Saccharomyces cerevisiae: a tool to evaluate the signaling pathways of adhesion-GPCRs.

Adhesion G-protein coupled receptors (aGPCRs) are the second largest family of GPCRs, with 33 homologues in humans. Despite the multitude of roles that they play, the majority of them are still orphans and the signaling pathways involved in their activations barely unknown. The aim of this study is to investigate the constitutive activity of different truncations of the N-terminus of the GPR56, GPR64 and GPR112 receptors that belong to the subfamily VIII of the aGPCRs, in order to shed light on their downstream pathways. Modified strains of Saccharomyces cerevisiae have been used for this study. These strains contain chimeric Gp1a/Gα proteins that are able to couple to a heterologous GPCR and give a response via the pheromone-response pathway. The constitutive activity has been evaluated as the growth of yeast cells in a medium lacking of histidine, since the production of this amino acid (essential for S. cerevisiae) is due to the activation of the receptor. During the initial screen the GPR112-7TM and the GPR112-GPS have been tested in all the strains, coupling different Gα proteins; this was necessary to optimise the assay in itself and to discriminate the strains to use later. In the following part of the study, all the constructs generated by the truncation of the N-termini have been tested in selected yeast strains, based on the initial screen and the information gathered in literature. Intriguingly, the receptors showed different responses among the different strains and a same receptor displayed a different mechanism of activation based on the signaling pathway involved. In fact, the GPR64-ZO1 seems to have interactions with other components on the extracellular region, the GPR56 shows a higher activity when the N-terminal fragment is cleaved out, the GPR112-GAIN (via Gα14) modulates the activity of the receptor, which is fully activated when the N-terminus is removed. Moreover, the GPR64-ZO1 modulates the activity of the receptor positively via Gα14 but negatively via Gα12, the three constructs of the GPR112 show the same level of activation via Gα16, Gα12 and Gα13, but via Gα14 the receptor is fully activated when the N-terminus is removed, the GPR56-7TM has a remarkable activity via Gα12 and Gα13

The cationic region of Rhes mediates its interactions with specific Gβ subunits

Ras homologue enriched in striatum (Rhes) is a small monomeric G protein which functions in a variety of cellular processes, including attenuation of G protein-coupled receptor (GPCR)signalling. There have been many studies into the effects of Rhes, but there is no molecular information about how Rhes might bring about these effects. Rhes shares striking sequence homology to AGS1 (activator of G protein signalling 1) and we considered whether the two proteins function in similar ways. AGS1 binds to the Gβ1 subunit of heterotrimeric G proteins and we have used yeast two-hybrid studies to show that Rhes binds selectively to Gβ1, Gβ2 and Gβ3 subunits. Binding to the Gβ subunits involves the cationic regions of AGS1 and Rhes, and we used Rhes-AGS1 chimeras to show that their different cationic regions determine the Gβ-specificity of the interactions. Possible implications of this interaction for the activity of Rhes are discussed

Intranuclear Signaling Cascades Triggered by Nuclear GPCRs

G protein-couped receptors (GPCRs) play a key role on cellular membranes, where they respond to a broad array of extracellular signals such as lipids, peptides, proteins and sensory agents. Intracellular biological responses triggered by these receptors include hormone secretion, muscle contraction, cellular metabolism a tyrosine kinase receptors transactivation. Recent results indicate that GPCRs localize to and signal also at nuclear level, thus regulating distinct signaling pathways which can also result from the integration of extracellular and intracellular stimuli. Nuclear GPCRs play a central role in many cellular processes, including regulation of gene transcription, cellular proliferation, neovascularization and RNA synthesis. On nuclear membranes and in nucleoplasm are present all the downstream signal transduction components of GPCRs, including G proteins, adenylyl cyclase, and second messengers such as Ca++, ERKs, p38MAPK and other protein kinases. Nuclear GPCRs may be constitutively active or may be activated by ligands internalized from the extracellular space or synthesized within the cell. The translocation of membrane receptors to the nucleus could be attributed to the presence of a Nuclear Localization Signal, which is present in the eighth helix or in the third intracellular loop of a limited number of GPCRs. However, several sequence motifs that do not resemble classical Nuclear Localization Signals can promote import of GPCRs. In this review we discuss the most recent results on nuclear localization and signaling of several GPCRS

Concepts of GPCR-controlled navigation in the immune system

G-protein-coupled receptor (GPCR) signaling is essential for the spatiotemporal control of leukocyte dynamics during immune responses. For efficient navigation through mammalian tissues, most leukocyte types express more than one GPCR on their surface and sense a wide range of chemokines and chemoattractants, leading to basic forms of leukocyte movement (chemokinesis, haptokinesis, chemotaxis, haptotaxis, and chemorepulsion). How leukocytes integrate multiple GPCR signals and make directional decisions in lymphoid and inflamed tissues is still subject of intense research. Many of our concepts on GPCR-controlled leukocyte navigation in the presence of multiple GPCR signals derive from in vitro chemotaxis studies and lower vertebrates. In this review, we refer to these concepts and critically contemplate their relevance for the directional movement of several leukocyte subsets (neutrophils, T cells, and dendritic cells) in the complexity of mouse tissues. We discuss how leukocyte navigation can be regulated at the level of only a single GPCR (surface expression, competitive antagonism, oligomerization, homologous desensitization, and receptor internalization) or multiple GPCRs (synergy, hierarchical and non-hierarchical competition, sequential signaling, heterologous desensitization, and agonist scavenging). In particular, we will highlight recent advances in understanding GPCR-controlled leukocyte navigation by intravital microscopy of immune cells in mice

The role of the RACK1 ortholog Cpc2p in modulating pheromone-induced cell cycle arrest in fission yeast

The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1) is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G1/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gβ-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G1 arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase

Targeting protein function: the expanding toolkit for conditional disruption

A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcripts, meaning that the effects of gene perturbation are exhibited over longer time frames than the process under investigation. There has been much activity over the last few years to address this fundamental problem. In the present review, we describe recent advances in disruption technologies acting at the level of the expressed protein, involving inducible methods of protein cleavage, (in)activation, protein sequestration or degradation. Drawing on examples from model organisms we illustrate the utility of fast-acting techniques and discuss how different components of the molecular toolkit can be employed to dissect previously intractable biochemical processes and cellular behaviours.</jats:p

<i>N</i>‐Palmitoylglycine and other <i>N</i>‐acylamides activate the lipid receptor G2A/GPR132

The G‐protein‐coupled receptor GPR132, also known as G2A, is activated by 9‐hydroxyoctadecadienoic acid (9‐HODE) and other oxidized fatty acids. Other suggested GPR132 agonists including lysophosphatidylcholine (LPC) have not been readily reproduced. Here, we identify N‐acylamides in particular N‐acylglycines, as lipid activators of GPR132 with comparable activity to 9‐HODE. The order‐of‐potency is N‐palmitoylglycine &gt; 9‐HODE ≈ N‐linoleoylglycine &gt; linoleamide &gt; N‐oleoylglycine ≈ N‐stereoylglycine &gt; N‐arachidonoylglycine &gt; N‐docosehexanoylglycine. Physiological concentrations of N‐acylglycines in tissue are sufficient to activate GPR132. N‐linoleoylglycine and 9‐HODE also activate rat and mouse GPR132, despite limited sequence conservation to human. We describe pharmacological tools for GPR132, identified through drug screening. SKF‐95667 is a novel GPR132 agonist. SB‐583831 and SB‐583355 are peptidomimetic molecules containing core amino acids (glycine and phenylalanine, respectively), and structurally related to previously described ligands. A telmisartan analog, GSK1820795A, antagonizes the actions of N‐acylamides at GPR132. The synthetic cannabinoid CP‐55 940 also activates GPR132. Molecular docking to a homology model suggested a site for lipid binding, predicting the acyl side‐chain to extend into the membrane bilayer between TM4 and TM5 of GPR132. Small‐molecule ligands are envisaged to occupy a “classical” site encapsulated in the 7TM bundle. Structure‐directed mutagenesis indicates a critical role for arginine at position 203 in transmembrane domain 5 to mediate GPR132 activation by N‐acylamides. Our data suggest distinct modes of binding for small‐molecule and lipid agonists to the GPR132 receptor. Antagonists, such as those described here, will be vital to understand the physiological role of this long‐studied target

Expression and purification of recombinant G protein-coupled receptors: A review

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques

Heterologous production, characterization and isolation of selected G protein-coupled receptors for structural studies

G protein-coupled receptors (GPCRs) play regulatory roles in many different physiological processes and they represent one of the most important class of drug targets. However, due to the lack of three-dimensional structures, structure based drug design has not been possible. The major bottleneck in getting three-dimensional crystal structure of GPCRs is to obtain milligram quantities of pure, homogenous and stable protein. Therefore, during my Ph.D. thesis, I focused on expression, characterization and isolation of three GPCRs namely human bradykinin receptor subtype 2 (B2R), human angiotensin II receptor subtype 1 (AT1aR), and human neuromedin U receptor subtype 2 (NmU2R). These receptors were heterologously produced in three different expression systems (i.e. Pichia pastoris, insect cells and mammalian cells), biochemically characterized and subsequently solubilized and purified for structural studies The human bradykinin receptor subtype 2 (B2R) is constitutively expressed in a variety of cells, including endothelial cells, vascular smooth muscle cells and cardiomyocytes. Activation of B2R is important in pathogenesis of inflammation, pain, tissue injury and cardioprotective mechanisms. During this study, recombinant B2R was produced in methylotrophic yeast Pichia pastoris (3.5 pmol/mg), insect cells (10 pmol/mg) and mammalian cells (60 pmol/mg). The recombinant receptor was characterized in terms of [3H] bradykinin binding, G protein coupling, localization, and glycosylation. Subsequently, it was solubilized and purified using affinity chromatography. Homogeneity and stability of purified B2R was monitored by gel filtration analysis. Milligram amounts of pure and stable receptor were obtained from BHK cells and Sf9 cells, which were used for three-dimensional crystallization attempts. The second receptor, which I worked on, is human angiotensin II receptor subtype 1 (AT1aR). AT1aR is distributed in smooth muscle cells, liver, kidney, heart, lung and testis. Activation of AT1aR is implicated in the regulation of blood pressure, hypertension and cardiovascular diseases. Recombinant AT1aR was produced at high levels in Pichia pastoris (167 pmol/mg), while at moderate levels in insect cells (29 pmol/mg) and mammalian cells (32 pmol/mg). The recombinant receptor was characterized in terms of [3H] angiotensin II binding, localization, and glycosylation. Subsequently, the receptor was solubilized and purified using affinity chromatography. Homogeneity and stability of purified AT1aR was monitored by gel filtration analysis. Milligram amounts of pure and stable receptor were obtained from Pichia pastoris, which were used for threedimensional crystallization attempts. In addition to B2R and AT1aR, I also attempted to produce and isolate the human neuromedin U receptor subtype 2 (NmU2R), which was deorphanized recently. It is found in highest abundance in the central nervous system, particularly the medulla oblongata, spinal cord and thalamus. The distribution of this receptor suggests its regulatory role in sensory transmission and modulation. During this study, recombinant NmU2R was produced in Pichia pastoris (6 pmol/mg) and BHK cells (9 pmol/mg). Recombinant receptor was characterized with regard to [125I] NmU binding, localization and glycosylation. Subsequently, the receptor was solubilized and purified using affinity chromatography. Due to its low expression level, further expression optimization is required in order to obtain milligram amounts for structural studies. The long-term goal of this study was to obtain three-dimensional crystal structure of recombinant GPCRs. However, 3-dimensional crystallization of human recombinant membrane proteins still remains a difficult task. On the other hand, recent advances in the solid-state NMR spectroscopy offer ample opportunities to study receptor-ligand systems, provided milligram quantities of purified receptor are available. Therefore, in parallel to 3-dimensional crystallization trials, purified B2R was also used for solid-state NMR analysis in order to investigate the receptor bound conformation of bradykinin. Preliminary results are promising and indicate significant structural changes in bradykinin upon binding to B2R. Further experiments are ongoing and will hopefully result in the structure of receptor bound bradykinin. One of the challenges in GPCR crystallization is the small hydrophilic surface area that is available to make crystal contacts. One possibility to overcome this problem can be the reconstitution of a GPCR complex with an interacting protein for cocrystallization. For this purpose, I coexpressed B2R and AT1aR, which form a stable heterodimer complex, in BHK cells. I could successfully isolate the heterodimer complex by using two-step affinity purification. Unfortunately, this complex was not stable over time and disassociates within three days of purification. However, during coexpression of B2R and AT1aR in BHK cells, I observed that B2R was localized in the plasma membrane in coexpressing cells while it was retained intracellularly when expressed alone. This coexpression of AT1aR with B2R resulted in a four-fold increase in [3H] bradykinin binding sites on the cell surface. In addition, these two receptors were cointernalized in response to their individual specific ligands. Interestingly, colocalization of B2R and AT1aR was also found in human foreskin fibroblasts (which endogenously express both receptors), in line with the possibility that heterodimerization may be required for surface localization of B2R in native tissues as well. This is the first report where surface localization of a peptide GPCR is triggered by a distantly related peptide GPCR. These data support the hypothesis that heterodimerization may be a prerequisite for cell surface localization of some GPCRs. A second approach that I followed to stabilize the purified B2R was to reconstitute the B2R-&#946;-arrestin complex. &#946;-arrestin is a cytosolic protein that participates in agonist mediated desensitization of GPCRs and therefore dampens the cellular responses initiated by the activation of GPCRs. I tried to reconstitute B2R-&#946;-arrestin complex in vitro by mixing purified B2R and purified &#946;-arrestin. But, no interaction of these two proteins was observed in the pull-down assays. However, a C-terminal mutant of B2R (where a part of the C-terminus of the B2R is exchanged with that of the vasopressin receptor) was found to interact with &#946;-arrestin in vitro as revealed by pull-down assays. In conclusion, this work establishes the production, characterization and isolation of three recombinant human GPCRs. Recombinant receptors were produced in milligram amounts and therefore, pave the way for structural analysis. The heterodimer complex of B2R-AT1aR and B2R-&#946;-arrestin complex can be of great help during crystallization. In addition, it was also found for the first time that the surface localization of a peptide GPCR can be triggered by heterodimerization with a distantly related peptide GPCR.Die G-Protein-gekoppelten Rezeptoren (GPCRs) stellen die größte Familie der Zelloberflächenrezeptoren dar. 1-5% des Wirbeltiergenoms kodiert für diese Rezeptorfamilie. Im Humangenom sind etwa 800-1000 Gene vertreten, die für GPCRs kodieren. Trotz der großen Unterschiede in ihrer Sequenz und Aktivierung haben alle GPCRs zwei Gemeinsamkeiten: (1) Ihre Architektur wird durch sieben Transmembranhelices beschrieben. (2) Ihre Funktion in der Signaltransduktion üben alle durch Aktivierung der heterotrimeren Guanylnukleotid-Bindeproteine (GProteine) aus. Die GPCRs sind an der Regulierung einer Vielzahl von physiologischen Prozessen beteiligt und stellen daher wichtige Ziele für die Medikamentenentwicklung dar. Bisher gibt es kaum Möglichkeiten zur strukturbasierenden Medikamentenentwicklung, da, bis auf das Rinder-Rhodopsin, nur sehr wenige Informationen zur dreidimensionalen Struktur von GPCRs verfügbar sind. Das Rinder-Rhodopsin nimmt allerdings unter den GPCRs eine Sonderstellung ein. Im Gegensatz zu allen übrigen GPCRs bindet es seinen Liganden, 11-cis Retinal, kovalent und liegt dann in der nicht-aktivierten Form vor. Zudem kann Rhodopsin in großen Mengen aus Rinderretina isoliert werden, wohingegen die übrigen GPCRs nur in geringen Mengen in ihren natürlichen Geweben vorkommen. Die vorliegende Arbeit verfolgt drei Ziele: Erstens sollen GPCRs durch heterologe Expression in hohen Ausbeuten hergestellt und biochemisch charakterisiert werden. Die Etablierung eines Solubilisierungs- und Aufreinigungsprotokolls stellt das zweite Ziel dar. Drittens soll die Interaktion von Ligand und Rezeptor mittels verschiedener Techniken untersucht werden. Grund für die erste Zielsetzung ist die geringe Verfügbarkeit reinen, homogenen und stabilen Proteins im Milligramm-Maßstab, welches die größte Hürde für strukturelle Untersuchungen von GPCRs darstellt. Hier wurden verschiedene Expressionssysteme zur heterologen Produktion von Membranproteinen etabliert. Die Wahl des Expressionssystems ist hierbei entscheidend, um posttranslationale Modifikationen wie Glykosylierung sowie die korrekte Faltung des Rezeptors zu gewährleisten. Neben E. coli haben sich hierbei vor allem eukaryotische Expressionssystems wie Pichia pastoris bewährt. In der vorliegenden Arbeit wurden drei GPCRs hergestellt und analysiert: der humane Bradykinin Rezeptor Typ 2 (B2R), der humane Angiotensin II Rezeptor Typ 1 (AT1aR) und der humane Neuromedin U Rezeptor Typ 2 (NmU2R). Diese drei Rezeptoren wurden in drei Expressionsystemen (Pichia pastoris, Insektenzellen und Säugerzellen) heterolog produziert und biochemisch charakterisiert. Für jedes der drei Proteine wurden Solubilisierungs- und Aufreinigungsprotokolle etabliert. Die aufgereinigten Proteine wurden anschließend für Kristallisationsexperimente, für Festkörper NMR Untersuchungen und weitere Experimente eingesetzt. Der erste untersuchte Rezeptor, B2R, kann vor allem in Endothelzellen, vaskulären glatten Muskelzellen und Kardiomyozyten nachgewiesen werden. Seine Aktivierung spielt bei der Entstehung von Entzündungen, Schmerz, Gewebsverletzung sowie herzschützenden Mechanismen eine Rolle. Im Rahmen der Doktorarbeit wurde B2R in der Hefe Pichia pastoris (3,5 pmol/mg), in BHK-Zellen (10 pmol/mg) und in Sf9-Zellen (60 pmol/mg) erfolgreich rekombinant produziert. Zur Charakterisierung wurde die Bindung des Liganden [3H] Bradykinin, die GProtein- Kopplung, zelluläre Lokalisierung sowie die Glykosylierung des Rezeptors untersucht. Der heterolog produzierte Rezeptor konnte in hoher Reinheit isoliert werden. Homogenität und Stabilität des aufgereinigten Proteins wurden mittels Gelfiltration analysiert. Aus BHK und Sf9 Zellen konnten Milligramm-Mengen reinen und stabilen Rezeptors isoliert werden, die zu Kristallisationsexperimenten verwendet wurden. Hier zeigten sich kristallartige Strukturen, die zur Zeit weiter charakterisiert werden. Der zweite untersuchte Rezeptor, AT1aR, kann in glatten Muskelzellen, Leber, Nieren, Herz, Lunge und Hoden nachgewiesen werden. Die Aktivierung dieses Rezeptors spielt eine Rolle bei der Regulation des Blutdrucks und bei cardiovaskulären Erkrankungen. Rekombinanter AT1aR konnte mit hoher Ausbeute (167 pmol/mg) in Pichia pastoris hergestellt werden. Die Ausbeute bei Produktion in Insektenzellen (29 pmol/mg) und Säugerzellen (32 pmol/mg) lag im mittleren Bereich. Der rekombinante Rezeptor wurde hinsichtlich der Bindung von [3H] Angiotensin II, der zellulären Lokalisierung und Glykosylierung charakterisiert. Im Anschluss wurde er erfolgreich mittels Affinitätschromatographie gereinigt. Homogenität und Stabilität des gereinigten AT1aR wurden mittels Gelfiltration analysiert. Aus Pichia pastoris konnte das Protein im Milligramm-Maßstab isoliert werden, so dass Kristallisationsexperimente möglich waren. Dem dritten Rezeptor, NmU2R, konnte erst kürzlich sein Ligand, Neuromedin U, zugeordnet werden. Der Rezeptor ist im zentralen Nervensystem, und hier insbesondere in der Medulla oblongata, dem Rückenmark und dem Thalamus lokalisiert. Aufgrund dieser Verteilung wird angenommen, dass er eine Rolle in der Regulation der Weiterleitung sensorischer Nervenimpulse sowie deren Modulation spielt. Während meiner Arbeit konnte ich bei der heterologen Produktion des Rezeptors Ausbeuten von 6 pmol/mg in Pichia pastoris und 9 pmol/mg in BHK Zellen erzielen. Der rekombinante Rezeptor wurde mittels Bindung eines Radioliganden ([125I] NmU) charakterisiert. Weiterhin wurde die zellulärer Lokalisierung und Glykosylierung des GPCRs untersucht. Obwohl der rekombinante NmU2R erfolgreich isoliert werden konnte, sind auf Grund der geringen Produktionsmengen zur Zeit keine Struktur untersuchungen möglich. Zur Analyse der pharmakologisch wichtigen Ligand-Rezeptor- Wechselwirkung wurde Festkörper NMR Spektroskopie eingesetzt. Durch die Verwendung von selektiv mit 13C und 15N markierten Peptiden können Konformationsänderung des Peptidliganden beim Binden des Rezeptors untersucht werden. Die Bestimmung der genauen Konformation des gebunden Liganden ist für die Medikamentenentwicklung von Bedeutung. In der vorliegenden Arbeit wurde mittels der Festkörper NMR Spektroskopie die Konformation des rezeptorgebunden Liganden, Bradykinin, untersucht. Die ersten Ergebnisse weisen auf signifikante Strukturänderungen Bradykinins hin, sobald es an den B2R bindet. Untersuchungen bezüglich Wechselwirkung von GPCRs mit anderen Protein sind auch für die Kristallisation relevant. Eine der Herausforderungen in der Kristallisation von GPCRs ist die kleine hydrophile Oberfläche, die zur Bildung von Kristallkontakten im Kristallgitter oft nicht ausreichend ist. Eine Möglichkeit, dieses Problem zu lösen, ist die Bildung eines stabilen Komplexes aus dem Rezeptor und einem interagierenden Protein. Zusätzlich kann der Rezeptor durch die Interaktion in eine weniger flexible Form überführt werden, was die Kristallisation und die spätere Strukturbestimmung erleichtern kann. Basierend auf diesem Ansatz wurden B2R und AT1aR, die einen stabilen heterodimeren Komplex bilden, in BHK Zellen ko-exprimiert. Bemerkenswert war hierbei dass B2R im Komplex mit AT1aR in der Plasmamembran vorzufinden war, während B2R alleine hauptsachlich in intrazellulären Membranen exprimiert wurde. Weiterhin führte die Koexpression der beiden Rezeptoren zu einem vierfachen Anstieg der [3H] Bradykinin Bindungsstellen auf der Zelloberfläche. Es konnte ebenfalls nachgewiesen werden, dass nach der Stimulation mit nur einem der beiden rezeptorspezifischen Liganden beide GPCRs zusammen internalisiert wurden. Dieses Phänomen wurde auch in menschlichen Vorhaut- fibroblasten nachgewiesen, in denen beide Rezeptoren vorkommen. Die erhaltenen Ergebnisse deuten darauf hin, dass auch in nativen Geweben die Anwesenheit des AT1aR für die Expression und den Transfer des B2R zur Plasmamembran nötig ist. Diese Daten unterstützen die Hypothese, dass Heterodimerisierung eine Voraussetzung für die Zelloberflächenlokalisierung bestimmter GPCRs ist. Der Ko-Komplex aus B2R und AT1aR konnte mittels dualer Affinitätschromatographie isoliert, wie durch SDS-PAGE Analyse, analytische Gelfiltration und Bindung von Radioliganden gezeigt werden konnte. „Pull-down“ Experimente, die drei Tage nach der Reinigung durchgeführt wurden, wiesen darauf hin, dass der Ko-Komplex nicht stabil war und zerfiel. Bei der Reinigung von Membranproteinen verursacht der Verlust von Lipiden während des Isolationsprozeßes oft eine Beeinträchtigung der Stabilität des Proteins. Auch das verwendete Detergenz beeinflusst die Stabilität von Membranproteinen. Experimente zur Verbesserung der Langzeitstabilität des Komplexes durch Zugabe von Lipiden und anderen Detergenzien sind in Vorbereitung. Die Bildung von Ko-Komplexen wurde zusätzlich mit Beta-Arrestin, einem Inhibitor der Kopplung von G-Proteinen und ihren Rezeptoren untersucht. Beta- Arrestin ist ein zytosolisches Protein, dass an der Desensibilisierung der Agoniststimulierten GPCRs beteiligt ist. Versuche, eine Ko-Komplexbildung aus gereinigtem B2R und gereinigtem Beta-Arrestin in vitro zu erzielen, schlugen fehl. In „Pull-Down“ Experimenten konnte keine Interaktion nachweisen werden. Wurde anstatt des nativen B2R eine C-terminale Mutante, bei welcher der C-Terminus des B2R gegen den des Vasopressinrezeptors ausgetauscht worden war, verwendet, konnte in vitro Ko- Komplexbildung mit Beta-Arrestin festgestellt werden. Mit Experimenten zur Bestimmung der Langzeitstabilität des Ko-Komplexes sowie zur Ko-Kristallisations wurde begonnen. Im Rahmen der vorliegenden Arbeit wurde die Produktion, Charakterisierung und Aufreinigung von drei rekombinanten humanen GPCRs etabliert. Die rekombinanten Rezeptoren wurden im Milligramm-Maßstab produziert. Damit ist die erste, wesentliche Hürde zur Strukturanalyse genommen. Der B2R-&#946;-Arrestin Komplex kann sich als vorteilhaft für die Kristallisation herausstellen. Zusätzlich konnte zum ersten Mal gezeigt werden, dass der Transfer eines GPCRs an die Zelloberfläche von der Heterodimerisierung mit einem anderen nicht-verwandten GPCR abhängig sein kann