Academic literature on the topic 'Receptor heteromerization'
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Journal articles on the topic "Receptor heteromerization"
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Albee, Lauren J., Xianlong Gao, and Matthias Majetschak. "Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells." PLOS ONE 16, no. 6 (June 24, 2021): e0253821. http://dx.doi.org/10.1371/journal.pone.0253821.
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Recently, we reported that the chemokine (C-X-C motif) receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) heteromerize with α1A/B/D-adrenoceptors (ARs) and arginine vasopressin receptor 1A (AVPR1A) in recombinant systems and in rodent and human vascular smooth muscle cells (hVSMCs). In these studies, we observed that heteromerization between two receptor partners may depend on the presence and the expression levels of other partnering receptors. To test this hypothesis and to gain initial insight into the formation of these receptor heteromers in native cells, we utilized proximity ligation assays in hVSMCs to visualize receptor-receptor proximity and systematically studied how manipulation of the expression levels of individual protomers affect heteromerization patterns among other interacting receptor partners. We confirmed subtype-specific heteromerization between endogenously expressed α1A/B/D-ARs and detected that AVPR1A also heteromerizes with α1A/B/D-ARs. siRNA knockdown of CXCR4 and of ACKR3 resulted in a significant re-arrangement of the heteromerization patterns among α1-AR subtypes. Similarly, siRNA knockdown of AVPR1A significantly increased heteromerization signals for seven of the ten receptor pairs between CXCR4, ACKR3, and α1A/B/D-ARs. Our findings suggest plasticity of seven transmembrane helix (7TM) receptor heteromerization in native cells and could be explained by a supramolecular organization of these receptors within dynamic clusters in the plasma membrane. Because we previously observed that recombinant CXCR4, ACKR3, α1a-AR and AVPR1A form hetero-oligomeric complexes composed of 2–4 different protomers, which show signaling properties distinct from individual protomers, re-arrangements of receptor heteromerization patterns in native cells may contribute to the phenomenon of context-dependent GPCR signaling. Furthermore, these findings advise caution in the interpretation of functional consequences after 7TM receptor knockdown in experimental models. Alterations of the heteromerization patterns among other receptor partners may alter physiological and pathological responses, in particular in more complex systems, such as studies on the function of isolated organs or in in vivo experiments.
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Johnstone, Elizabeth K. M., Heng B. See, Rekhati S. Abhayawardana, Angela Song, K. Johan Rosengren, Stephen J. Hill, and Kevin D. G. Pfleger. "Investigation of Receptor Heteromers Using NanoBRET Ligand Binding." International Journal of Molecular Sciences 22, no. 3 (January 22, 2021): 1082. http://dx.doi.org/10.3390/ijms22031082.
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Receptor heteromerization is the formation of a complex involving at least two different receptors with pharmacology that is distinct from that exhibited by its constituent receptor units. Detection of these complexes and monitoring their pharmacology is crucial for understanding how receptors function. The Receptor-Heteromer Investigation Technology (Receptor-HIT) utilizes ligand-dependent modulation of interactions between receptors and specific biomolecules for the detection and profiling of heteromer complexes. Previously, the interacting biomolecules used in Receptor-HIT assays have been intracellular proteins, however in this study we have for the first time used bioluminescence resonance energy transfer (BRET) with fluorescently-labeled ligands to investigate heteromerization of receptors on the cell surface. Using the Receptor-HIT ligand binding assay with NanoBRET, we have successfully investigated heteromers between the angiotensin II type 1 (AT1) receptor and the β2 adrenergic receptor (AT1-β2AR heteromer), as well as between the AT1 and angiotensin II type 2 receptor (AT1-AT2 heteromer).
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Rozenfeld, Raphael, and Lakshmi A. Devi. "Receptor heteromerization and drug discovery." Trends in Pharmacological Sciences 31, no. 3 (March 2010): 124–30. http://dx.doi.org/10.1016/j.tips.2009.11.008.
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Pelassa, Simone, Diego Guidolin, Arianna Venturini, Monica Averna, Giulia Frumento, Letizia Campanini, Rosa Bernardi, et al. "A2A-D2 Heteromers on Striatal Astrocytes: Biochemical and Biophysical Evidence." International Journal of Molecular Sciences 20, no. 10 (May 17, 2019): 2457. http://dx.doi.org/10.3390/ijms20102457.
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Our previous findings indicate that A2A and D2 receptors are co-expressed on adult rat striatal astrocytes and on the astrocyte processes, and that A2A-D2 receptor–receptor interaction can control the release of glutamate from the processes. Functional evidence suggests that the receptor–receptor interaction was based on heteromerization of native A2A and D2 receptors at the plasma membrane of striatal astrocyte processes. We here provide biochemical and biophysical evidence confirming that receptor–receptor interaction between A2A and D2 receptors at the astrocyte plasma membrane is based on A2A-D2 heteromerization. To our knowledge, this is the first direct demonstration of the ability of native A2A and D2 receptors to heteromerize on glial cells. As striatal astrocytes are recognized to be involved in Parkinson’s pathophysiology, the findings that adenosine A2A and dopamine D2 receptors can form A2A-D2 heteromers on the astrocytes in the striatum (and that these heteromers can play roles in the control of the striatal glutamatergic transmission) may shed light on the molecular mechanisms involved in the pathogenesis of the disease.
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Woods, Amina S., Francisco Ciruela, Kjell Fuxe, Luigi F. Agnati, Carmen Lluis, Rafael Franco, and Sergi Ferré. "Role of Electrostatic Interaction in Receptor–Receptor Heteromerization." Journal of Molecular Neuroscience 26, no. 2-3 (2005): 125–32. http://dx.doi.org/10.1385/jmn:26:2-3:125.
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Canals, Meritxell, Daniel Marcellino, Francesca Fanelli, Francisco Ciruela, Piero de Benedetti, Steven R. Goldberg, Kim Neve, et al. "Adenosine A2A-Dopamine D2Receptor-Receptor Heteromerization." Journal of Biological Chemistry 278, no. 47 (August 21, 2003): 46741–49. http://dx.doi.org/10.1074/jbc.m306451200.
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Bono, Federica, Veronica Mutti, Chiara Fiorentini, and Cristina Missale. "Dopamine D3 Receptor Heteromerization: Implications for Neuroplasticity and Neuroprotection." Biomolecules 10, no. 7 (July 9, 2020): 1016. http://dx.doi.org/10.3390/biom10071016.
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The dopamine (DA) D3 receptor (D3R) plays a pivotal role in the control of several functions, including motor activity, rewarding and motivating behavior and several aspects of cognitive functions. Recently, it has been reported that the D3R is also involved in the regulation of neuronal development, in promoting structural plasticity and in triggering key intracellular events with neuroprotective potential. A new role for D3R-dependent neurotransmission has thus been proposed both in preserving DA neuron homeostasis in physiological conditions and in preventing pathological alterations that may lead to neurodegeneration. Interestingly, there is evidence that nicotinic acetylcholine receptors (nAChR) located on DA neurons also provide neurotrophic support to DA neurons, an effect requiring functional D3R and suggesting the existence of a positive cross-talk between these receptor systems. Increasing evidence suggests that, as with the majority of G protein-coupled receptors (GPCR), the D3R directly interacts with other receptors to form new receptor heteromers with unique functional and pharmacological properties. Among them, we recently identified a receptor heteromer containing the nAChR and the D3R as the molecular effector of nicotine-mediated neurotrophic effects. This review summarizes the functional and pharmacological characteristics of D3R, including the capability to form active heteromers as pharmacological targets for specific neurodegenerative disorders. In particular, the molecular and functional features of the D3R-nAChR heteromer will be especially discussed since it may represent a possible key etiologic effector for DA-related pathologies, such as Parkinson’s disease (PD), and a target for drug design.
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Rozenfeld, Raphael, and Lakshmi A. Devi. "Exploring a role for heteromerization in GPCR signalling specificity." Biochemical Journal 433, no. 1 (December 15, 2010): 11–18. http://dx.doi.org/10.1042/bj20100458.
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The critical involvement of GPCRs (G-protein-coupled receptors) in nearly all physiological processes, and the presence of these receptors at the interface between the extracellular and the intracellular milieu, has positioned these receptors as pivotal therapeutic targets. Although a large number of drugs targeting GPCRs are currently available, significant efforts have been directed towards understanding receptor properties, with the goal of identifying and designing improved receptor ligands. Recent advances in GPCR pharmacology have demonstrated that different ligands binding to the same receptor can activate discrete sets of downstream effectors, a phenomenon known as ‘ligand-directed signal specificity’, which is currently being explored for drug development due to its potential therapeutic advantage. Emerging studies suggest that GPCR responses can also be modulated by contextual factors, such as interactions with other GPCRs. Association between different GPCR types leads to the formation of complexes, or GPCR heteromers, with distinct and unique signalling properties. Some of these heteromers activate discrete sets of signalling effectors upon activation by the same ligand, a phenomenon termed ‘heteromer-directed signalling specificity’. This has been shown to be involved in the physiological role of receptors and, in some cases, in disease-specific dysregulation of a receptor effect. Hence targeting GPCR heteromers constitutes an emerging strategy to select receptor-specific responses and is likely to be useful in achieving specific beneficial therapeutic effects.
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Toneatti, Rudy, Jong M. Shin, Urjita H. Shah, Carl R. Mayer, Justin M. Saunders, Miguel Fribourg, Paul T. Arsenovic, et al. "Interclass GPCR heteromerization affects localization and trafficking." Science Signaling 13, no. 654 (October 20, 2020): eaaw3122. http://dx.doi.org/10.1126/scisignal.aaw3122.
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Membrane trafficking processes regulate G protein–coupled receptor (GPCR) activity. Although class A GPCRs are capable of activating G proteins in a monomeric form, they can also potentially assemble into functional GPCR heteromers. Here, we showed that the class A serotonin 5-HT2A receptors (5-HT2ARs) affected the localization and trafficking of class C metabotropic glutamate receptor 2 (mGluR2) through a mechanism that required their assembly as heteromers in mammalian cells. In the absence of agonists, 5-HT2AR was primarily localized within intracellular compartments, and coexpression of 5-HT2AR with mGluR2 increased the intracellular distribution of the otherwise plasma membrane–localized mGluR2. Agonists for either 5-HT2AR or mGluR2 differentially affected trafficking through Rab5-positive endosomes in cells expressing each component of the 5-HT2AR–mGluR2 heterocomplex alone, or together. In addition, overnight pharmacological 5-HT2AR blockade with clozapine, but not with M100907, decreased mGluR2 density through a mechanism that involved heteromerization between 5-HT2AR and mGluR2. Using TAT-tagged peptides and chimeric constructs that are unable to form the interclass 5-HT2AR–mGluR2 complex, we demonstrated that heteromerization was necessary for the 5-HT2AR–dependent effects on mGluR2 subcellular distribution. The expression of 5-HT2AR also augmented intracellular localization of mGluR2 in mouse frontal cortex pyramidal neurons. Together, our data suggest that GPCR heteromerization may itself represent a mechanism of receptor trafficking and sorting.
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Zrein, Adel, Amina M. Bagher, Alexander P. Young, Eileen M. Denovan-Wright, and Melanie E. M. Kelly. "Endothelin receptor heteromerization inhibits β-arrestin function in HEK293 cells." Canadian Journal of Physiology and Pharmacology 98, no. 8 (August 2020): 531–40. http://dx.doi.org/10.1139/cjpp-2019-0620.
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The endothelin receptor A (ETA) and endothelin receptor B (ETB) are G protein-coupled receptors that are co-expressed in vascular smooth muscle cells. Endothelin-1 (ET-1) activates endothelin receptors to cause microvascular vasoconstriction. Previous studies have shown that heteromerization between ETA and ETB prolongs Ca2+ transients, leading to prolongation of Gαq-dependent signaling and sustained vasoconstriction. We hypothesized that these effects are in part mediated by the resistance of ETA/ETB heteromers to β-arrestin recruitment and subsequent desensitization. Using bioluminescence resonance energy transfer 2 (BRET2), we found that ETB has a relatively equal affinity to form either homomers or heteromers with ETA when co-expressed in the human embryonic kidney 293 (HEK293) cells. When co-expressed, activation of ETA and ETB by ET-1 caused a heteromer-specific reduction and delay in β-arrestin-2 recruitment with a corresponding reduction and delay in ET-1-induced ETA/ETB co-internalization. Furthermore, the co-expression of ETA and ETB inhibited ET-1-induced β-arrestin-1-dependent extracellular signal-regulated kinase (ERK) phosphorylation while prolonging ET-1-induced Gαq-dependent ERK phosphorylation. ETA/ETB heteromerization mediates the long-lasting vasoconstrictor response to ET-1 by the prolongation of Gαq-dependent signaling and inhibition of β-arrestin function.
Dissertations / Theses on the topic "Receptor heteromerization"
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Hatcher-Solis, Candice N. "PHARMACOLOGICAL IMPLICATIONS OF ADENOSINE 2A RECEPTOR- DOPAMINE TYPE 2 RECEPTOR HETEROMERIZATION." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4458.
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G protein-coupled receptors (GPCRs) are heptahelical, transmembrane proteins that mediate a plethora of physiological functions by binding ligands and releasing G proteins that interact with downstream effectors. GPCRs signal as monomers, complexes of the same receptor subtype (homomers), or complexes of different receptor subtypes (heteromers). Recently, heteromeric GPCR complexes have become attractive targets for drug development since they exhibit distinct signaling and cell-specific localization from their homomeric counterparts. Yet, the effect of heteromerization on the pharmacology of many GPCR homomers remains unknown. Therefore, we have undertaken the task to examine the effect of heteromerization on Gs signaling through the adenosine 2A receptor (A2AR) and Gi signaling through the dopamine type 2 receptor (D2R) since the A2AR-D2R heteromer is an emerging therapeutic target for Parkinson’s disease (PD). We examined the effect of heteromerization on A2AR and D2R homomeric signaling using electrophysiology and the Xenopus laevis oocyte heterologous expression system. G protein-coupled inwardly rectifying potassium channels (GIRKs) were used as reporters for Gi signaling because activation leads to direct Gbeta-gamma (Gβγ)-mediated stimulation of the GIRK current. We also coupled GIRK channels to Gs signaling by overexpressing Gαs and signaling throughGαsβγ. Our electrophysiological assay is innovative because it allows us to optimize the conditions of heteromerization and directly observe GPCR signaling at the G protein level. Our data demonstrate that heteromer formation alone decreases dopamine-elicited Gi signaling through the D2R and CGS-21680-elicited Gs signaling through the A2AR. Furthermore, this reciprocal antagonism was predominately due to changes in efficacy versus potency. We also examined crosstalk observing that applying agonists or antagonists to the adjacent receptor further modulate this inhibition with the combination of agonists and antagonists relieving inhibition. Mutating the A2AR-D2R heteromer interface abrogated all of the aforementioned ligand-induced effects on G protein signaling through the A2AR-D2R heteromer. We are currently aiming to validate our results from the oocyte experiments with an in vivo model. Our data further elucidate the effect of various ligands on G protein signaling through the A2AR- D2R heteromer, which may facilitate future studies that examine A2AR-D2R heteromer signaling.
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Younkin, Jason W. "Allosteric Effects of G-Protein Coupled Receptor Heteromerization: Relevance to Psychosis." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4457.
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G-protein coupled receptors (GPCRs) implicated in disease are the predominant pharmaceutical targets. Growing evidence suggests that GPCRs form homo- and heteromeric complexes, resulting in allosteric functional changes. Ligands targeting one receptor can alter the function of the other receptor or receptors. Knowledge of these functional changes will provide unique opportunities to treat diseases. We examined two GPCR heteromers implicated in psychosis: mGlu2R-5HT2AR and D2R-5HT2AR. Using whole-cell patch clamp, we studied HEK-293 cells stably transfected with mGlu2R and 5HT2AR. Maximal heteromer formation allows inverse agonists to increase the G-protein activity of the opposite receptor, while sub-maximal heteromer formation does not. However, similar results are obtained in sub-maximal heteromer cells by applying a combination of a mGlu2R synthetic agonist with a 5HT2AR anti-psychotic drug. These results confirm our oocyte results, now in a mammalian cell line. Using two-electrode voltage clamp, we also investigated the allosteric changes upon heteromerization of D2R-5HT2AR in oocytes injected with appropriate cRNAs. Heteromer formation in the presence of dopamine or serotonin results in an increase in G-protein activity of each receptor while the simultaneous presence of both neurotransmitters further increases the G-protein activity. The addition of synthetic agonists or anti-psychotics decreases the G-protein activity of the opposite receptor while agonizing or antagonizing its target receptor, respectively. Maximal allosteric effects upon D2R-5HT2AR formation only occur at a specific cRNA injection ratio, but partial effects exist at other ratios. Our data suggest that allosteric functional changes upon heteromerization are physiologically relevant and are mostly different when comparing mGlu2R-5HT2AR to D2R-5HT2AR.
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Leskelä, T. (Tarja). "Human δ opioid receptor Phe27 and Cys27 variants:the role of heteromerization and pharmacological chaperones in receptor processing and trafficking." Doctoral thesis, Oulun yliopisto, 2011. http://urn.fi/urn:isbn:9789514296970.
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Abstract The opioid receptors (δ, κ and μ) are family A G protein-coupled receptors (GPCRs) that have an important role in the regulation of pain. Like all GPCRs they have a common structure that consists of seven transmembrane domains with an extracellular amino (N)-terminus and an intracellular carboxyl-terminus. The human δ opioid receptor (h(δOR) has two polymorphic variants. A single-nucleotide polymorphism causes replacement of Phe with Cys at the amino acid position 27 in the receptor N-terminus. The allelic frequency of hδORCys27, the less common variant, is about 10% in Caucasians. In this study, the two hδOR variants were expressed in heterologous expression systems and their biosynthesis was characterized in detail using various cell biological and biochemical techniques. In particular, the role of receptor heteromerization and opioid receptor pharmacological chaperones in processing, maturation and trafficking of the variants was assessed. The hδOR variants showed significant differences in maturation and trafficking. The hδORCys27 had a significantly lower maturation efficiency compared with hδORPhe27. In addition, long-term receptor expression led to the accumulation of hδORCys27 in the endoplasmic reticulum (ER) and also impaired receptor targeting to ER-associated degradation. The hδOR variants also differed at the cell surface, as the hδORCys27 variant was internalized constitutively in a faster and more extensive manner than hδORPhe27. However, the variants had similar pharmacological properties and activated G proteins in an identical manner. This study also showed that hδORCys27 acted in a dominant negative manner and redirected some hδORPhe27 precursors to degradation. This resulted in impaired plasma membrane expression of hδORPhe27 in co-transfected cells. The hδOR variants were found to form heteromers early in the secretory pathway, which is the most likely reason for the dominant negative behavior of hδORCys27 on hδORPhe27. The mechanism of action of opioid receptor pharmacological chaperones, membrane-permeable opioid ligands, was investigated in detail using hδORCys27 and its mutant form hδORCys27-(Asp95Ala) as models. Opioid antagonists were found to be able to bind to and stabilize receptor precursors in the ER and enhance their dissociation from the ER molecular chaperone calnexin. This led to an increase in the number of receptors at the plasma membrane. In addition, hδORPhe27, like hδORCys27, was responsive to antagonist treatment whether the variants were expressed together or individuallyTiivistelmä Opioidireseptorit kuuluvat G-proteiinikytkentäisiin reseptoreihin, ja niillä on tärkeä rooli kipuaistimuksen säätelyssä. Ne ovat solukalvoproteiineja, joiden aminohappoketju läpäisee kalvon seitsemän kertaa. Reseptorien aminoterminaalipää sijaitsee solun ulkopuolella ja karboksiterminaalipää solun sisällä. Ihmisen δ-opioidireseptori esiintyy kahtena polymorfisena muotona, Phe27:nä ja Cys27:nä, joissa aminohappo 27 on joko fenyylialaniini (Phe) tai kysteiini (Cys). Cys27 on harvinaisempi muoto, ja sen yleisyys on noin 10 % eurooppalaista alkuperää olevalla väestöllä. Tämän väitöskirjan tavoitteena oli tutkia δ-opioidireseptorin varianttimuotojen biosynteesiä reseptoriproteiinia tuottavissa heterologisissa solumalleissa (HEK293- ja SH-SY5Y-solut) solubiologisilla ja biokemiallisilla menetelmillä.. Väitöskirja osoittaa, että δ-opioidireseptorin varianttimuotojen välillä on eroa prosessoinnissa. Cys27-varianttia kuljetetaan endoplasmakalvostosta solun pinnalle vähemmän kuin Phe27-varianttia, ja pitkäaikainen reseptorituotanto johtaa vastasyntetisoituneiden reseptorien kerääntymiseen solun sisälle. Samalla reseptorien ohjaus proteasomihajotukseen heikkenee. Soluissa, jotka tuottavat molempia varianttimuotoja samanaikaisesti, Cys27-variantin havaittiin ohjaavan myös Phe27-varianttia proteasomihajotukseen vähentäen sen kuljetusta solun pinnalle. Tämä Cys27-variantin dominanttinegatiivinen ominaisuus johtuu todennäköisesti siitä, että variantit muodostavat dimeerisen rakenteen endoplasmakalvostossa. Havaittiin myös, että Cys27-varianttireseptorit ohjataan solun pinnalta lysosomihajotukseen tehokkaammin kuin vastaavat Phe27-varianttimuodot. Prosessointieroista huolimatta variantit eivät poikkea toisistaan farmakologisilta ominaisuuksiltaan, ja ne aktivoivat G proteiineja samalla tavalla. Väitöskirjassa tutkittiin myös farmakologisten kaperonien toimintamekanismeja käyttämällä mallina δ-opioidireseptorin Cys27-varianttia ja sen pistemutaatiota (Asp95Ala). Farmakologisten kaperonien eli reseptorispesifisten ligandien todettiin sitoutuvan reseptoreihin endoplasmakalvostossa ja stabiloivan niiden rakennetta, mikä vähentää reseptorin ja proteiinien laadunvalvontaan osallistuvan kaperonin, kalneksiinin, välistä vuorovaikutusta. Tämä johtaa reseptorien määrän kasvuun solun pinnalla
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Nanekar, R. (Rahul). "Biochemical and biophysical studies on adenosine receptors and their interaction partners." Doctoral thesis, Oulun yliopisto, 2016. http://urn.fi/urn:isbn:9789526211022.
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Abstract Adenosine receptors are heterotrimeric guanine nucleotide-binding (G protein)-coupled receptors (GPCRs) that mediate the effects of the endogenous agonist adenosine. The adenosine A3 receptor (A3R) is the least explored among the four human adenosine receptor subtype members (A1, A2A, A2B and A3) and it is implicated in both neuroprotective and neurodegenerative effects. During the course of this work, the production of the recombinant human A3R in yeast and insect cells was evaluated and heteromerization between the human adenosine A2A receptor (A2AR) and the dopamine D2 receptor (D2R) was studied. A3R with carboxyl-terminal GFP tag was expressed in the yeast Saccharomyces cerevisiae upto 15 mg per litre of culture. Another yeast Pichia pastoris increased the expression up to 108 mg/L of the same receptor when grown in bioreactors. Despite the very high expression levels, purification of A3R from both yeasts was a daunting task, as the aggregation of the receptor could not be averted. In this study, insect cells have been found out to be more suitable host for A3R expression: 10µg of the monomeric A3R could be purified from one liter of insect cell culture. For successful crystallization thermostability of the A3R was to be improved. This work has demonstrated that insertion of T4L, a fusion protein, in the third intracellular loop of A3R increased the thermostability of the receptor by 10°C. As a next step, the combination of point mutations based on alanine-scanning mutagenesis and a fusion protein approach could be useful to stabilize and further crystallize the A3R. This work has demonstrated that the amounts of A3R expressed in insect cells and the final yield of the receptor isolated by affinity purifications, forms a good basis for the beginning of biochemical characterization Receptor heteromerization is a mechanism used by GPCRs to diversify their signaling properties and functions. The human A2AR and D2R heteromers exist in the GABAergic enkephalinergic neurons. The domains responsible for forming intermolecular contacts were purified from Escherichia coli (E. coli). Using biochemical/biophysical techniques such as native-PAGE and mass spectrometry, It was validated that purified carboxyl-terminus of the A2AR and the 3rd intracellular loop of D2R form heterodimers. The investigation of purified calmodulin protein binding to the 3rd intracellular loop of D2R showed that the protein-protein interactions are calcium dependentTiivistelmä Adenosiinireseptorit kuuluvat G-proteiinikytkeiset reseptorit (GPCR:t) proteiiniperheeseen. Adenosiinireseptorit välittävät endogeenisen ligandinsa adenosiinin vaikutuksia solukalvolta solunsisäisiin signaalijärjestelmiin. Adenosiini A3 reseptori (A3R) on adenosiinireseptorien neljästä alatyypistä (A1, A2A, A2B ja A3) vähiten tutkittu. Aikaisempien tutkimusten perusteella A3 reseptori yhdistetään sekä hermosoluja suojaaviin että rappeuttaviin tapahtumiin. Tässä työssä arvioitiin sekä ihmisen rekombinantti-A3R:n tuottumista hiiva- ja hyönteissoluissa että tutkittiin ihmisen adenosiini A2A reseptorin (A2AR) ja dopamiini D2 reseptorin (D2R) heteromerisoitumista. Rekombinantti A3 reseptori- vihreä fluoresoiva proteiini (GFP) fuusioproteiinia tuotettiin Saccharomyces cerevisiae -hiivassa 15 mg litrassa kasvatusliuosta. Pichia pastoris -hiivakanta taas kasvatti saman reseptorin tuottumista aina 108 mg/l saakka, kun tuotto tehtiin bioreaktorissa. Hyvin korkeasta tuottotasosta huolimattaA3R:n puhdistus hiivasta oli ylitsepääsemätön tehtävä, sillä reseptorin saostumista ei voinut välttää. Työssä havaittiin, että hyönteissolut sopivat paremmin A3R:n tuottoon: noin 10 µg monomeerista A3R:a voitiin puhdistaa litran hyönteissoluviljelmästä. Reseptorin stabiilisuuden lisääminen helpottaa reseptorin biokemiallista ja biofysikaalista karakterisointia. Tässä työssä osoitettiin, että T4L-proteiinin lisääminen A3R:n kolmannen solunsisäisen silmukan paikalle lisää reseptorin lämpöstabiilisuutta 10 °C. Jatkotutkimuksissa voitaisiin käyttää alaniiniskannausmutageneesiin perustuvien pistemutaatioiden ja fuusioproteiinin yhdistelmää A3R:n lisästabilointiin ja kiteytykseen. Tämän työn perusteella määrät, joilla A3R tuottuu hyönteissoluissa ja jotka saadaan eristettyä affiniteettipuhdistuksilla, muodostavat hyvän perustan proteiinin biokemialliselle karakterisoinnille. Reseptorin heteromerisoituminen on GPCR:en käyttämä mekanismi signalointiominaisuuksien ja toimintojen monipuolistamiseksi. Ihmisessä A2AR ja D2R heteromeereja on GABAergisissä enkefalinergisissä hermosoluissa. Molekyylien välisiin kontakteihin osallistuvat domeenit puhdistettiin Escherichia coli (E. coli) -bakteerista. Biokemiallisia ja biofysikaalisia tekniikoita kuten natiivi-PAGE:a ja massaspektrometriaa käyttäen vahvistettiin, että puhdistettu A2AR:n karboksiterminaalinen osa ja D2R:n kolmas solunsisäinen silmukka muodostavat heterodimeereja. Myös tutkittaessa puhdistetun kalmoduliini-proteiinin sitoutumista D2R:n kolmanteen solunsisäiseen silmukkaan osoitettiin proteiini-proteiini -vuorovaikutuksen olevan kalsiumista riippuvainen
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Kabli, Noufissa. "Uncovering the Functional Implications of Mu- and Delta-opioid Receptor Heteromerization in the Brain." Thesis, 2013. http://hdl.handle.net/1807/65509.
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Opioid Receptors (ORs) are involved in the pathophysiology of several neuropsychiatric conditions yet remain an untapped therapeutic resource. Although only mu-, delta-, and kappa-OR types have been cloned, additional subtypes result from complexes generated by direct receptor-receptor interactions. Mu- and delta-ORs form a heteromeric receptor complex with unique pharmacological and signalling properties distinct from those of mu- and delta-OR homomers. In these studies, we sought to characterize the ligand binding pocket and agonist-induced internalization profile of the mu-delta heteromer, to investigate mu-delta heteromer-specific signalling in brain, and to interrogate the contribution of this receptor complex to opioid-mediated behavioural effects.
In competition radioligand binding studies, delta-agonists displaced high affinity mu-agonist binding from the mu-delta heteromer but not the muOR homomer, suggestive of delta-agonists occupying or allosterically modulating the muOR ligand binding pocket within the heteromer. Delta-agonists induced internalization of the mu-delta heteromer in a dose-dependent, pertussis toxin resistant, and muOR- and deltaOR-dependent manner from the cell surface via the clathrin and dynamin endocytic machinery. Agonist-induced internalization of the mu-delta heteromer persisted following chronic morphine treatment conditions which desensitized the muOR homomer.
Using Galpha-specific GTPgammaS binding assays, we demonstrated that mu-delta heteromer signalling previously characterized in cell lines was present in the striatum and hippocampus, and did not desensitize following prolonged morphine treatment conditions which desensitized muOR homomer-mediated signalling.
Since delta-agonists which also target the mu-delta heteromer possess antidepressant-like and anxiolytic-like properties, we investigated the role of this receptor complex in mood regulation. We devised a strategy to selectively analyze the effects of the mu-delta heteromer by dissociating it using a specific interfering peptide aimed at a sequence implicated in mu-delta heteromerization. The interfering peptide abolished the unique pharmacological and trafficking properties of delta-agonists at the mu-delta heteromer and dissociated this receptor complex in vitro. Intra-accumbens administration of the interfering peptide disrupted the mu-delta interaction in vivo and allowed for isolation of the mu-delta heteromer contribution to the mood-regulatory effects of a delta-agonist with activity at the heteromer. Activation of the mu-delta heteromer in the nucleus accumbens produced antidepressant-like and anxiolytic-like actions in animal models of depression and anxiety.
Book chapters on the topic "Receptor heteromerization"
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Fuxe, Kjell, Sergi Ferré, Maria Torvinen, Jöelle Hillion, Ingrid Strömberg, Ove Franzén, Carlos Ibanéz, et al. "Heteromerization of Adenosine and Dopamine Receptor Subtypes: Relevance for Neuronal Integration in Normal and Pathological States." In Catecholamine Research, 199–204. Boston, MA: Springer US, 2002. http://dx.doi.org/10.1007/978-1-4757-3538-3_46.
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Fernández-Dueñas, Víctor, Thierry Durroux, and Francisco Ciruela. "Time-Resolved Fluorescence Resonance Energy Transfer Using Fluorescent Ligands to Study Native G Protein-Coupled Receptor Heteromerization in Brain." In Neuromethods, 107–15. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1522-5_8.
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Ferre, Sergi. "Receptor Heteromerization." In Catecholamine Research in the 21st Century, 91. Elsevier, 2014. http://dx.doi.org/10.1016/b978-0-12-800044-1.00078-7.
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Pfleger, Kevin Donald, Sanam Mustafa, Heng Boon See, Ruth Marie Seeber, and Mohammed Akli Ayoub. "Profiling Novel Pharmacology Resulting from G Protein-Coupled Receptor Heteromerization." In BASIC/TRANSLATIONAL - G Protein-Coupled Receptor Biology, P1–178—P1–178. The Endocrine Society, 2011. http://dx.doi.org/10.1210/endo-meetings.2011.part1.p8.p1-178.
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Maggio, Roberto, Cristina Rocchi, and Marco Scarselli. "Experimental Strategies for Studying G Protein-Coupled Receptor Homo- and Heteromerization with Radioligand Binding and Signal Transduction Methods." In Methods in Enzymology, 295–310. Elsevier, 2013. http://dx.doi.org/10.1016/b978-0-12-391862-8.00016-8.
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Gomes, Ivone, Wakako Fujita, Moraje V. Chandrakala, and Lakshmi A. Devi. "Disease-Specific Heteromerization of G-Protein-Coupled Receptors That Target Drugs of Abuse." In Progress in Molecular Biology and Translational Science, 207–65. Elsevier, 2013. http://dx.doi.org/10.1016/b978-0-12-386931-9.00009-x.
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