was observed. Stimulation of Gq/11-coupled M1, M3, M5 muscarinic, V1 vasopressin, α1a adrenergic, B2 bradykinin receptors, but not Gi/o-coupled M2 and M4 muscarinic receptors, also led to paracrine transactivation of CB1 receptors. These data suggest that, in addition to their retrograde neurotransmitter role, endocannabinoids have much broader paracrine mediator functions during activation of Gq/11-coupled receptors