Orthogonal Peptide-Templated Labeling Elucidates Lateral ETAR/ETBR Proximity and Reveals Altered Downstream Signaling |
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Authors: | Philipp Wolf Alexander Mohr Georgina Gavins Victoria Behr Karin Mörl Prof Oliver Seitz Prof Annette G Beck-Sickinger |
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Affiliation: | 1. Faculty of Life Sciences, Institute of Biochemistry, Leipzig University, Brüderstrasse 34, 04103 Leipzig, Germany;2. Faculty of Mathematics and Natural Sciences, Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany |
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Abstract: | Fine-tuning of G protein-coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one-step labeling method of multiple membrane-embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this approach to four receptors of the cardiovascular system: the endothelin receptor A and B (ETAR and ETBR), angiotensin II receptor type 1, and apelin. Wild type-like G protein activation after N-terminal modification was demonstrated for all receptor species. Using FRET-competent dyes, a constitutive proximity between hetero-receptors was limited to ETAR/ETBR. Further, we demonstrate, that ETAR expression regulates the signaling of co-expressed ETBR. Our orthogonal peptide-templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling. |
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Keywords: | bioorganic chemistry membrane proteins protein modification receptors signal transduction |
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