Journal: Nature

Article Title: GPCR antagonism via rewiring of receptor trafficking and degradation

doi: 10.1038/s41586-026-11088-0

Figure Lengend Snippet: (a) Schematic of native versus engineered GPCR trafficking (left). Native agonists typically induce GPCR internalization, followed by recycling or degradation. GTACs direct GPCRs for degradation by leveraging rapid, constitutive TfR1-mediated internalization. GTAC design (right): bifunctional antibody with GPCR-binding domain(s) and TfR1-binding domain(s) fused to an Fc. The control antibody lacks TfR1-binding domain(s). (b) Cell-surface GPCR levels (anti-FLAG flow cytometry) after treatment with GTACs or control antibodies. Cell-surface AT1R levels in FLAG-AT1R-GFP + TfR1 + Expi293 cells after 6 h treatment; cell-surface RXFP1 levels in FLAG-RXFP1 + TfR1 + Expi293 cells after 2 h treatment; cell-surface APJ levels in FLAG-APJ + TfR1 + HEK293T cells after 2 h treatment; and cell-surface A2AR levels in FLAG-A2AR + TfR1 + Expi293 cells after 6 h treatment. Data represent mean ± standard deviation (s.d.) from n = 3 replicates. (c) NFAT-luciferase activation in FLAG-AT1R + TfR1 + NFAT-Luc + Expi293 cells, after 2 h pre-treatment with 50 nM (left) or varying concentration (right) AT1R GTAC or Nb-Fc, followed by 4 h stimulation with varying concentration (left) or 9 nM (right) AngII. Data represent mean ± s.d. from n = 4 biological replicates. (d) β-arrestin recruitment in FLAG-AT1R-Tango + TfR1 + HTLA cells (luminescence) after pre-treatment for 2 h with 50 nM (left) or varying concentration (right) AT1R GTAC or Nb-Fc, followed by 14 h activation with varying concentrations (left) or 200 nM (right) AngII. Data represent mean ± s.d. from n = 3 biological replicates for both experiments. (e) RLN2-induced cAMP signaling in FLAG-RXFP1 + TfR1 + GloSensor + HEK293T cells (luminescence) after 2 h pre-treatment with 10 nM (left) or varying concentration (right) RXFP1 GTAC or control, followed by 30 min stimulation with RLN2 at varying concentrations (left) or 1 nM (right). Data represent mean ± s.d. from n = 3 biological replicates.

Article Snippet: Cells were transfected at 70-80% confluency with NFAT-Luciferase (1ug/well using Lipofectamine3000) and incubated 16 h. Cells were trypsinized, seeded in a 96-well plate (50,000 cells/well), and induced with 1 ug/mL dox for 24 h. Cells were treated with Ab/GTAC for 2 h, and subsequently AngII (MedChemExpress #HY-13948) was added (9 nM AngII was used to generate Ab/GTAC dose-response curves and obtain IC50 values).

Techniques: Binding Assay, Control, Flow Cytometry, Standard Deviation, Luciferase, Activation Assay, Concentration Assay

Journal: Nature

Article Title: GPCR antagonism via rewiring of receptor trafficking and degradation

doi: 10.1038/s41586-026-11088-0

Figure Lengend Snippet: (a) Schematic of FLAG GTACs depleting FLAG-GPCRs. (b) Cell-surface AT1R (left, anti-HA flow cytometry) and total AT1R (right, GFP fluorescence) in FLAG-HA-AT1R-GFP + TfR1 + Expi293 after 6 h FLAG GTAC or Fab-Fc treatment. Data represent mean ± s.d. from n = 3 replicates. β-arrestin recruitment in FLAG-AT1R-Tango + TfR1 + HTLA cells (luminescence) after (c) 2 h pre-treatment with FLAG GTAC or Fab-Fc, then AngII stimulation (14 h, 200 nM), and (d) 2 h pre-treatment with 50 nM FLAG GTAC or Fab-Fc, then AngII (14 h). Data represent mean ± s.d. from n = 3 biological replicates. (e) Total AT1R quantification (left, GFP/cell) and live-cell confocal microscopy of FLAG-AT1R-GFP + TfR1 + Expi293 (right) after FLAG GTAC or Fab-Fc treatment. Scale bar: 20 μm. Centers: medians; box bounds: 25th-75th percentiles; whiskers: minima and maxima. Data represent mean ± s.d. from n = 4 cells. (f) Schematic showing BILF1 constitutively activating NF-κB and downregulating MHC-I. (g) Cell-surface BILF1 (left, anti-HA) and NF-κB reporter activity (right, GFP) in FLAG-HA-BILF1 + TfR1 + NF-κB-GFP + HEK293T after FLAG GTAC or Fab-Fc treatment. Data represent mean ± s.d. from n = 3 biological replicates. (h) Surface-bound FLAG antibody (anti-FLAG) on inducible FLAG-BILF1 + Raji after FLAG GTAC or control treatment (2 h, 25 nM, 0 C or 37 C). Statistical comparisons via unpaired two-tailed t tests. Data represent mean ± s.d. from n = 3 biological replicates. (i) Cell-surface MHC-I in FLAG-BILF1 + Raji after FLAG GTAC or control treatment (left: anti-HLA-ABC, % uninduced; right: % change from induced to uninduced). Statistical comparisons via unpaired two-tailed t tests. Data represent mean ± s.d. from n = 4 biological replicates. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Exact p-values in SI Table 7 .

Article Snippet: Cells were transfected at 70-80% confluency with NFAT-Luciferase (1ug/well using Lipofectamine3000) and incubated 16 h. Cells were trypsinized, seeded in a 96-well plate (50,000 cells/well), and induced with 1 ug/mL dox for 24 h. Cells were treated with Ab/GTAC for 2 h, and subsequently AngII (MedChemExpress #HY-13948) was added (9 nM AngII was used to generate Ab/GTAC dose-response curves and obtain IC50 values).

Techniques: Inhibition, Activity Assay, Flow Cytometry, Fluorescence, Confocal, Microscopy, Control, Two Tailed Test

Journal: Nature

Article Title: GPCR antagonism via rewiring of receptor trafficking and degradation

doi: 10.1038/s41586-026-11088-0

Figure Lengend Snippet: (a) Schematics showing potential determining factors of GTAC activity. (b) Cell-surface AT1R (anti-FLAG flow cytometry) and total AT1R (GFP fluorescence) in FLAG-AT1R-GFP + TfR1 + Expi293 cells following 1.5 h pre-treatment with 1 μM MG132, 250 nM bafilomycin A1, or 25 μg/mL cycloheximide, then AT1R GTAC or Nb-Fc treatment (6 h, 6.25 nM). Data represent mean ± s.d. from n = 3 biological replicates. (c) Cell-surface AT1R (anti-HA) and total AT1R (GFP) over 24 h in FLAG-HA-AT1R-GFP + TfR1 + Expi293 cells after treatment with 1 μM AngII or 50 nM FLAG GTAC. Data represent mean ± s.d. from n = 3 biological replicates. (d) Cell-surface AT1R (anti-HA) in TfR1 + Expi293 cells expressing FLAG-HA-AT1R-GFP with AT1R WT, T282M, or TSTS/A after 1 h treatment with 1 μM AngII, 50 nM FLAG GTAC, or 10 nM AT1R GTAC. Data represent mean ± s.d. from n = 3 replicates. Panels b-d: statistical comparisons via unpaired two-tailed t tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Exact p-values are listed in SI Table 7 . (e) Confocal microscopy of CCR6-GFP + HeLa cells with 24 h TfR1 HIGH GTAC treatment in addition to bafilomycin (n = 8 cells), chloroquine (n = 6 cells) or MG132 (n = 5 cells). (Untreated: n = 8 cells; No inhibitor: n = 6 cells). (f) pH-sensitive binding of CCR6 Fab, probed with H31A mutation. (g) CCR6 Fab light chain CDR sequence with H31 (yellow). (h) Cell surface binding of 5 nM CCR6-Fab-Fc or 125 nM H31 CCR6-Fab-Fc to CCR6 + and CCR6 − Jurkat cells. Data represent mean ± s.d. from n = 3 biological replicates. (i) Western blot analysis and quantification of total CCR6 levels in CCR6 + Jurkat cells after 18 h H31A CCR6-TfR1 HIGH GTAC treatment (n = 3 biological replicates).

Article Snippet: Cells were transfected at 70-80% confluency with NFAT-Luciferase (1ug/well using Lipofectamine3000) and incubated 16 h. Cells were trypsinized, seeded in a 96-well plate (50,000 cells/well), and induced with 1 ug/mL dox for 24 h. Cells were treated with Ab/GTAC for 2 h, and subsequently AngII (MedChemExpress #HY-13948) was added (9 nM AngII was used to generate Ab/GTAC dose-response curves and obtain IC50 values).

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Expressing, Two Tailed Test, Confocal, Microscopy, Binding Assay, Mutagenesis, Sequencing, Western Blot, Analysis

Journal: Nature

Article Title: GPCR antagonism via rewiring of receptor trafficking and degradation

doi: 10.1038/s41586-026-11088-0

Figure Lengend Snippet: (a) Schematics of AT1R GTAC, AT1R Nb-Fc, FLAG GTAC, and FLAG Fab-Fc constructs (left). AT1R GTACs and Nb-Fc binding to FLAG-AT1R + TfR1 + or FLAG-AT1R ” Expi293 cells (right, Protein A flow cytometry). Data represent mean ± standard deviation from n = 3 replicates. (b) Cell surface AT1R (left, anti-FLAG), and total AT1R (right, GFP) in FLAG-AT1R-GFP + TfR1 + Expi293 cells after 1 h or 6 h treatment with AT1R GTAC or Nb-Fc. Data represent mean ± s.d. from n = 3 biological replicates. (c) Cell surface AT1R (left, anti-FLAG) and total AT1R (right, GFP) in FLAG-AT1R-GFP + TfR1 + Expi293 cells over a 24 h time course after treatment with 10 nM AT1R GTAC or 10 nM Nb-Fc. Data represent mean ± s.d. from n = 3 biological replicates. (d) Cell surface APJ (anti-FLAG) in FLAG-APJ + TfR1 + HEK293T cells after treatment with 50 nM APJ GTAC or Nb-Fc over 24 h. Data represent mean from n = 2 biological replicates. (e) Cell surface AT1R (left, anti-FLAG) and total AT1R (right, GFP) in FLAG-AT1R-GFP + TfR1 + Expi293 cells after 6 h treatment with 25 nM AT1R, RXFP1, and CCR6 GTACs and control antibodies. Statistical comparisons via unpaired two-tailed t tests. Data represent mean ± standard deviation from n = 3 biological replicates. (f) Cell surface AT1R (left, anti-FLAG) and total AT1R (right, GFP) in FLAG-AT1R-GFP + TfR1 + Expi293 cells following treatment with AngII for 1 h, 6 h, or 24 h. Data represent mean ± s.d. from n = 3 biological replicates. (g) Cell surface AT1R (left, anti-FLAG) and total AT1R (right, GFP) in FLAG-AT1R-GFP + TfR1 + Expi293 cells after 6 h treatment with AT1R GTAC or Nb-Fc, following 1.5 h pre-treatment with either 250 ng/mL bafilomycin A1, 25 μg/mL cycloheximide, or no pre-treatment, measured by flow cytometry. Data represent mean ± s.d. from n = 3 biological replicates. (h) Cell surface AT1R (left, anti-FLAG) and cell surface TfR1 (middle, anti-v5) in FLAG-AT1R-GFP + TfR1-v5 + Expi293 cells after treatment with 10 nM AT1R GTAC or Nb-Fc across 24 h. Same data represented in line plot (right). Data represent mean ± s.d. from n = 3 biological replicates. Statistical comparisons via unpaired two-tailed t tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Exact p-values are listed in SI Table 7 .

Article Snippet: Cells were transfected at 70-80% confluency with NFAT-Luciferase (1ug/well using Lipofectamine3000) and incubated 16 h. Cells were trypsinized, seeded in a 96-well plate (50,000 cells/well), and induced with 1 ug/mL dox for 24 h. Cells were treated with Ab/GTAC for 2 h, and subsequently AngII (MedChemExpress #HY-13948) was added (9 nM AngII was used to generate Ab/GTAC dose-response curves and obtain IC50 values).

Techniques: Binding Assay, Construct, Fc Binding, Flow Cytometry, Standard Deviation, Control, Two Tailed Test

Journal: Nature

Article Title: GPCR antagonism via rewiring of receptor trafficking and degradation

doi: 10.1038/s41586-026-11088-0

Figure Lengend Snippet: (a) Multiplexed four-color live-cell confocal microscopy of HeLa cells expressing Rab11-mCherry, CCR6-GFP, and TfR1-BFP with 24 h treatment of 10 nM GTAC conjugated to Alexa Fluor 647 or 50 nM CCL20. (b) Pearson colocalization analysis of CCR6-GFP & Rab7-mCherry (late endosomes), CCR6-GFP and Rab11-mCherry (recycling endosomes), or (c) TfR1-BFP and Rab11-mCherry at different times with GTAC treatment. Data are presented as mean ± s.d. n of cells for analyses are summarized in SI Table 4 . Statistical comparisons performed via unpaired two-tailed t-tests. (d) Live-cell confocal microscopy of FLAG-AT1R-GFP + TfR1 + Expi293 cells after 30 min or 6 h treatment with 1 μM AngII, 25 nM AT1R GTAC, or 25 nM FLAG GTAC. Scale bar: 20 μm. (e) Quantification of intracellular AT1R, measured as the ratio of intracellular to total GFP fluorescence per cell. Statistical comparisons performed via unpaired two-tailed t tests. Data represent mean ± s.d. from n = 4 cells. (f) Confocal microscopy images showing RXFP1 localization in FLAG-RXFP1-GFP + TfR1 + Hela cells after 6 h treatment with 50 nM bivalent RXFP1 GTAC, Nb-Fc, or untreated controls. Arrows indicate intracellular puncta. Scale bar: 20 μm. Images represent results of 2 independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Exact p-values are listed in SI Table 7 .

Article Snippet: Cells were transfected at 70-80% confluency with NFAT-Luciferase (1ug/well using Lipofectamine3000) and incubated 16 h. Cells were trypsinized, seeded in a 96-well plate (50,000 cells/well), and induced with 1 ug/mL dox for 24 h. Cells were treated with Ab/GTAC for 2 h, and subsequently AngII (MedChemExpress #HY-13948) was added (9 nM AngII was used to generate Ab/GTAC dose-response curves and obtain IC50 values).

Techniques: Confocal, Microscopy, Expressing, Analysis, Two Tailed Test, Fluorescence

Journal: Nature

Article Title: GPCR antagonism via rewiring of receptor trafficking and degradation

doi: 10.1038/s41586-026-11088-0

Figure Lengend Snippet: (a) Angiotensin II (AngII) increases beat-to-beat variation of the contraction waveform (single representative spot). Phase-warped, amplitude-normalized mechanical contraction waveforms for the 10 beats immediately before (blue) and 10 after (red) injection of Ang II. Thin lines: individual beats; bold line: across-beat mean; shaded band: ± 1 s.d. The shape variation (mean across-beat SD over the cycle) increases from 0.041 (Pre) to 0.073 (Post), + 80%. (b) Mechanical beat shape variation before vs. after 1 μM AngII or DMSO injection. Each beat was phase-warped and amplitude-normalized; variation is the across-beat SD of the normalized waveform (lower = more reproducible shape). Values are normalized by each recording's median pre-injection variation. Beating interval (c) and normalized mechanical beat-shape variation (d) in response to 1 μM Ang II injection across four drug treatments: an untreated cardiomyocyte culture (same data as "Untreated" in Fig. 5d and Extended Data Fig. 11b ) and three pre-treated cultures: VHHA-based AT1R GTAC (same data as “70 nM GTAC” in Fig. 5d and Extended Data Fig. 11b ), AT1R Nb-Fc, and TfR1 Nb-Fc. (e) Per-beat contraction amplitude (from onset to peak of the band-passed mechanical signal, a.u.) over the 10 beats immediately before vs. after injection of 1 μM Ang II or DMSO vehicle, on untreated hiPSC-cardiomyocytes. (f) Per-beat time-to-peak (TTP, s) over the 10 beats immediately before vs. after injection, on untreated cells. (g) Per-beat electromechanical delay (EMD) before vs. after injection. In contrast to beat interval and mechanical waveform variation, AngII did not significantly alter contraction amplitude, time-to-peak, or electromechanical delay under the conditions tested. Bar graphs: medians; error bars: interquartile ranges. Statistical comparisons performed via unpaired two-tailed t-tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Exact p-values are listed in SI Table 7 .

Article Snippet: Cells were transfected at 70-80% confluency with NFAT-Luciferase (1ug/well using Lipofectamine3000) and incubated 16 h. Cells were trypsinized, seeded in a 96-well plate (50,000 cells/well), and induced with 1 ug/mL dox for 24 h. Cells were treated with Ab/GTAC for 2 h, and subsequently AngII (MedChemExpress #HY-13948) was added (9 nM AngII was used to generate Ab/GTAC dose-response curves and obtain IC50 values).

Techniques: Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Chronic kidney disease promotes anxiety susceptibility through an angiotensin II–central amygdala axis

doi: 10.64898/2026.08.16.744184

Figure Lengend Snippet: (A) Serum Ang II levels in Control, CKD, SUS and CKD-SUS mice (n=10 per group).(B-C) Quantification and representative images of fluorescence accumulation in the CeA (n = 5; scale bars: left 100 μm and right 50 μm).(D) Representative immunofluorescence images of AT1R (red) and c-Fos (white) expression in CeA sections, with DAPI nuclear staining (blue) (scale bar: 50 μm).(E) Representative co-localization curve corresponding to the region indicated by the green arrow (pointing) in panel (D).Data are presented as mean ± SEM. Statistics used one-way analysis of variance followed by Bonferroni post hoc tests. *p < 0.05, **p < 0.01.

Article Snippet: Serum and CSF Ang II concentrations were measured using a Mouse Angiotensin II ELISA Kit (RayBiotech, Cat. No. EIA-ANGII, Guangzhou, China).

Techniques: Control, Fluorescence, Immunofluorescence, Expressing, Staining

Journal: bioRxiv

Article Title: Chronic kidney disease promotes anxiety susceptibility through an angiotensin II–central amygdala axis

doi: 10.64898/2026.08.16.744184

Figure Lengend Snippet: (A) Cerebrospinal fluid (CSF) Ang II levels. Due to limited CSF volume, each data point represents pooled samples from 3–5 mice (detection limit: 0.1 pg/mL).(B) Representative immunofluorescence images of Ang II in the central amygdala (CeA) (scale bars: 100 μm and 50 μm).(C) Quantification of Ang II fluorescence-positive area in the CeA (n = 4).(D) Representative images of viral transduction and AT1R expression in the CeA (n = 3). Green: virus; red: AT1R; blue: DAPI (scale bar: 100 μm).(E) Quantification of AT1R expression in the CeA after viral transduction (n = 3).Data are presented as mean ± SEM. Statistics used two-tailed unpaired t tests.“ns” indicates no significant difference, **p < 0.01.

Article Snippet: Serum and CSF Ang II concentrations were measured using a Mouse Angiotensin II ELISA Kit (RayBiotech, Cat. No. EIA-ANGII, Guangzhou, China).

Techniques: Immunofluorescence, Fluorescence, Transduction, Expressing, Virus, Two Tailed Test