TY - JOUR A1 - Niklaus, Marius A1 - Klingler, Philipp A1 - Weber, Katja A1 - Koessler, Angela A1 - Kuhn, Sabine A1 - Boeck, Markus A1 - Kobsar, Anna A1 - Koessler, Juergen T1 - Platelet Toll-Like-Receptor-2 and -4 Mediate Different Immune-Related Responses to Bacterial Ligands JF - TH Open N2 - Background  Like immune cells, platelets express toll-like receptors (TLRs) on their surface membrane. TLR2 and TLR4 are able to recognize bacterial antigens and have the potential to influence hemostatic functions and classical intracellular signaling pathways. This study investigated the role of TLR2 and TLR4 for immune-related functions in human platelets. Materials and Methods  Washed platelets and neutrophils were prepared from fresh human peripheral blood. Basal-, Pam3CSK4- (as TLR2 agonist) and Lipopolysaccharides (LPS; as TLR4 agonist) -induced CD62P expression, fibrinogen binding and TLR2 or TLR4 expression, intracellular reactive oxygen species (ROS) production in H2DCFDA-loaded platelets and uptake of fluorescence-labeled TLR ligands, and fluorophore-conjugated fibrinogen were evaluated by flow cytometry. Analysis of platelet–neutrophil complexes was performed after coincubation of washed platelets and neutrophils in the presence and absence of TLR2 or TLR4 agonists on poly-L-lysine coated surfaces, followed by immunostaining and immunofluorescence imaging. Results  Pam3CSK4 rapidly and transiently increased TLR2 and TLR4 expression. Over the course of 30 minutes after activation with Pam3CSK4 and LPS, the expression of both receptors decreased. Pam3CSK4-stimulated intracellular ROS production and the uptake of TLR ligands or fibrinogen much stronger than LPS. Besides, TLR4 activation led to a significant increase of platelet–neutrophil contacts. Conclusion  Stimulation leads to rapid mobilization of TLR2 or TLR4 to the platelet surface, presumably followed by receptor internalization along with bound TLR ligands. After activation, platelet TLR2 and TLR4 mediate different immune-related reactions. In particular, TLR2 induces intracellular responses in platelets, whereas TLR4 initiates interactions with other immune cells such as neutrophils. KW - receptors KW - immunity KW - cell-cell interactions KW - platelet physiology Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301401 VL - 6 IS - 3 SP - e156 EP - e167 ER - TY - JOUR A1 - Reinhold, A. K. A1 - Batti, L. A1 - Bilbao, D. A1 - Buness, A. A1 - Rittner, H. L. A1 - Heppenstall, P. A. T1 - Differential Transcriptional Profiling of Damaged and Intact Adjacent Dorsal Root Ganglia Neurons in Neuropathic Pain JF - PLoS ONE N2 - Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG) using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS). Two fluorescent tracers, Fluoroemerald (FE) and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropinreleasing hormone (CRH), providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and "bystanders," thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG. KW - peripheral nerve injury KW - sensory neurons KW - rat KW - involvement KW - mechanisms KW - receptors KW - inhibition KW - expression KW - sciatic nerve KW - inflammatory pain Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143290 VL - 10 IS - 4 ER - TY - JOUR A1 - Afonso-Grunz, Fabian A1 - Hoffmeier, Klaus A1 - Müller, Sören A1 - Westermann, Alexander J. A1 - Rotter, Björn A1 - Vogel, Jörg A1 - Winter, Peter A1 - Kahl, Günter T1 - Dual 3'Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells JF - BMC Genomics N2 - Background: The interaction of eukaryotic host and prokaryotic pathogen cells is linked to specific changes in the cellular proteome, and consequently to infection-related gene expression patterns of the involved cells. To simultaneously assess the transcriptomes of both organisms during their interaction we developed dual 3'Seq, a tag-based sequencing protocol that allows for exact quantification of differentially expressed transcripts in interacting pro-and eukaryotic cells without prior fixation or physical disruption of the interaction. Results: Human epithelial cells were infected with Salmonella enterica Typhimurium as a model system for invasion of the intestinal epithelium, and the transcriptional response of the infected host cells together with the differential expression of invading and intracellular pathogen cells was determined by dual 3'Seq coupled with the next-generation sequencing-based transcriptome profiling technique deepSuperSAGE (deep Serial Analysis of Gene Expression). Annotation to reference transcriptomes comprising the operon structure of the employed S. enterica Typhimurium strain allowed for in silico separation of the interacting cells including quantification of polycistronic RNAs. Eighty-nine percent of the known loci are found to be transcribed in prokaryotic cells prior or subsequent to infection of the host, while 75% of all protein-coding loci are represented in the polyadenylated transcriptomes of human host cells. Conclusions: Dual 3'Seq was alternatively coupled to MACE (Massive Analysis of cDNA ends) to assess the advantages and drawbacks of a library preparation procedure that allows for sequencing of longer fragments. Additionally, the identified expression patterns of both organisms were validated by qRT-PCR using three independent biological replicates, which confirmed that RELB along with NFKB1 and NFKB2 are involved in the initial immune response of epithelial cells after infection with S. enterica Typhimurium. KW - complete genome sequence KW - secretion systems KW - RNA-Seq KW - deepSuperSAGE KW - transcriptome KW - gene expression KW - serovar Typhimurium KW - human macrophages KW - epithelial cells KW - infection KW - SuperSAGE KW - receptors KW - Dual 3'seq KW - MACE KW - tag based KW - simultaneous KW - genome wide KW - gene expression profiling KW - host pathogen interaction KW - Salmonella enterica Typhimurium strain SL1344 Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143230 VL - 16 IS - 323 ER - TY - THES A1 - Messerer, Regina T1 - Synthesis of Dualsteric Ligands for Muscarinic Acetylcholine Receptors and Cholinesterase Inhibitors T1 - Synthese von dualsteren Liganden für muskarinerge Acetylcholinrezeptoren sowie Inhibitoren der Cholinesterasen N2 - The study is dealing with the synthesis and pharmacological investigation of newly designed dualsteric ligands of muscarinic acetylcholine receptors belonging to the superfamily of G protein-coupled receptors. Such bipharmacophoric ligands combine the advantages of the orthosteric binding site (high-affinity) and of the topographically distinct allosteric binding site (subtype-selectivity) resulting in compounds with reduced side effects. This opens the way to a new therapeutic approach in the treatment of e.g. chronic pain, drug withdrawal, Parkinson`s and Alzheimer`s disease. Furthermore, the newly synthesized dualsteric compounds were pharmacologically investigated in order to get a better understanding of the activation and signaling processes in muscarinic acetylcholine receptors, especially with regard to partial agonism. The development of the “dynamic ligand binding” concept offers new perspectives for ligand binding and signaling at G protein-coupled receptors. GPCRs are no longer considered as simple on/off switches. Dualsteric ligands can bind in a dualsteric pose, reflecting an active receptor state as well as in a purely allosteric binding pose, characterized by an inactive receptor state resulting in partial agonism. The degree of partial agonism depends on the ratio of active versus inactive receptor populations. On this basis, orthosteric/orthosteric hybrid ligands consisting of the antagonist atropine and scopolamine, respectively, as well as of the agonist iperoxo and isoxazole, respectively, linked via different alkyl chain length were synthesized in order to investigate partial agonism (Figure 1). Figure 1: Structures of the synthesized iperoxo/isoxazole-atropine/scopolamine-hybrids. Furthermore, different sets of quaternary and tertiary homodimers consisting either of two iperoxo or two acetylcholine units were synthesized in order to study their extent on partial agonism (Figure 2). The two agonists were connected by varying alkyl chain length. Binding studies on CHO-hM2 cells of the quaternary compounds revealed that dimerization of the agonist results in a loss of potency. The iperoxo-dimers reached higher maximum effects on the Gi- as well as on the Gs pathway in comparison to the acetylcholine-dimers. Besides the choice of the orthosteric building block (potency of the agonist), the alkyl chain length is also crucial for the degree of partial agonism. Figure 2: Structures of the synthesized quat./tert. iperoxo/acetylcholine-homodimers. Quinolone-based hybrids connected to the superagonist iperoxo and to the endogenous ligand acetylcholine, respectively, linked through an alkyl chain of different length were synthesized in order to develop further partial agonists (Figure 3). FRET studies confirmed M1 subtype-selectivity as well as linker dependent receptor response. The greatest positive FRET signal was observed with quinolone-C6-iper resulting from a positive cooperativity between the two separated moieties, alloster and orthoster. However, the corresponding hybrids with a longer linker led to an inverse FRET signal indicating a different binding mode, e.g. purely allosteric, in contrast to the shorter linked hybrids. Furthermore, the flexible alkyl spacer was replaced by a rigidified linker resulting in the hybrid quinolone-rigid-iperoxo (Figure 3). FRET studies on the M1 receptor showed reduced FRET kinetics, resulting from interactions between the bulky linker and the aromatic lid, located between the orthosteric and allosteric binding site. A bitopic binding mode of the rigidified hybrid is presumed. For further clarity, mutational studies are necessary. Figure 3: M1-selective hybrid compounds. Another aim of this work was the design and synthesis of new hybrid compounds, acting as agonists at the M1 and M2 receptor and as inhibitors for AChE and BChE in the context of M. Alzheimer. Several sets of hybrid compounds consisting of different pharmacophoric units (catalytic active site: phthalimide, naphthalimide, tacrine; peripheric anionic site: iperoxo, isoxazole) linked through a polymethylene chain of varying length were synthesized. Tac-C10-iper (Figure 4), consisting of tacrine and the superagonist iperoxo linked by a C10 polymethylene spacer, was found to have excellent anticholinesterase activity for both AChE (pIC50 = 9.81) and BChE (pIC50 = 8.75). Docking experiments provided a structural model to rationalize the inhibitory power towards AChE. Additionally, the tacrine related hybrids showed affinity to the M1 and M2 receptor. Such compounds, addressing more than one molecular target are favorable for multifactorial diseases such as Alzheimer. Figure 4: Structure of the most active compound regarding anticholinesterase activity. In summary, the choice of the pharmacophoric units, their connecting point as well as the nature, length, and flexibility of the linker play an important role for the activity of designed bivalent ligands. A shorter linker length cannot bridge both binding sites simultaneously in contrast to longer linker chains. On the other hand, too long linker chains can result in unwanted steric interactions. Further investigations with respect to structural variations of hybrid compounds, with or without quaternary ammonium groups, are necessary in the light of drug development. N2 - Die vorliegende Studie beschäftigt sich mit der Synthese und der pharmakologischen Untersuchung von neu entwickelten dualsteren Liganden des muskarinischen Acetylcholinrezeptors, welcher zur Superfamilie der G-Proteine gehört. In derartigen bipharmakophoren Liganden sind die Vorteile des orthosteren Bindemodus und des räumlich davon getrennten allosteren Bindemodus vereint. Der orthostere Bindemodus bewirkt eine hohe Affinität zum Rezeptor, während der allostere Bindemodus Subtypselektivität vermittelt. Dadurch weisen diese Verbindungen weniger Nebenwirkungen auf. Dies eröffnet einen neuen Therapieansatz in der medikamentösen Behandlung von z.B. chronischen Schmerzen, Drogenentzug, Morbus Parkinson und Morbus Alzheimer. Die neu synthetisierten, dualsteren Verbindungen wurden pharmakologisch untersucht, um ein besseres Verständnis über das Bindungsverhalten und die Signalweiterleitung an muskarinischen Acetylcholinrezeptoren zu erhalten, besonders in Hinblick auf Partialagonismus. Die Entwicklung des Konzeptes der „dynamischen Ligandenbindung“ bietet neue Perspektiven in Hinblick auf das Bindungsverhalten und die Signalweiterleitung an G-Protein gekoppelten Rezeptoren. Somit werden GPCRs nicht mehr nur in ihrem aktiven oder inaktiven Zustand betrachtet. Vielmehr können dualstere Liganden sowohl einen dualsteren Bindemodus, welcher den aktiven Rezeptorzustand widerspiegelt, als auch einen rein allosteren Bindemodus, welcher durch einen inaktiven Rezeptorzustand charakterisiert ist, einnehmen, was schließlich zu Partialagonismus führt. Die Stärke des resultierenden Partialagonismus hängt vom Verhältnis zwischen aktiver und inaktiver Rezeptorbesetzung ab. Auf Basis dessen wurden orthostere/orthostere Hybridverbindungen, bestehend aus einem Antagonisten, Atropin oder Scopolamin, und einem Agonisten, Iperoxo oder Isoxazol, die über eine Alkylkette unterschiedlicher Länge miteinander verknüpft sind, synthetisiert, um mit deren Hilfe den Partialagonismus zu steuern (Abbildung 1). Abbildung 1: Strukturen der synthetisierten Iperoxo/Isoxazol-Atropin/Scopolamin-Hybride. Es wurden verschiedene quartäre sowie tertiäre Homodimere, welche entweder aus zwei Iperoxo-Einheiten oder aus zwei Acetylcholin-Einheiten bestehen, synthetisiert, um deren Ausmaß in Bezug auf Partialagonismus untersuchen zu können (Abbildung 2). Die beiden Agonisten wurden über unterschiedlich lange Alkylketten miteinander verknüpft. Bindungsstudien an CHO-hM2 Zellen der quartären Verbindungen zeigten, dass die Dimerisierung eines Agonisten zu einer verringerten Wirkstärke führt. Die Dimere von Iperoxo erreichten sowohl auf dem Gi- als auch auf dem Gs-Signalweg höhere Maximaleffekte als die Dimere von Acetylcholin. Neben der Wahl des orthosteren Bausteins (Wirkstärke des Agonisten) spielt auch die Länge der Alkylkette eine entscheidende Rolle für die Stärke des Partialagonismus. Abbildung 2: Strukturen der synthetisierten quart./tert. Iperoxo/Acetylcholin-Homodimere. Um weitere Partialagonisten zu entwickeln, wurden Chinolon-basierte Verbindungen, die mit dem Superagonisten Iperoxo oder mit dem endogenen Liganden Acetylcholin über eine Alkylkette mit unterschiedlicher Länge verknüpft sind, synthetisiert (Abbildung 3). FRET-Messungen bestätigen, dass es sich bei den Hybriden um M1-subtypselektive Substanzen handelt und das FRET-Signal von der Länge der Zwischenkette abhängig ist. Das stärkste positive FRET-Signal wurde mit der Verbindung Chinolon-C6-Iper erzielt, welches durch positive Kooperativität zwischen den beiden Liganden, Alloster und Orthoster, zustande kommt. Im Gegensatz zu den kurzkettigen Hybriden beobachtete man bei den langkettigen Hybriden ein inverses FRET-Signal, welches auf einen anderen Bindemodus zum Rezeptor hindeutet, z.B. könnte es sich um eine rein allostere Bindung handeln. Außerdem wurde die flexible Alkylkette durch einen starren Linker ersetzt, welches im Hybrid Chinolon-rigide-Iperoxo verwirklicht ist (Abbildung 3). FRET-Messungen dieser starren Hybridverbindung am M1-Rezeptor zeigten eine verzögerte FRET-Kinetik, welche vermutlich auf Wechselwirkungen zwischen dem starren Linker und dem aromatischen Deckel, der sich zwischen der orthosteren und der allosteren Bindestelle befindet, zurückzuführen ist. Es wird vermutet, dass das starre Hybrid bitopisch in den Rezeptor bindet. Um diese Annahme bestätigen zu können, müssten Mutationsstudien durchgeführt werden. Abbildung 3: M1-selektive Hybridverbindungen. Ein weiteres Ziel dieser Arbeit war das Wirkstoffdesign und die Synthese von neuen Hybridverbindungen, die als Agonisten am M1- und am M2-Rezeptor sowie als Inhibitoren der AChE als auch der BChE im Hinblick auf die Alzheimer`sche Krankheit wirken sollen. Verschiedenartige Hybridverbindungen, bestehend aus unterschiedlichen pharmakophoren Gruppen (katalytische, aktive Seite: Phthalimid, Naphthalimid, Tacrin; periphere, anionische Seite: Iperoxo, Isoxazol), die über eine Polymethylenkette unterschiedlicher Länge miteinander verknüpft sind, wurden synthetisiert. Tac-C10-Iper (Abbildung 4), bestehend aus Tacrin und dem Superagonisten Iperoxo, welche über eine C10 Polymethylenkette miteinander verknüpft sind, zeigte exzellente Anticholinesterase-Aktivitäten sowohl für die AChE (pIC50 = 9.81) als auch für die BChE (pIC50 = 8.75). Docking-Experimente lieferten ein Strukturmodell, welches die inhibitorische Aktivität in Bezug auf die AChE begründet. Zusätzlich zeigten die aus Tacrin bestehenden Hybride Affinität zum M1- als auch zum M2-Rezeptor. Solche Verbindungen, die mehr als ein Zielmolekül adressieren, sind für multifaktorielle Krankheiten, wie z.B. die Alzheimer`sche Krankheit, von Vorteil. Abbildung 4: Struktur der aktivsten Substanz in Bezug auf die Anticholinesterase-Aktivität. Zusammenfassend kann festgestellt werden, dass sowohl die Wahl des Pharmakophors, deren Verbindungsstelle als auch die Zusammensetzung, Länge und Flexibilität des Linkers eine große Rolle für die Aktivität der entwickelten bivalenten Verbindungen spielen. Kurzkettige Linker können im Gegensatz zu längeren Zwischenketten nicht beide Bindestellen gleichzeitig überbrücken. Andererseits können zu lange Zwischenketten unerwünschte sterische Wechselwirkungen hervorrufen. Weitere Untersuchungen in Bezug auf strukturelle Veränderungen der Hybridverbindungen, mit oder ohne quartäre Ammoniumgruppen, sind in Bezug auf die Arzneimittelentwicklung notwendig.   KW - Cholinesteraseinhibitor KW - Muscarinrezeptor KW - Ligand KW - GTP-bindende Proteine KW - dualsteric ligands KW - muscarinic acetylcholine receptor KW - cholinesterase inhibitors KW - receptors KW - coupled KW - gprotein KW - inhibitors KW - cholinesterase Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-149007 ER - TY - JOUR A1 - Gutknecht, Lise A1 - Araragi, Naozumi A1 - Merker, Sören A1 - Waider, Jonas A1 - Sommerlandt, Frank M. J. A1 - Mlinar, Boris A1 - Baccini, Gilda A1 - Mayer, Ute A1 - Proft, Florian A1 - Hamon, Michel A1 - Schmitt, Angelika G. A1 - Corradetti, Renato A1 - Lanfumey, Laurence A1 - Lesch, Klaus-Peter T1 - Impacts of Brain Serotonin Deficiency following Tph2 Inactivation on Development and Raphe Neuron Serotonergic Specification JF - PLoS One N2 - Brain serotonin (5-HT) is implicated in a wide range of functions from basic physiological mechanisms to complex behaviors, including neuropsychiatric conditions, as well as in developmental processes. Increasing evidence links 5-HT signaling alterations during development to emotional dysregulation and psychopathology in adult age. To further analyze the importance of brain 5-HT in somatic and brain development and function, and more specifically differentiation and specification of the serotonergic system itself, we generated a mouse model with brain-specific 5-HT deficiency resulting from a genetically driven constitutive inactivation of neuronal tryptophan hydroxylase-2 (Tph2). Tph2 inactivation (Tph2-/-) resulted in brain 5-HT deficiency leading to growth retardation and persistent leanness, whereas a sex- and age-dependent increase in body weight was observed in Tph2+/- mice. The conserved expression pattern of the 5-HT neuron-specific markers (except Tph2 and 5-HT) demonstrates that brain 5-HT synthesis is not a prerequisite for the proliferation, differentiation and survival of raphe neurons subjected to the developmental program of serotonergic specification. Furthermore, although these neurons are unable to synthesize 5-HT from the precursor tryptophan, they still display electrophysiological properties characteristic of 5-HT neurons. Moreover, 5-HT deficiency induces an up-regulation of 5-HT\(_{1A}\) and 5-HT\(_{1B}\) receptors across brain regions as well as a reduction of norepinephrine concentrations accompanied by a reduced number of noradrenergic neurons. Together, our results characterize developmental, neurochemical, neurobiological and electrophysiological consequences of brain-specific 5-HT deficiency, reveal a dual dose-dependent role of 5-HT in body weight regulation and show that differentiation of serotonergic neuron phenotype is independent from endogenous 5-HT synthesis. KW - lacking KW - knock-out mice KW - energy expenditure KW - locomotor activity KW - 5-HT transporter KW - anxiety like KW - receptors KW - behavior KW - tryptophan KW - nucleus Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133728 VL - 7 IS - 8 ER - TY - JOUR A1 - Jesaitis, A. J. A1 - Klotz, Karl-Norbert T1 - Cytoskeletal regulation of chemotactic receptors: Molecular complexation of N-formyl peptide receptors with G proteins and actin N2 - Signal transduction via receptors for N-formylmethionyl peptide chemoattractants (FPR) on human neutrophils is a highly regulated process. It involves direct interaction of receptors with heterotrimeric G-proteins and may be under thc control of cytoskeletal clemcnts. Evidencc exists suggesting that thc cytoskeleton and/or the membrane ske1eton determines the distribution of FPR in the plane of the plasma membrane, thus controlling FPR accessibility to different protcins in functionally distinct membrane domains. In desensitized cells, FPR are restricted to domains which are depleted of G proteins but enriched in cytoskeletal proteins such as actin and fodrin. Thus, the G protein signal transduction partners of FPR become inacccssible to the agonist-occupied receptor, preventing cell activation. We are investigating the molecular basis for the interaction of FPR with the membrane skeleton, and our results suggest that FPR, and possibly other receptors, may directly bind to cytoskeletal proteins such as actin. KW - Immunologie KW - chemotaxis KW - formyl peptides KW - receptors KW - actin KW - G proteins KW - cytoskeleton KW - membrane skeleton Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-79673 ER - TY - JOUR A1 - Waelbroeck, M. A1 - Camus, J. A1 - Tastenoy, M. A1 - Lambrecht, G. A1 - Mutschler, E. A1 - Tacke, Reinhold A1 - Christophe, J. T1 - Stereoselectivity of procyclidine binding to muscarinic receptor subtypes M\(_1\), M\(_2\) and M\(_4\) N2 - The goals of the present study were: (1) to investigate thc binding properlies oi (R)- and (S)-procyclidine and two aehiral derivatives of muscarinic M\(_1\)• M\(_2\) and M\(_4\) receptor subtypes and (2) to identify the interaetions which allow these receptors to diseriminate between the two stereoisomers. (R)-Procyclidine showed a higher affinity for human neuroblastoma NB-OK 1 muscarinie M\(_1\) and rat striatum musearinie M\(_4\) receptors. a~ compared to rat cardiac M\(_2\) receptors. (S)-Procyclidine had a 130-iold lower affinity than (R)-procyclidine for M\(_1\) and M\(_4\) receptors. and a 40-fold lower affinity for M\(_2\) receptors. Pyrrinol. the aehiral diphenyl derivative with the eyclohexyl g.roup of (S}-procyclidine replaeed by a phenyl group, has an eight-fold lower affinity for M\(_1\) and M\(_4\) receptors. as eompared to (R)-procyclidine, and a three-fold lower affinity for M\(_2\) receptors. Hexahydro-procyclidine. the eorresponding achiral dicyclohexyl compound, had a 10- to 20-fold lower affinity than (R)-procyclidine for the three reeeptors. The inerease in binding free energy, which is observed when the phenyl and eyclohexyl groups of procyelidine are separately replaeed by cyclohexyJ and phenyl groups, respectively. was additive in the ease of M\(_1\)• M\(_2\) and M\(_4\) receptcrs. This indicates that the musearinic reeeptor s!ereoseleetivity was based on the eoexistence of two binding sites, one preferring a phenylrather than eyclohexyl group and the seeond preferring a cyclohexyl rather than a phenyl group. In addition. there were aiso binding sites for the hydroxy moiety and the protonated amino group of the ligands. The greater affinity and stereoselectivity of M\(_1\) and M\(_4\) muscarinic receptors for (R)-procyelidine reflected the better fit of the eyclohexyl group of (R)-procyclidine to the subsite of M\(_1\) and M\(_4\) as compared to M\(_2\) receptors. KW - Anorganische Chemie KW - Musearlnie M1 KW - receptors KW - Muscarinie M2 receptors KW - Musearinic M4 receptors KW - Pyrrinol KW - Hexahydro-procyclidine KW - Muscarinic receptors Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-64034 ER - TY - THES A1 - Hartung, Anke T1 - Localization of BMP receptors in distinct plasma membrane domains and its impact on BMP signaling T1 - Lokalisierung von BMP Rezeptoren in speziellen Plasmamembrandomänen und deren Auswirkung auf den BMP Signalweg N2 - Endocytosis of growth factor receptors plays an important role in the activation and propagation as well as the attenuation of signaling pathways. Its malfunctioning can cause several pathologies, e.g. by controlling the level of receptors at the cell surface. BMPs are members of the TGF-ß superfamily and are involved in the regulation of proliferation, differentiation, chemotaxis and apoptosis. BMP signaling is initiated at two types of transmembrane serine/threonine kinases, BRI and BRII. BMP receptor activation occurs upon ligand binding to preformed complexes (PFCs) or BMP2-induced signaling complexes (BISCs) composed of BRI and BRII. Binding of BMP2 to PFCs results in activation of the Smad pathway, whereas BISCs initiate the activation of Smad-independent pathways via p38 resulting in the induction of Alkaline phosphatase (ALP). BMP receptor endocytosis has not been extensively studied and the potential role of localization to different regions of the plasma membrane in determining the signaling pathways activated by PFCs and BISCs was not explored so far. In the present work, the localization of BMP receptors in distinct membrane domains and the consequential impact on BMP signaling were investigated. By separating detergent-resistant membranes (DRMs) from cell lysates and subsequent gradient ultracentrifugation, it could be demonstrated that BRI and BRII cofractionate with cav-1, the marker protein of caveolae. Moreover, both receptor types interacted with cav-1 and showed a partially colocalization with cav-1 at the plasma membrane. Although these results point to a caveolar localization, BMP receptors cofractionated also with DRMs in cells exhibiting no caveolae, suggesting an additional non-caveolar raft localization. Beyond that, BRII could also be localized to clathrin-coated pits (CCPs) by means of immuno-electronmicroscopy studies. The second part of this thesis demonstrated that both membrane regions influence BMP signaling in distinct ways. Smad1/5 was shown to be phosphorylated independently of endocytic events at the cell surface. On the one hand, disruption of DRM regions by cholesterol depletion inhibited specifically BMP2-mediated ALP production, while Smad signaling was unaffected. On the other hand, inhibition of clathrin-mediated endocytosis by specific inhibitors affected BMP2-induced Smad signaling as well as the induction of ALP, suggesting that both Smad-dependent and Smad-independent signaling pathways are required for BMP2 induced ALP production. These findings propose an important regulatory impact of different endocytic routes and membrane regions on BMP signaling as well as that a distinct membrane localization of BMP receptors account for specific signaling properties initiated at PFCs or BISCs. N2 - Endozytose von Wachstumsfaktor-Rezeptoren spielt eine entscheidende Rolle bei Aktivierung und Übertragung wie auch bei der Schwächung von Signalen. Störungen der Endozytose können schwere Krankheitsbilder hervorrufen, z.B. durch ihren Einfluss auf die Regulation der Rezeptormenge an der Zelloberfläche. BMPs sind Mitglieder der TGF-ß Superfamilie und sind involviert in die Regulation von Proliferation, Differenzierung, Chemotaxis und Apoptose. Zwei Arten von Transmembranproteinen, die Serin/Threonin-Kinase Aktivität besitzen, sind bedeutend für den BMP Signalweg – die BMP Rezeptoren BRI und BRII. Die Aktivierung von BRI und BRII erfolgt durch Ligandenbindung an präformierte Komplexe (PFCs) oder BMP2-induzierte Signalkomplexe (BISCs), die aus beiden Rezeptorarten bestehen. Wenn BMP2 an PFCs bindet, wird die Smad-Signalkaskade initiiert, wohingegen BISCs Smad-unabhängige Signale über p38 weiterleiten, was schließlich zur Produktion von alkalischer Phosphatase (ALP) führt. Das Feld der BMP Rezeptor Endozytose wurde noch nicht sehr ausführlich untersucht, genauso wenig wie die potentielle Rolle, die unterschiedliche Rezeptorlokalisierungen in verschiedenen Plasmamembran-Regionen bei der Initiierung der Signalwege, die durch PFCs bzw. BISCs aktiviert werden, spielen könnten. In der vorliegenden Arbeit wurden die Lokalisierung von BMP Rezeptoren in speziellen Membrandomänen sowie deren Einfluss auf die BMP Signalkaskade untersucht. Mittels Reinigung von Detergenz-resistenten Membranen (DRMs) aus Zelllysaten und anschließender Gradientenultrazentrifugation konnte gezeigt werden, dass BRI und BRII mit dem caveolären Markerprotein cav-1 kofraktionieren. Darüber hinaus interagieren beide Rezeptorarten mit cav-1 und kolokalisieren auch teilweise mit cav-1 an der Plasmamembran. Obwohl diese Ergebnisse auf ein eindeutiges Vorkommen der Rezeptoren in Caveolae schließen lassen, kofraktionieren sie auch mit DRMs in Zellen, die von Natur aus keine Caveolae ausbilden, woraus man eine zusätzliche nichtcaveoläre Raft-Lokalisierung schlussfolgern kann. Des Weiteren konnte BRII mittels Immun- Elektronenmikroskopie in „clathrin-coated pits“ (CCPs) lokalisiert werden. Im zweiten Teil der Arbeit wurde gezeigt, dass beide untersuchten Membranregionen die BMP Signalkaskade auf unterschiedliche Art und Weise beeinflussen. Es wurde bewiesen, dass Smad1/5 unabhängig von endozytotischen Vorgängen an der Plasmamembran phosphoryliert wird. Einerseits führte die Zerstörung von DRM-Regionen durch Cholesterindepletion zur spezifischen Inhibierung der BMP2-vermittelten ALP Produktion, ohne gleichzeitig die BMP Signalkaskade über Smads zu beeinflussen. Andererseits bewirkte eine spezifische Blockierung der Clathrin-vermittelten Endozytose eine Inhibition des BMP2-induzierten Smad-Signalwegs und auch der ALP Produktion, was auf ein Zusammenspiel von Smad-unabhängigen und Smad-abhängigen Signalwegen bei der ALP-Induzierung schließen lässt. Die Ergebnisse der vorliegenden Studie lassen die Schlussfolgerung zu, dass verschiedene endozytotische Wege und Membranregionen einen bedeutenden, regulatorischen Einfluss auf die BMP Signalkaskade ausüben. Weiterhin wurde festgestellt, dass die Membranlokalisierung von BMP Rezeptoren für das Einschlagen verschiedener Signalwege ausgehend von PFCs und BISCs verantwortlich ist. KW - Knochen-Morphogenese-Proteine KW - Rezeptor KW - Endocytose KW - BMP KW - Rezeptoren KW - Endozytose KW - Lipid Raft KW - Caveolae KW - BMP KW - receptors KW - endocytosis KW - lipid raft KW - caveolae Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-18360 ER -