Peptide-templated bioconjugation of proteins on and in live cells for studies of GPCR trafficking and crosstalk

g蛋白偶联受体（gpcr）的功能特性取决于其定位、表面表达密度及其在细胞膜中的迁移能力。一些gpcr在单体和二聚体形式之间经历动态平衡。显微成像已经成为研究活细胞上和活细胞内荧光标记的gpcr不可缺少的工具。然而，必须考虑到荧光标记会影响GPCR的功能特性，如向细胞膜输出、配体结合和GPCR内化。在之前的资助期内，我们开发了一种标记方法，该方法只需要小的标签序列，具有高标签特异性，在几分钟（而不是几小时）内进行，并且可以引入几乎任何报告组。标记的受体在配体刺激下保持活性并内化。在目前阶段，我们的方法仅限于细胞外蛋白区域的标记。我们的目标是开发一种蛋白质标记反应，既能维持我们方法的有利特性（小标签尺寸，模块化），又能提供细胞内靶标（AdipoR1受体，阻滞蛋白，g蛋白信号蛋白的调节因子），又能提供多路蛋白质成像（通过多路肽定向酰基转移）。iii)使活细胞表达量化（通过用PNA标记GPCR后的活细胞滚圈扩增）和iv)允许控制GPCR的寡聚化状态（通过将PNA标记的GPCR与DNA/PNA杂交）。在联合研究项目中开发的方法将使我们能够研究人类Y， NPFF， CRF和胃饥饿素受体的动态。我们将分析受体串扰，量化配体可接近的受体，并以动态（通过FRET）或时间分辨（通过脉冲追踪实验）的方式阐明细胞内通路和相互作用伙伴。此外，我们将把概念扩展到其他标签，创建半合成受体（例如体内脂化或聚乙二醇化受体结构）以研究其功能。

The functional properties of G-protein coupled receptors (GPCRs) depend on localization, 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 internalization. In the previous funding period, we developed a labeling 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 labeled receptors maintained their activity and internalized upon stimulation with the ligands. At the current stage our method is restricted to the labeling of extracellular protein regions. It is the aim to develop a protein labeling reaction which sustains the favorable 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 oligomerization state of GPCRs (via hybridization 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 analyze 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.