Throughout the last decade label-free analytical approaches capable of recording the integral biological response of mammalian cells to chemical or biological stimuli have received considerable interest in GPCR-induced signal transduction. Whereas molecular assays are highly specific for one particular interaction or the concentration of one specific signalling molecule (cAMP, Ca2+), they can not capture the integrated response of the cells which might involve several or even unexpected signal transduction pathways. In contrast, label-free monitoring of cell-based assays integrates non-specifically over the entire cell body and returns a fingerprint-like time profile of the physical quantity being measured.
It is the objective of this project to extend the information content of existing label-free monitoring techniques (electrical, optical) during GPCR-stimulation by systematically applying more complex data acquisition modes and tailoring the experimental parameters such that highly specific response profiles are generated for a given stimulus. In the longrun the project aims to identify the signal transduction pathways that have been triggered by a given GPCR agonist from the fingerprint profile to unravel possible functional selectivities. From a meth-odological viewpoint the project is located at the the interface between medicinal and analytical chemistry. It comprises cell culture work, state-of-the-art impedimetric and optical biosensing, confocal microscopy and classical cytological assays.
Research stay abroad
Data analysis of the SPR readout will be supported by theoretical modeling of the cell-sensor junction in cooperation with the lab of Prof. Jiri Homola (Institute of Photonics and Electronics Prague, Czech Republik) who will host the graduate for 3-4 months. Due to the real-time data recorded in both approaches, readouts might assist in guiding the rationale design of specific GPCR-ligands with well-defined bioresponse profiles.
Functional selectivity or ligand biased signalling has been observed for a growing number of GPCRs. Thus, receptor agonists may trigger different signalling pathways dependent on the cell background. The histamine H4 receptor, for example, which is expressed on several types of immune cells, is supposed to exert pro-inflammatory activity via activation of Gi/o proteins. In some cell types, such as mouse mast cells however, ligand binding may also result in G protein independent signalling via β-arrestin recruitment. Apart from the facultative interaction of the receptor with G proteins or β-arrestin, functional selectivity can also develop further downstream of the signal pathway by cross talk. Functional selectivity is also highly dependent on the ligand structure. It is assumed that for example different hH4R ligands stabilize different receptor conformations which then activate different patterns of G-protein dependent and -independent signalling pathways.
Likewise it has been shown that D2R-ligands exert functional selectivity by activation of different types of G-proteins. Functional selectivity is considered as an important cause of unexpected pharmacological properties and efficacy of newly developed drug candidates. Therefore, in the present project targeted proteome analysis will be applied to address the downstream pluridimensionality of hH4R- and D2R-ligand efficacy.
The main objective of this project is to develop multiplexed targeted proteome assays to monitor downstream signalling events resulting from H4R- and D2R-ligand binding in order to address ligand-biased H4R-and D2R-signalling in a pluridimensional manner. The goal is to develop a relatively fast and flexible assay system based on proteomic tools, such as (nano) UHPLC-ESI-MS/MS in the multiple reaction monitoring mode and metabolic stable isotope labelling (SILAC) of treated and untreated cells. The assay system will be included in the process of ligand development; in a long term, the expected information will help to predict pharmacological properties and drug efficacy at an early stage of drug development. Furthermore, this information will help to understand correlation of drug structure and functional selectivity.
Research stay abroad
The work plan will be complemented by a research stay in the Kislinger´s group at the Ontario Cancer Institute in Toronto, where untargeted expression proteomics by multidimensional protein identification technology (MudPIT) will be applied. On a longer term, it is the goal to complement the present approach with untargeted methods for the selection of effector proteins, which optimally reflect different ligand-biased signalling pathways.
WILL BE TERMINATED SOON
State of the art: Recent observations suggest that GPCR heteromerization leads to new binding properties of ligands and functional selectivity. A functional interaction between the neuropetidergic neurotensin (NT) and dopaminergic pathways is known for several years. Previous studies showed that NT antagonizes dopamine effects mediated by D2 receptors via the interaction between the neurotensin 1 receptor (NTS1) and the D2 receptor. The main objective of this project is to test the hypothesis if signalling of D2 receptor ligands is altered by NTS1/D2 receptor heteromers on the level of ion channel activity and to use our methodology for the dis-covery of novel functionally selective GPCR ligands. A broad set of techniques ranging from calcium imaging, patch-clamp technique to confocal microscopy, co-immunoprecipitation and iterative drug design will be applied to address these questions.
Research stay abroad
To extend the findings obtained in cell models, the PhD student is supposed to spend 3-4 months in the laboratory of Prof. Dr. Lucas Pozzo-Miller, University of Alabama in Birmingham (USA) to investigate functional selectivity of D2 ligands and NTS1/D2 receptor heteromerization in acute cortical brain slices using two-photon microscopy and patchclamp techniques.
Whilst GPCRs have traditionally been considered to act as monomeric transducers of extracellular signals to intracellular compartments, growing evidence suggest they can also form homo- and heterodimers or even higher order oligomers within the cell membrane. Besides enabling cross-talk between individual signaling networks, receptor dimerization can induce activation of alternative signaling pathways, influence ligand pharmacology and is critical for receptor trafficking and function. Among other receptors, dopamine D2-like receptors have been found to form homo- and heterodimers, and altered cooperativity between individual protomers has been associated with the pathophysiology of different central nervous diseases.
Very recently, we were able to demonstrate that tailor-made bivalent ligands consisting of dopaminergic pharmacophores linked to the peptide NT(8-13) by an appropriate spacer are able to bind to D2R/NTS1R receptor heterodimers with ultra-high affinity and selectivity over the respective monomeric receptors (H. Hübner et al., Nature Communications, 2016).
To understand and exploit molecular interactions of bivalent ligands with receptor dimers, the project aims investigate highly specific bivalent ligands targeting either homo- or heterodimers of dopamine D2-like receptors. As a first step, in-vitro affinity profiles of tailor-made tool compounds will be generated. Radioligand binding studies on membrane preparations of cells coexpressing the two receptor subtypes forming heterodimers will be compared to the respective monoexpressing controls. Moreover, implications on the function of the involved receptors will be studied on the level of canonical G protein signaling as well as alternate pathways, such as β-arrestin recruitment ultimately leading to receptor internalization. Pharmacological assays will be adapted to the needs of studying homo- and heterodimerization and diagnostic receptor mutants will be implemented to decipher individual receptor contributions to the signaling outcome of bivalent ligand/receptor interactions.
Research stay abroad
The qualification program will facilitate that the doctoral student complements his/her scientific investigations in the laboratory of a collaborating international research group.
Megakaryoblastic Leukemia 1 (MKL1, MAL, MRTF-A) is a transcriptional coactivator of the Serum Response Factor (SRF), which regulates the transcription of genes involved in cell proliferation, cell motility, adhesion and differentiation and triggers hepatocellular carcinoma (HCC) formation (Olson et al., 2010, Muehlich et al., 2012, Hampl et al., 2013, Hermanns et al., 2017).
Recent data from the Cancer Genome Atlas (TCGA) revealed that mutations or aberrant expression of GPCRs, including LPA receptors, have been found in 20% of all cancers (https://cancergenome.nih.gov),. However, a functional link between LPA receptor signaling, MKL transcriptional activation and HCC cell proliferation has not yet been investigated in detail.
In this project we aim to decipher how LPA receptor activation affects MKL/SRF transcriptional activity and tumor cell proliferation. Given the pathophysiological relevance of MKL1 in tumorigenesis, our results may be of utmost importance to develop a novel pharmacological strategy for tumor therapy.
1) Olson EN, Nordheim A. (2010) Linking actin dynamics and gene transcription to drive cellular motile functions. Nat Rev Mol Cell Biol. 11(5):353-65.
2) Muehlich S, Hampl V, Khalid S, Singer S, Frank N, Breuhahn K, Gudermann T, Prywes R. (2012) The transcriptional coactivators megakaryoblastic leukemia 1/2 mediate the effects of loss of the tumor suppressor deleted in liver cancer 1. Oncogene Aug 30;31(35):3913-23.
3) Hampl V, Martin C, Aigner A, Hoebel S, Singer S, Frank N, Sarikas A, Ebert O, Prywes R,
Gudermann T, Muehlich S. (2013) Depletion of the transcriptional coactivators Megakaryoblastic Leukemia 1 and 2 abolishes hepatocellular carcinoma xenograft growth by inducing oncogene-induced senescence. EMBO Molecular Medicine Sep;5(9):1367-82.
4) Hermanns C, Hampl V, Holzer K, Aigner A, Penkava J, Frank N, Martin DE, Maier KC,
Waldburger N, Roessler S, Margarete Goppelt-Struebe M, Akrap I, Thavamani A, Singer S, Nordheim A, Gudermann T, Muehlich S. (2017) The novel MKL target gene myoferlin modulates expansion and senescence of hepatocellular carcinoma. Oncogene 36(24):3464-3476.