Cellular signaling involves a series of interacting protein complexes formed after induction by external or internal triggers. The formation and function of these complexes is tightly regulated. Genetic or induced alterations of regulatory factors may deviate cell fate and lead to pathological responses. We study normal and pathological protein complexes in the membrane (interleukin-2 and -15 receptors, MHC I) and in the nucleus (AP-1 transcription factor, nuclear receptors). A combination of modern microscopy techniques (FRET, fluorescence (cross)correlation spectroscopy (FCS, FCCS), stimulated emission depletion (STED) microscopy), modeling and molecular biological tools are used to dissect interactions and regulation of these complexes. We also develop different modalities of FRET and FCS techniques for the study protein-protein and protein-DNA interactions.
1. Assembly and function of interleukin-2/15 receptors on T cells
Interleukin-2 and -15 (IL-2, IL-15) are cytokines critically involved in controlling T cell activation and function. Their multisubunit receptors share the β and γc chains responsible for one set of ligand-induced signal transduction events, but both have their “private” α chains ensuring high-affinity binding of the appropriate cytokine and specificity of the immune response. Earlier we have shown that IL-2/15 receptor complexes are enriched in lipid rafts and form co-clusters with MHC I and MHC II glycoproteins (Figure 1). We study the factors influencing the assembly and function of these receptors in collaboration with Thomas A. Waldmann’s group (National Cancer Institute, Bethesda, USA).
The membrane potential can affect the general properties of the cell membrane and the conformation of transmembrane proteins having a dipole moment. During their development and function T cells migrate to tissues that depolarize their cell membrane; this may occur e.g. in inflamed tissues or hypoxic tumor microenvironments. We study the effect of membrane potential changes on the signaling properties, mobility and interactions of IL-2/15 receptors.
MHC I is the most highly expressed member of the IL-2R/IL-15R/MHC I protein supercluster. We investigated the effect of MHC I knockdown on receptor assembly and clustering by FRET, FCS and STED superresolution microscopy. We found that the expression level of MHC I regulates the mobility of IL-2/15 receptors, and influences the size of co-clusters.
We are also interested whether IL-2/15R subunits assemble only in the plasma membrane or they can be preassembled in the ER and the Golgi. We follow the assembly of fluorescent-protein-tagged subunits along the secretory pathway. In IL-2 producing T cells, signaling might take place in the cell before receptor subunits are expressed in the plasma membrane. This may have clinical importance in resistance to antagonist antibody therapies (e.g. to humanized anti-IL-2Ralpha, Daclizumab) targeting receptor subunits at the cell surface.
The HLA B27 allele of MHC I is linked to several autoinflammatory and autoimmune diseases such as Bechterew’s disease or celiac disease. This allele is capable of forming nonphysiological MHC I heavy chain dimers via disulfide bonds. In collaboration with the Department of Rheumatology we study the clustering of MHC I with IL-2/15R as well as the signaling properties of the receptors on T cells from Bechterew’s patients.
2. Studying nuclear receptor activation in single cells
We study the molecular details of the activation process of nuclear receptors (RAR: retinoic acid receptor, RXR: rexinoid receptor, PPARγ: peroxisome proliferator-activated receptor gamma, VDR: vitamin D receptor, LXR: liver X receptor) in live cells by fluorescence microscopy (in collaboration with László Nagy’s group at the Department of Biochemistry and Molecular Biology, and Katalin Tóth’s group at the German Cancer Research Center, Heidelberg, Germany). RAR, PPARγ, VDR and LXR form heterodimers with RXR; in addition, RXR also heterodimerizes with several other nuclear receptors. According to the molecular switch model, in the absence of ligand NRs bind to the promoter/enhancer of their target genes in a corepressor complex and suppress transcription. Upon agonist binding, corepressor is released and coactivator is bound activating transcription. By using FCS we have shown that the majority of receptors are freely diffusing in the absence of ligand, and their affinity to DNA increases upon ligand binding, reflected by a stark increase in the fraction of bound receptors (Figure 2). This shift of mobility is dependent on coactivator binding. ChIP-seq also showed that the number of RXR binding sites and their occupancy increased upon ligand activation. We study how ligand binding, the presence of coactivators and chromatin binding influence homo- and heterodimer formation of RXR by using FRET and 2D fluorescence cross-correlation spectroscopy. A common feature of the studied membrane (IL-2/15R) and nuclear receptors is that they share interaction partners with other signaling elements. For the mechanism of subunit sharing between IL-2R and IL-15R we have proposed a model previously. We investigate if nuclear receptors use a similar mechanism.
3. Interactions of the AP-1 complex
The AP-1 transcription factor regulates gene expression in response to various stimuli, including signaling by IL-2 and IL-15 (Figure 3). It plays a role in proliferation, apoptosis, differentiation, cancer formation, etc. Two of the members of the AP-1 family, c-Jun and c-Fos can form heterodimers, and c-Jun can also form homodimers. In contrast, the c-Fos homodimer was found to be instable in earlier in vitro studies. By using FCS and FRET microscopy we have proven that c-Fos can homodimerize and this homodimer can bind to chromatin in live cells overexpressing the protein. We also determined the dissociation constant of the homodimer and the c-Fos-c-jun heterodimer in live cells by a FRET titration method that we developed. We plan to investigate the formation of Fos homodimers in tumor cells overexpressing c-Fos, which may have a role in tumorigenesis.
4. Development of microscopy techniques for studying protein interactions
We develop FRET, FCS, FCCS techniques and analysis methods to investigate the dynamics, (co)mobility and interactions of proteins.