Orthogonal Peptide‐Templated Labeling Elucidates Lateral ET(A)R/ET(B)R Proximity and Reveals Altered Downstream Signaling

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 (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.