Orthogonal Peptide‐Templated Labeling Elucidates Lateral ET_AR/ET_BR Proximity and Reveals Altered Downstream Signaling

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 onestep 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 (ET A R and ET B R), 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 ET A R/ET B R. Further, we demonstrate, that ET A R expression regulates the signaling of co-expressed ET B R. Our orthogonal peptide-templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling.

Bind&Bite: covalently stabilized heterodimeric coiled-coil peptides for the site-selective, cysteine-free chemical modification of proteins

“…This concept has recently been demonstrated by using two orthogonal sets of coiled-coils as templates for the transfer of fluorophores from thioester-linked peptides to the cysteine-modified complementary peptides fused to the proteins of interest. 19–21…”

“…14 Their interaction is driven by the formation of an intermolecular hydrophobic core involving leucine and isoleucine residues, and stabilized by salt bridges between positively (arginine and lysine) and negatively (glutamate and aspartate) charged residues. 15,16 Covalent stabilization of coiled-coils has thus far been achieved through sophisticated reactions involving additional cysteine residues flanking the coiled-coil sequences, 17 in conjunction with the use of a hetero-bivalent cross-linker 18 and native chemical ligation (NCL) using coiled-coils as tag-carriers, 19–21 respectively. Here, we set out to explore the feasibility of covalently stabilizing coiled-coils through isopeptide bonds bridging the side chains of glutamate and lysine residues.…”

Dynamics of the Second Extracellular Loop Control Transducer Coupling of Peptide-Activated GPCRs

“…In the first set of experiments, we performed receptor titration experiments to determine the receptor-to-arrestin-ratio that ensures saturation of the luminescence donor (Nluc-arr3) by the fluorescence acceptor (receptor-eYFP) and hence, results in reproducible measurement window for ligand concentration-response curves. Accordingly, we chose to transfect 30 ng of a modified Nluc-tagged bovine arr-3 construct [71], 3900 ng receptor construct, and 70 ng of an empty pcDNA3.1 vector. After the transfection, 150,000 cells/well were re-seeded into solid white 96-well plates in technical triplicate using a phenol red-free culture medium.…”