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

A hepatitis C virus (HCV) fertőzés pathogenesise: a genetikai és az immunológiai tényezők, valamint az oxidativ stress szerepe és a kórokozó virus sajátosságai, különös tekintettel a HCV-okozta betegségekben és a tünetmentes virushordozó állapotban = Pathogenesis of hepatitis C virus (HCV) infection: the role of the genetic and immunological factors, the oxidative stress and the viral features in the HCV-related diseases and in the symptomfree virus carriers

Genetikai, immunológiai és környezeti tényezők potenciális szerepe krónikus hepatitis C virus (HCV) infekcióban: összehasonlitó vizsgálatok aktiv HCV hepatitises betegekben és tünetmentes "egészséges" virushordozókban. 1. Sem a haemochromatosis gen mutációk, sem a CTLA4 polymorfizmusok nem befolyásolják a HCV-okozta májbetegség aktivitását, az angiotensin convertalo enzym (ACE) gen deletio azoban kedvező hatásu az anti-HCV terapia kimenetelére. 2. Az NK és B sejek felszinén levő CD81 molekula overexpressioja, valamint a regulatoros T sejtek termelte transformáló növekedési faktor (TGFbeta1), és az utóbbi által a cytotoxikus sejteken downregulált NKG2D aktiváló receptor - meghatározó szerepet játszanak a HCV infecióban kulcsfontosságu károsodott cellularis immunválaszban 3. A plasma TGFbeta1, a hyaluronsav és a procollagen-III-peptid szintek - mint a fibrogenesis markerei - kórosan emelkedettek krónikus aktiv HCV hepatitisben, de nem a tünetmentes virus-hordozókban. Az interferon + ribavirin antiviralis terapia gátolhatja a fibrogenesist, függetlenül a virológiai választól. 4. Mint additiv környezeti tényező, a SEN virus ko-infekció, gyakran előfordul HCV fertőzésben, és jelenléte negativan befolyásolja az anti-HCV terapia hatását krónikus C hepatitisben. | Genetic, immunologic and environmental factors and their potential role in the pathogenesis of chronic hepatitis C virus (HCV) infection: a comparison between active HCV hepatitis and symptomfree 'healthy' virus carriers. 1. Neither haemochromatosis gene mutations, nor CTLA4 polymorphisms influence the activity of HCV-related liver disease, however, ACE gene deletion has a favourable effect on the outcome of anti-HCV treatment in chronic hepatitis C. 2. Overexpression of CD81 molecule on NK and B cells, as well as transforming growth factor beta1 (TGFbeta1) secreted by regulatory T cells, and downregulated NKG2D activating receptor on cytotoxic effector cells, may play a privotal role in the impaired cell mediated immunity in active HCV infection 3. Plasma TGFbeta1, hyalurinic acid and procollagene-III-peptide levels - as markers of fibrogenesis - are elevated in active hepatitis C patients, but not in symptomfree carriers. Interferon + ribavirin therapy may inhibit fibrogenesis independently of virological response in chronic HCV hepatitis. 4. SEN virus co-infection, - as an additive environmetal factor - frequently occuring in HCV patients, decreases the effect of anti-HCV treatment

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

Altered agonist sensitivity of a mutant V2 receptor suggests a novel therapeutic strategy for nephrogenic diabetes insipidus.

Loss of function mutations of the type 2 vasopressin receptor (V2R) in kidney can lead to nephrogenic diabetes insipidus (NDI). We studied a previously described, but uncharacterized mutation of V2R (N321K missense mutation) of an NDI patient. The properties of the mutant receptor were evaluated. We constructed a highly sensitive Epac based BRET (bioluminescence resonance energy transfer) biosensor to perform real-time cAMP measurements after agonist stimulation of transiently transfected HEK293 cells with V2Rs. beta-arrestin binding of the activated receptors was examined with luciferase-tagged beta-arrestin and mVenus-tagged V2Rs using BRET technique. Cell surface expressions of HA-tagged receptors were determined with flow cytometry using anti-HA-Alexa488 antibodies. Cellular localization examinations were implemented with fluorescent tagged receptors visualized with confocal laser-scanning microscopy. The effect of various vasopressin analogues on V1R was tested on mouse arteries by wire myography. N321K mutant V2R showed normal cell surface expression but the potency of AVP for cAMP generation was low, while the clinically used desmopressin (dDAVP) was not efficient. The beta-arrestin binding and internalization properties of the mutant receptor were also different compared to the wild type. Function of the mutant receptor can be rescued with administration of V2R receptor agonist dVDAVP, which had no detectable side effects on V1R in the effective cAMP generating concentrations. Based on the findings we could propose a therapeutical strategy for NDI patients carrying the N321K mutation, since our in vivo experiments suggest that dVDAVP could rescue the function of the N321K-V2R without significant side effect on V1R