Membrán mikrodomének szerepe az angiotenzinreceptor működésében = The role of membrane microdomains in angiotensin receptor function

Kísérleteink során biolumineszcencia rezonancia energiaenergiatranszfer (BRET) módszereket állítottunk be az AT1-es angiotenzinreceptor jelátviteli folyamata során a membrán mikrodoménekben létrejövő fehérje-fehérje kölcsönhatások nyomon követésére. Konfokális mikroszkóppal és BRET módszerrel végzett vizsgálatokkal igazoltuk, hogy az AT1-receptorral ellentétben az AT2-receptor angiotenzin II hatására nem kapcsolódik össze ß-arresztin fehérjékkel, mely arra utal, hogy az AT2-receptor tartós aktiválás esetén nem deszenzitizálódik. Kimutattuk továbbá, hogy AT1-receptort és CB1 kannabinoid receptor együtt expresszáló sejtekben angiotenzin II hatására a CB1-receptor is aktiválódik. Adataink arra utalnak, hogy e válasz közvetítésében az angiotenzin II hatására a membránban található lipidekből felszabaduló endokannabinoidok játszanak szerepet. Bizonyítékot szolgáltattunk arra, hogy a dimerizált AT1-receptorok működése során az egyik receptor gátlása a vele molekuláris kapcsolatban lévő másik receptor működését is gátolhatja. Eredményeink alapján arra következtethetünk, hogy az AT1-receptor terápiás célból történő gátlásakor más receptorok működésében is alapvető változások jöhetnek létre. Tekintettel arra, hogy az angiotenzin II AT1-receptorokon keresztül létrehozott hatásainak gátlása fontos terápiás célpont számos keringési betegség gyógyításakor, e mechanizmusok felismerése fontos támpontot jelenthet a terápiás beavatkozások pontos hatásmechanizmusának megértése szempontjából. | We have established methods based on bioluminescence energy transfer (BRET) in order to detect protein-protein interactions, which occur in membrane microdomains during the signal transduction mediated by activation of AT1 angiotensin receptors. We have provided evidence using confocal microscopy and BRET methods that the ß-arrestin binding properties of AT1 and AT2 angiotensin receptors are different. In contrast to AT1 receptors, which interact with ?-arrestin molecules upon angiotensin II stimulation, AT2 receptors do not show this interaction under similar conditions, which suggests that continuous activation of AT2 receptors do not lead to their desensitization. We have also shown that in cells co-expressing AT1 angiotensin and CB1 cannabinoid receptors, angiotensin II can induce CB1 receptor activation. Our data suggest that this response is mediated by angiotensin II-stimulated endocannabinoid release from membrane lipids. We have also demonstrated that in AT1 receptor dimers inhibition of one receptor can inhibit the signaling of the other receptor. In conclusion, our findings suggest that during therapeutic inhibition of AT1 receptors in patients the activity of other receptor systems can also be affected. Considering that AT1 receptors are a major target in the treatment of several cardiovascular diseases, elucidation of these mechanisms is potentially very important for the understanding of the consequences of these therapeutic interventions

␤-Arrestin-and Dynamin-Dependent Endocytosis of the AT 1 Angiotensin Receptor

ABSTRACT The major mechanism of agonist-induced internalization of G protein-coupled receptors (GPCRs) is ␤-arrestin-and 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 ␤-arrestin1(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 ␤-arrestin-and dynamin-dependent endocytosis of the AT 1 receptor via clathrin-coated vesicles at physiological angiotensin II concentrations

Elektron transzfer rendszerek élettani szerepe = The physiological role of electron transfer systems

Fagocitákban leírtuk a NADPH oxidázt szabályozó két különböző GTPáz aktiváló fehérje szabályozását és a kísérő K+ transzport baktérium ölő hatását. Agyi mitokondriumokban (mito) a légzési lánc I. komplexének szubsztrátjai membránpotenciál (Em) függően reaktív oxigénszármazékot (ROS) képeznek. Az alfa-glicerofoszfát (aGP) ROS-t képez az I. komplexen és az aGP-dehidrogenáz enzimen, utóbbit a Ca2+ aktivája. Idegvégződésekben a mito ROS képzését az Em nem befolyásolja. A mito-k elektromos szincíciumot képeznek, de a Ca2+ diffúziója korlátozott. Alacsony O2.- szint a Ca2+ -mobilizáló agonista Ca2+ jel képző hatását glomerulóza sejtben gátolja. A ROS támadáspontja a belső raktárból történő Ca2+ felszabadulás. UV hatására a mito Ca2+ felvétele is csökkent. Angiotenzin II -vel ingerelt H295R sejtben a mito Ca2+ jel képzés sebessége a mito és az endoplazmás retikulum (ER) közelségével korrelál. A p38 MAPK és az újtípusú PKC izoformák egyidejű gátlása a Ca2+ jelnek a citoszolból a mito-ba történő áttevődését gátolja és a fenti korrelációt megszünteti. Az ER lumenében a tiol/diszulfid rendszertől elkülönülő NAD(P)+/NAD(P)H rendszer működik. Redox állapotát a glukóz-6-foszfát transzporter és az intraluminális oxidoreduktázok határozzák meg. A redukált állapot fenntartása szükséges a glukokortikoidok prereceptoriális aktiválásához, s egyes sejtekben antiapoptotikus hatású. Jellemeztük az ER szulfát transzporterét, valamint a transzlokon peptid csatorna anion permeabilitását. | We described in phagocytes the regulation of two GTPase activating proteins, terminating the activity of plasmalemmal NADPH oxidase and the role of K+ movements in bacterial killing. In brain mitochondria complex I dependent substrates show a membrane potential (Em) dependent reactive oxygen species (ROS) formation. ROS production by alpha-glycerophosphate (aGP) occured at complex I and on the aGP-dehydrogenase enzyme. The latter is activited by Ca2+. Mitochondria form an electric syntitium but the diffusion of Ca2+ is limited. In glomerulosa cells, at low [O2.-] angiotensin-induced Ca2+ signalling is attenuated, the site of ROS action is Ca2+ release from the internal stores. The rate of mitochondrial Ca2+ uptake in angiotensin-stimulated cells correlates with the vicinity of the mitochondrion and the endoplasmic reticulum (ER). Simultaneous activation of p38 MAPK and the novel isoforms of PKC attenuates the transfer of cytosolic Ca2+ signal into the mitochondria and abolishes this correlation. In the ER we observed a novel NAD(P)+/NAD(P)H system different from the thiol/disulphide system. Its reduced state is tuned by the glucose-6-phosphate transporter and the luminal oxidoreductases and is required for the prereceptorial activation of glucocorticoids. We have characterized the sulphate transport in the ER, and the contribution of the translocon peptide channel to the membrane permeation of small anions

Improved methodical approach for quantitative BRET analysis of G protein coupled receptor dimerization

G Protein Coupled Receptors (GPCR) can form dimers or higher ordered oligomers, the process of which can remarkably influence the physiological and pharmacological function of these receptors. Quantitative Bioluminescence Resonance Energy Transfer (qBRET) measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V2 vasopressin and CaSR calcium sensing receptors, whereas our data argue against the heterodimerization of these receptors with other studied GPCRs, including type I and II angiotensin, β2 adrenergic and CB1 cannabinoid receptors

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

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

Regulation of endocannabinoid release by G proteins: A paracrine mechanism of G protein-coupled receptor action.

In the past years, the relationship between the endocannabinoid system (ECS) and other hormonal and neuromodulatory systems has been intensively studied. G protein-coupled receptors (GPCRs) can stimulate endocannabinoid (eCB) production via activation of G(q/11) proteins and, in some cases, G(s) proteins. In this review, we summarize the pathways through which GPCR activation can trigger eCB release, as well as the best known examples of this process throughout the body tissues. Angiotensin II-induced activation of AT(1) receptors, similar to other G(q/11)-coupled receptors, can lead to the formation of 2-arachidonoylglycerol (2-AG), an important eCB. The importance of eCB formation in angiotensin II action is supported by the finding that the hypertensive effect of angiotensin II, injected directly into the hypothalamic paraventricular nucleus of anaesthetized rats, can be abolished by AM251, an inverse agonist of CB(1) cannabinoid receptors (CB(1)Rs). We conclude that activation of the ECS should be considered as a general consequence of the stimulation of G(q/11)-coupled receptors, and may mediate some of the physiological effects of GPCRs