Fluoreszenzmikroskopische Untersuchungen zur Interaktion G-Protein gekoppelter Rezeptoren

G-Protein gekoppelte Rezeptoren (GPCR) sind Rezeptoren mit 7 Transmembrandomänen. Nach Bindung ihres Liganden werden über die Kopplung von G-Proteinen rezeptorspezifisch Signaltransduktionswege aktiviert. Ein bislang nicht ausreichend verstandener Prozess für die Funktion von GPCR ist deren Oligomerisierung. Für einige GPCR konnte gezeigt werden, dass die Oligomerisierung den Rezeptortransport und/oder die Dynamik der Rezeptoraktivierung moduliert. Dabei ist noch nicht aufgeklärt, ob die entsprechenden GPCR ausschließlich als Oligomere oder in einem bestimmten Monomer-Dimer Verhältnis (M/D) vorliegen und welcher Dynamik dieses Verhältnis unterliegt. In dieser Arbeit wurde die Homo-Oligomerisierung des Endothelin-B-Rezeptors (ETBR), des Vasopressin-V2-Rezeptors (V2R) und der Corticotropin-Releasing-Factor-Rezeptoren Typ 1 (CRF1R) und Typ 2(a) (CRF2(a)R) analysiert. Im Anschluss an diese Untersuchungen wurde das M/D der GPCR bestimmt. Zur Detektion der Protein-Protein Interaktionen wurden die biophysikalischen Methoden Fluoreszenz-Resonanz-Energie-Transfer (FRET) und Fluoreszenz-Kreuzkorrelations-Spektroskopie (FCCS) eingesetzt. Mit Hilfe der FCCS konnte das spezifische M/D der GPCR bestimmt und über FRET ein Unterschied in der Interaktions-Dynamik zwischen den GPCR der Familie 1 (am Bsp. des V2R) und der Familie 2 (am Bsp. des CRF1R) ermittelt werden. Des Weiteren lieferten die genutzten Methoden den Nachweis, dass der zum CRF1R homologe CRF2(a)R ausschließlich als Monomer vorliegt. Zusätzliche Untersuchungen an Signalpeptidmutanten des CRF1R und des CRF2(a)R weisen darauf hin, dass das Pseudosignalpeptid des CRF2(a)R, welches bislang einzigartig in der Superfamilie der GPCR ist, die Oligomerisierung des Rezeptors verhindert. Zusätzlich zu diesen neuen Daten konnte in dieser Arbeit erstmals ein Zusammenhang zwischen Rezeptorinteraktion und G-Protein Selektivität für den CRF1R und den CRF2(a)R festgestellt werdenThe heptahelical G protein-coupled receptors (GPCRs) are important drug targets. Following activation by their ligands, they exert their function via the binding of G proteins and activation of specific signal transduction cascades. To date, the functional significance of the oligomerization of GPCRs is not completely understood. For some GPCRs it could be shown that the oligomerization modulates receptor transport and/or the dynamics of receptor activation. Most importantly, it is not clear whether the GPCRs exist exclusively as oligomers or in a certain monomer-dimer ratio (M/D) or whether a given ratio is dynamic. In this work, the homo-oligomerization of the endothelin-B-receptor (ETBR), the vasopressin-V2-receptor (V2R) and the corticotropin-releasing-factor-receptors type 1 (CRF1R) and type 2(a) (CRF2(a)R) was analysed. In addition, the M/D of these GPCRs was determined. For the detection of the protein-protein interactions, the following biophysical methods were established: fluorescence-resonance-energy-transfer (FRET) and fluorescence-crosscorrelation-spectroscopy (FCCS). With the help of FCCS, a specific M/D could be determined for each of the GPCRs. Using FRET, differences in the interaction dynamics between family 1 (V2R) and family 2 GPCRs (CRF1R) could be described. Moreover, it was experimentally verified that the CRF2(a)R is exclusively expressed as a monomer, in contrast to the other GPCRs and even the highly homologous CRF1R. Using signal peptide swap experiments, it could be demonstrated that the N-terminal pseudo signal peptide of the CRF2(a)R, which is so far unique in the superfamily of GPCRs, prevents oligomerization of the receptor. In addition, a relation of receptor oligomerization and G protein coupling selectivity was established for the CRF1R and the CRF2(a)R which is novel for the GPCR protein family

The Pseudo Signal Peptide of the Corticotropin-releasing Factor Receptor Type 2A Prevents Receptor Oligomerization

N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF2(a)R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF2(a)R and the homologous corticotropin-releasing factor receptor type 1 (CRF1R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF2(a)R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general

The Specific Monomer/Dimer Equilibrium of the Corticotropin-releasing Factor Receptor Type 1 Is Established in the Endoplasmic Reticulum

G protein-coupled receptors (GPCRs) represent the most important drug targets. Although the smallest functional unit of a GPCR is a monomer, it became clear in the past decades that the vast majority of the receptors form dimers. Only very recently, however, data were presented that some receptors may in fact be expressed as a mixture of monomers and dimers and that the interaction of the receptor protomers is dynamic. To date, equilibrium measurements were restricted to the plasma membrane due to experimental limitations. We have addressed the question as to where this equilibrium is established for the corticotropin-releasing factor receptor type 1. By developing a novel approach to analyze single molecule fluorescence cross-correlation spectroscopy data for intracellular membrane compartments, we show that the corticotropin-releasing factor receptor type 1 has a specific monomer/dimer equilibrium that is already established in the endoplasmic reticulum (ER). It remains constant at the plasma membrane even following receptor activation. Moreover, we demonstrate for seven additional GPCRs that they are expressed in specific but substantially different monomer/dimer ratios. Although it is well known that proteins may dimerize in the ER in principle, our data show that the ER is also able to establish the specific monomer/dimer ratios of GPCRs, which sheds new light on the functions of this compartment

Quantitative modelling of amyloidogenic processing and its influence by SORLA in Alzheimer's disease

The extent of proteolytic processing of the amyloid precursor protein (APP) into neurotoxic amyloid‐β (Aβ) peptides is central to the pathology of Alzheimer's disease (AD). Accordingly, modifiers that increase Aβ production rates are risk factors in the sporadic form of AD. In a novel systems biology approach, we combined quantitative biochemical studies with mathematical modelling to establish a kinetic model of amyloidogenic processing, and to evaluate the influence by SORLA/SORL1, an inhibitor of APP processing and important genetic risk factor. Contrary to previous hypotheses, our studies demonstrate that secretases represent allosteric enzymes that require cooperativity by APP oligomerization for efficient processing. Cooperativity enables swift adaptive changes in secretase activity with even small alterations in APP concentration. We also show that SORLA prevents APP oligomerization both in cultured cells and in the brain in vivo, eliminating the preferred form of the substrate and causing secretases to switch to a less efficient non‐allosteric mode of action. These data represent the first mathematical description of the contribution of genetic risk factors to AD substantiating the relevance of subtle changes in SORLA levels for amyloidogenic processing as proposed for patients carrying SORL1 risk alleles

Quantitative modelling of amyloidogenic processing and its influence by SORLA in Alzheimer\u27s disease

The extent of proteolytic processing of the amyloid precursor protein (APP) into neurotoxic amyloid-β (Aβ) peptides is central to the pathology of Alzheimer\u27s disease (AD). Accordingly, modifiers that increase Aβ production rates are risk factors in the sporadic form of AD. In a novel systems biology approach, we combined quantitative biochemical studies with mathematical modelling to establish a kinetic model of amyloidogenic processing, and to evaluate the influence by SORLA/SORL1, an inhibitor of APP processing and important genetic risk factor. Contrary to previous hypotheses, our studies demonstrate that secretases represent allosteric enzymes that require cooperativity by APP oligomerization for efficient processing. Cooperativity enables swift adaptive changes in secretase activity with even small alterations in APP concentration. We also show that SORLA prevents APP oligomerization both in cultured cells and in the brain in vivo, eliminating the preferred form of the substrate and causing secretases to switch to a less efficient non-allosteric mode of action. These data represent the first mathematical description of the contribution of genetic risk factors to AD substantiating the relevance of subtle changes in SORLA levels for amyloidogenic processing as proposed for patients carrying SORL1 risk alleles

Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS

The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 ± 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other

Es wird einmal

Ausstellungsband zur Ausstellung " Die Lange Nacht der Kreativität". Ausgestellt wurden Abschlussarbeiten und herausragende Werke ehemaliger Studierender der Illustration an dem Department Design der HAW Hamburg

A cellular census of human lungs identifies novel cell states in health and in asthma.

Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (TH2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a TH2-dominated interactome in asthmatic lungs.This work was funded by OpenTargets, an open innovation public-private partnership (http://www.opentargets.org), a GlaxoSmithKline collaborative agreement with University Medical Center Groningen, Wellcome (WT206194), EMBO and HFSP Long Term fellowships to R. Vento-Tormo, the Marie Curie ENLIGHT-TEN training network for Tomas Gomes, the Lung Foundation Netherlands (projects no 5.1.14.020 and 4.1.18.226), and Health-Holland, Top Sector Life Sciences & Health. LMS acknowledges funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 753039, HBS acknowledges funding by the Helmholtz Association and the German Center for Lung Research (DZL). F.J.T. acknowledges financial support by the German Research Foundation (DFG) within the Collaborative Research Centre 1243, Subproject A17, by the Helmholtz Association (Incubator grant sparse2big, grant number ZT-I-0007) and by the Chan Zuckerberg Initiative DAF (advised fund of Silicon Valley Community Foundation), grant number 182835