Grundmann, Manuel: A multifaceted approach to investigate signal transduction at the receptor and post-receptor level focusing on the modulation of the free fatty acid receptor family. - Bonn, 2015. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
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author = {{Manuel Grundmann}},
title = {A multifaceted approach to investigate signal transduction at the receptor and post-receptor level focusing on the modulation of the free fatty acid receptor family},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2015,
month = nov,

note = {Knowledge about the function and dysfunction of cellular signal transduction is at the very heart of biomedical research since every physiological and pathophysiological process is intimately associated with adequate or defective signaling. The advent of innovative methodological techniques paved the way for major breakthroughs in deciphering cell signaling in the life sciences. Granting this, it is not technology per se but the proactive adjustment and attentive implementation of the various methodological approaches that lay the foundation for rewarding research efforts. It is becoming increasingly clear that data obtained from a single assay readout are generally insufficient to mirror the multidimensionality of biological signaling networks. Thus it was an aim of this study by synopsis of different technological, biochemical and pharmacological approaches to provide new insights into the anatomy of signaling events.
G protein-coupled receptors (GPCRs) do not only represent the largest group of drug targets but are also prototypic as microprocessing units in the cellular signaling machinery and therefore ideally suited to study the biology of signal transduction. This class of receptors represent a cornucopia of signaling diversity since a plethora of signal transduction concepts are mirrored herein. Hence, GPCRs are supremely favored research objects and examined in the majority of the publications communicated in this thesis.
Section I explained the principle of dynamic mass redistribution (DMR). The optical biosensor-based technique allows the detection of cellular events as an integrated whole cell readout and its label-free nature is capable to reflect signaling events in a non-invasive fashion without using any interfering labels. A set of different cellular backgrounds was used to demonstrate the versatility and applicability of the assay. The integration of recombinant, native and primary cells highlighted the potential of this technique to glean in vivo relevant biological information about investigated substances already in early stages of the drug discovery process. Since DMR provides unbiased insights into cell activation a testimony of multiple concurrent pathways triggered by a stimulus is accessible and helps to understand the underlying biology. Moreover, the first chapter described the possibility to untangle the GPCR signaling repertoire by means of selective pathway modulators and explained why the real time measuring mode allows the exploration of kinetic aspects in the cell response.
Chapter 2 opens section II of this thesis, in which the biology of the free fatty acid receptor FFA2 is detailed. With a multifaceted approach exploiting the advantages of holistic label-free readouts it was possible to uncover an unprecedented mode-of-action of a GPCR modulating ligand. 4 CMTB previously classified as positive allosteric modulator and allosteric agonist (ago-PAM) at the FFA2 receptor was disclosed as the first sequentially activating ligand (SEAL) reported so far. This ligand binds and activates a first recognition site of the receptor and is then repositioned to another site, where a second signaling impulse is generated. The association of multiple binding events with multiple signaling events therein is a key difference between SEALs and classical bitopic ligands that simultaneously bind the receptor. This mechanism extends the possibility to encrypt biological information by a temporal dimension. In this concept, GPCRs could function as transceivers that de- and encode the information in an adjustable manner.
Rationally correlating certain signaling events and the resulting phenotype within a complex biological environment remains a prime challenge in signal transduction research. The invention of designer receptors bettered the prospects to approach this question. By isolating a certain receptor signaling behavior and introduce this signaling machinery into an in vivo context scientists might be able to assess the relevance of a certain activation pattern for the individual phenotype. Chapter 3 reports on a strategy how to rationally design and generate a designer FFA2 receptor. A receptor activated solely by a synthetic ligand (RASSL) form of FFA2 was found based on species differences in agonist selectivity between the human and bovine FFA2 receptor and consequently did not respond to the endogenous short-chain fatty acid ligands but was selectively activated by stimulation with sorbic acid. Future studies involving designer receptors can help elucidating the in vivo role of the FFA2 receptor, since the ubiquitous availability of fatty acids in the body and overlapping ligand recognition pattern of FFA2 and FFA3 challenged a clear definition of FFA2 mediated phenotypes.
Section III deals with the human free fatty acid receptor FFA1 and is introduced by Chapter 4 that presents a study that investigated the interplay between FFA1 and non-esterified fatty acids (NEFAs) in the disease context of diabetes type 2 (TD2). The deleterious effects of NEFAs on pancreatic beta cell function and survival were shown to be dependent of FFA1 activation, a hypothesis that has been claimed by previous reports. In the present study, however, the activation of FFA1 with a selective small molecule agonist proved even beneficial, while antagonists deteriorated β-cell health, a finding that is also corroborated by other studies. A single nuclear polymorphisms (SNP) in the gene of the FFA1 was identified to modulate the sensitivity against (gluco)lipotoxic effects of fasting levels of non-esterified fatty acid (NEFA) level in humans confirming a modulatory role of FFA1 in the pathophysiology of TD2. The second publication in chapter 4 focused again on a methodological issue and notes the importance to diligently validate the experimental tools used to determine localization and amount of protein expression, a prerequisite of reliable data acquisition. In doing so, several inconsistent results regarding the study of FFA1 receptor biology were questioned.
In Chapter 5 we finally reported on the chemistry and optimization of synthetic FFA1 ligands. Because of the aim to develop highly selective and potent FFA1 agonists, the unbiased DMR assay proofed valuable to detect any off-target effects of the investigated compounds, as well as confirming their competence to activate the receptor with appropriate potency and efficacy. The three publications within this chapter present a strategic chemical approach to tailor the structures for their employment as pharmacologically validated substances. The availability of those ligands is inevitable to study receptor biology and was thus a prerequisite to conduct research as depicted in other chapters.
In the last part of the thesis, section IV, two chapters describe the analysis of signal transduction at the post-receptor level. Chapter 6 provides further evidence for a bona fide second messenger role of the non-canonical cyclic nucleotides cCMP and cUMP by delineating the biological effectors. The label-free DMR assay disclosed cNMP target proteins and shed light into the signal transduction network of cNMPs in human cells, thereby emphasizing the applicability of this technique that is not restricted to the analysis of signal transduction generated from cell surface receptors but also initiated at the post-receptor level. Chapter 7, finally, characterized and defined the mode-of-action of a previously published pan-G protein inhibitor (BIM46187). By stabilizing the empty-pocket conformation of Gα subunits, which are in the activation process, BIM46187 is the first reported GTP entry inhibitor and was furthermore redefined as a compound that selectively targets Gαq proteins in a cell-type dependent manner.
In conclusion, this thesis comprises several studies covering multiple aspects of cellular signal transduction providing a range from biological-pharmacological, technological and chemical perspectives on the subject. The bulk of the publications deals with the analysis of GPCR biology, especially the exploration of the free fatty acid receptor FFA1 and FFA2. These are complemented by a technology-centered, conceptual section and a section analyzing signal transduction at the post-receptor level.},

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