PP 1623: Peptide-Templated Bioconjugation with Proteins on and in Living Cells for Investigations of GPCR Transport and Cross-Reactivity

The functional properties of G-protein coupled receptors (GPCRs) depend on localisation, density of surface expression and their mobility in the cell membrane. Some GPCRs undergo dynamic equilibria between monomeric and dimeric forms. Microscopic imaging has become an indispensable tool to study fluorescence labelled GPCRs on and in live cells. However, it has to be considered that the fluorescent tag can affect GPCR functional properties such as export to the cell membrane, ligand binding and GPCR internalisation. In the previous funding period, we developed a labelling method that requires only small tag sequences, occurs with high tag specificity, proceeds within minutes (rather than hours) and enables the introduction of virtually any reporter group. The labelled receptors maintained their activity and internalised upon stimulation with the ligands. At the current stage our method is restricted to the labelling of extracellular protein regions. It is the aim to develop a protein labelling reaction which sustains the favourable properties (small tag size, modularity) of our method yet i) provides access to intracellular targets (AdipoR1 receptor, arrestins, regulators of G-protein signaling proteins), ii) makes available multiplexed protein imaging (via multiplexed peptide-directed acyl transfer), iii) enables live cell quantification of expression (via live cell rolling circle amplification after labeling of GPCR with PNA) and iv) allows for control over the oligomerisation state of GPCRs (via hybridisation of PNA-labeled GPCRs with DNA/PNA). The methods developed in the joined research project will allow us to investigate the dynamics of human Y, NPFF, CRF and ghrelin receptors. We will analyse receptor crosstalk, quantify the receptors accessible by the ligand and elucidate the intracellular pathways and interaction partners in a dynamic (by FRET) or time-resolved (by pulse-chase experiments) manner. Furthermore, we will expand the concept to other tags, create semi-synthetic receptors (e.g. in vivo lipidated or pegylated receptor constructs) for studying their function.

Principal investigators
Seitz, Oliver Prof. Dr. rer. nat. (Details) (Organic and Bioorganic Chemistry III)

Participating external organisations

DFG: Sachbeihilfe

Duration of project
Start date: 02/2016
End date: 06/2019

Research Areas
Biochemistry, Biological und Biomimetic Chemistry

Last updated on 2022-08-09 at 21:06