Figure 1.
Schematic illustration of signal activation of human GPCRs by membrane-tethered peptide ligands.
(A) Overview of this study. The membrane-tethered peptide activates human GPCR, which is heterologously produced in yeast, thereby activating the chimeric Gα proteins. This promotes the mitogen-activated protein kinase (MAPK) cascade and transcription factor Ste12p. Phosphorylated Ste12p induces transcription of the GFP reporter gene by binding to a pheromone response element in the FIG1 promoter. (B) Functional domains encoded by the membrane-tethered peptide plasmids. After processing by the secretory pathway, the signal sequence and glycosyl-phosphatidylinositol (GPI) targeting sequence are cleaved and the peptide sequence, which contains a free N-terminus, is tethered on the plasma membrane by GPI covalently linked to the C-terminus.
Figure 2.
Comparison of green fluorescence proteins (ZsGreen and EGFP).
Yeast strain BY4741 was transformed with pGK416-ZsGreen, pGK416-EGFP or pGK416 (empty vector). All transformants were grown in SD selective medium for 18 h. (A) Visualization of the green fluorescence (ZsGreen and EGFP). Cells were examined using the 40× objective lens of a fluorescence microscope. Exposure time was 1 s. The left photographs are fluorescence micrographs, and the right photographs are bright-field micrographs. (B) The mean GFP fluorescence of 10,000 cells was measured by flow cytometry. Error bars represent the standard deviation from three separate runs (n = 3); ***, p < 0.001, by one-way ANOVA, Tukey’s post test.
Figure 3.
Activation of human somatostatin receptor subtype-5 (hSSTR5) produced in yeast by exogenously-added somatostatin.
Yeast strains IMFD-70 (a, b); IMFD-72 (c, d); IMFD-70Zs (e, f); IMFD-72Zs (g, h); IMFD-70ZsD (i, j) and IMFD-72ZsD (k, l) were transformed with either pGK421 (empty vector) (a, c, e, g, i, k) or pGK421-SSTR5 (b, d, f, h, j, l). All transformants were grown in SD selective medium for 18 h. The cells then were incubated for another 4 h in pH-adjusted SD selective medium with or without 10 μM somatostatin (SST, 14 aa peptide). (A) The GFP fluorescence of 10,000 cells was measured by flow cytometry. Mean values of the green fluorescence signal of 10,000 cells. Error bars represent the standard deviation from three separate runs (n = 3); ***, p < 0.001, by one-way ANOVA, Tukey’s post test. (B, C) Visualization of the green fluorescence. (B) Cells were examined using the 40× objective lens of a fluorescence microscope. Exposure time was 1 s. The left photographs are fluorescence micrographs, and the right photographs are bright-field micrographs. (C) Cells were examined using the 100× objective lens of a fluorescence microscope. Exposure time was 0.2 s.
Figure 4.
Activation of human somatostatin receptor subtype-5 (hSSTR5) by membrane-tethered somatostatin.
Yeast strains IMFD-72 and IMFD-72ZsD, which coexpress pGK421-SSTR5 and either pGK426-S1442 (SST) or pGK426-alpha42 (α-factor), were incubated in pH-adjusted SD selective medium. (A) The GFP fluorescence of 10,000 cells was measured by flow cytometry. Mean values of the green fluorescence signal of 10,000 cells. Error bars represent the standard deviations (n = 3); ***, p < 0.001, by one-way ANOVA, Tukey’s post test. (B, C) Fluorescence microscopy analysis of cells incubated for 9 h. (B) Cells were examined using the 40× objective lens of a fluorescence microscope. Exposure time was 0.67 s. The left photographs are fluorescence micrographs, and the right photographs are bright-field micrographs. (C) Cells were examined using the 100× objective lens of a fluorescence microscope. Exposure time was 0.14 s.
Figure 5.
Activation of human somatostatin receptor subtype-2 (hSSTR2) by exogenously-added somatostatin.
Yeast strains IMFD-70, IMFD-72, IMFD-70ZsD and IMFD-72ZsD were transformed with pGK421-SSTR2. All transformants were grown in SD selective medium for 18 h. The cells then were incubated for another 4 h in pH-adjusted SD selective medium with or without 10 μM somatostatin (SST, 14 aa peptide). (A) The GFP fluorescence of 10,000 cells was measured by flow cytometry. Mean values of the green fluorescence signal of 10,000 cells. Error bars represent the standard deviations (n = 3); ***, p < 0.001, by one-way ANOVA, Tukey’s post test. (B, C) Visualization of the green fluorescence. (B) Cells were examined using the 40× objective lens of a fluorescence microscope. Exposure time was 1 s. The left photographs are fluorescence micrographs, and the right photographs are bright-field micrographs. (C) Cells were examined using the 100× objective lens of a fluorescence microscope. Exposure time was 0.33 s.
Figure 6.
Activation of human somatostatin receptor subtype-2 (hSSTR2) by membrane-tethered somatostatin.
Yeast strain IMFD-72ZsD which coexpresses pGK421-SSTR2 and either pGK426-S1442 (SST) or pGK426-alpha42 (α-factor) was incubated in pH-adjusted SD selective medium. (A) The GFP fluorescence of 10,000 cells was measured by flow cytometry. Mean values of the green fluorescence signal of 10,000 cells. Error bars represent the standard deviations (n = 3); ***, p < 0.001, by one-way ANOVA, Tukey’s post test. (B) Fluorescence microscopy analysis of the cells incubated for 9 h. Cells were examined using the 40× objective lens of a fluorescence microscope. Exposure time was 0.25 s. The left photographs are fluorescence micrographs, and the right photographs are bright-field micrographs.
Figure 7.
Activation of human neurotensin receptor subtype-1 (hNTSR1) produced in yeast following exogenously-added neurotensin.
Yeast strains IMFD-70, IMFD-72, IMFD-70ZsD and IMFD-72ZsD were transformed with pGK421-NTSR1. All transformants were grown in SD selective medium for 18 h. The cells then were incubated for another 4 h in pH-adjusted SD selective medium with or without 10 μM neurotensin (NTS, 13 aa peptide). (A) The GFP fluorescence of 10,000 cells was measured by flow cytometry. Mean values of the green fluorescence signal of 10,000 cells. Error bars represent the standard deviation from three separate runs (n = 3); ***, p < 0.001, by one-way ANOVA, Tukey’s post test. (B, C) Visualization of the green fluorescence. (B) Cells were examined using the 40× objective lens of a fluorescence microscope. Exposure time was 1 s. The left photographs are fluorescence micrographs, and the right photographs are bright-field micrographs. (C) Cells were examined using the 100× objective lens of a fluorescence microscope. Exposure time was 0.5 s.
Figure 8.
Quantitative digital image analysis of ZsGreen fluorescence using fluorescence microscopy and ImageJ software.
Yeast strain IMFD-72ZsD was transformed with pGK421-NTSR1. The transformant was grown in SD selective medium for 18 h. The cells then were incubated for another 4 h in pH-adjusted SD selective medium with neurotensin (NTS, 13 aa peptide) at several concentrations. (A) Visualization of the green fluorescence. Cells were examined using the 40× objective lens of a fluorescence microscope. Exposure time was 1 s. The upper photographs are fluorescence micrographs, and the lower photographs are bright-field micrographs. (B) The GFP fluorescence of 100 cells was measured as integrated density (IntDen, ImageJ software). Mean values of the green fluorescence signal of 100 cells. IntDen % was represented by relative IntDen normalized with the values of maximal effects of NTS-specific dose-responses. Error bars represent the standard deviation from four separate runs (n = 4); ***, p < 0.001, by one-way ANOVA, Tukey’s post test.
Figure 9.
Activation of human neurotensin receptor subtype-1 (hNTSR1) by membrane-tethered neurotensin.
(A) Amino acid sequences of membrane-tethered peptides. (B, C) Yeast strain IMFD-72ZsD, which coexpresses pGK421-NTSR1 and either pGK426-NTS42 (NTS), pGK426-NTS(8-13)42 (NTS(8-13)), pGK426-NMN42 (NMN) or pGK426-alpha42 (α-factor), was incubated in pH-adjusted SD selective medium. (B) The GFP fluorescence of 10,000 cells was measured by flow cytometry. Mean values of the green fluorescence signal of 10,000 cells. Error bars represent the standard deviations (n = 3); *, p < 0.05, and ***, p < 0.001, by one-way ANOVA, Tukey’s post test. (C) Fluorescence microscopy analysis of the cells incubated for 24 h. Cells were examined using the 40× objective lens of a fluorescence microscope. Exposure time was 0.67 s. The left photographs are fluorescence micrographs, and the right photographs are bright-field micrographs.