Fig. 2

Peptide-GPCR pairs exhibit tunable response characteristics, are naturally orthogonal, and peptides are functionally secreted. a Experimental framework for GPCR characterization. Performance of each peptide-GPCR pair was evaluated by recording its dose-response to synthetic cognate peptides, using fluorescence as a read-out. Parameter values for basal and maximal activation, fold change, EC₅₀, dynamic range (given through Hill slope) were extracted by fitting each curve to a four-parameter non-linear regression model using PRISM GraphPad. Experiments were done in triplicates and errors represent the SD. Dose-response curves of GPCRs (Sc.Ste2, Fg.Ste2, Zb.Ste2, Sj.Ste2, Pb.Ste2) with different response behaviors are featured. b The GPCRs cover a wide range of response parameters. The EC₅₀ values of peptide-GPCR pairs are plotted against fold change in activation. Experiments were done in triplicate and parameter errors can be found in Supplementary Table 5. c GPCRs are naturally orthogonal across non-cognate synthetic peptide ligands. A 30 × 30 orthogonality matrix was generated by testing the response of 30 GPCRs across all 30 peptide ligands. The test concentration was set at 10 µM of a given peptide ligand. The fluorescence signal for maximum activation of each GPCR (not necessarily its cognate ligand) was set to 100% activation and the threshold for categorizing cross-activation was set to be ≥ 15% activation of a given GPCR by a non-cognate ligand. Experiments were performed in triplicate. GPCRs are organized according to a phylogenetic tree of the protein sequences. d Orthogonality of peptide-GPCR pairs when peptides are secreted. The 15 best performing pairs (marked in red in panels a–c) were chosen for secretion. Experiments were performed by combinatorial co-culturing of strains constitutively secreting one of the indicated peptides and strains expressing one of the indicated GPCRs using GPCR-controlled fluorescent as read-out. Experiments were performed in triplicate and results represent the mean