A neurotensin receptor type 1-derived pepducin acts as a biased allosteric modulator to regulate target receptor function

Figures

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  Figure 1. PP-001 activates G protein signaling pathways but does not recruit s to NTSR1. (a) Principle of the BRET2 G protein dissociation assay. Agonist-induced G protein activation leads to a loss of BRET2 signal, as donor-tagged (RlucII) Gα proteins and acceptor-tagged (GFP10) Gγ proteins dissociate. (b) Principle of the BRET2 recruitment assay. The BRET2 signal increases as donor-tagged (RlucII) cytosolic s are recruited to the acceptor-tagged (GFP10) receptor, after agonist binding. (c) Gα protein dissociation in CHO-K1 cells transiently expressing the NTSR1 receptor, monitored by BRET2, in response to increasing concentrations of NT(8-13), PP-001, NP-001, or PP-SCR-001. (d) Recruitment in CHO-K1 cells, monitored by BRET2, in response to increasing concentrations of NT(8-13), PP-001, NP-001, or PP-SCR-001. Data represent the mean ± SEM of at least three independent experiments, tested in triplicate. Luminescence was measured 30 min after pepducin stimulation. BRET2 ratios were normalized to NT(8-13): data from vehicle-treated cells were set as 0% activation (or recruitment) and data from NT(8-13) (1 μmol/L)-treated cells were set to 100% activation (or recruitment). Schemas were…

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  Figure 2. PP-001 allosterically inhibits NT binding and NT-promoted recruitment, but not NT-promoted G protein activation. (a) Displacement of 125I-radiolabeled NT following incubation of CHO-hNTSR1 membranes with increasing concentrations of unlabeled NT(8-13), PP-001, NP-001, or PP-SCR-001 in a competitive radioligand binding assay. (b, c) NT-induced G protein dissociation and recruitment in CHO-K1 cells expressing NTSR1, pre-treated (10 min) with increasing concentrations (0, 1, 10, 32, and 100 μmol/L) of PP-001, as monitored by BRET2. (d, e) NT-induced Gα13 dissociation and 1 and 2 recruitment in CHO-K1 cells pre-treated (10 min) with increasing concentrations (0, 1, 10, and 100 μmol/L) of NP-001 and PP-SCR-001 (e), as monitored by BRET2. Luminescence was measured 30 min after pepducin stimulation, 20 min following NT(8-13) stimulation. Data represent the mean ± SEM of at least three independent experiments, tested in triplicate. Gamma radiation counts were normalized according to NT(8-13): data from untreated samples were set to 0% specific binding and data from NT(8-13) (1 μmol/L)-treated samples were set to 100% specific binding. BRET2 ratios were normalized according to NT(8-13),…

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  Figure 3. PP-001 does not promote NTSR1 receptor internalization on its own but inhibits NT-induced receptor internalization. (a) Principle of receptor internalization assay. Agonist-induced receptor endocytosis leads to a loss of BRET2 signal, as the donor-tagged (RlucII) receptor is isolated from the acceptor-tagged, PM-localized CAAX-rGFP sensor. (b, c) Kinetic assays monitoring NTSR1 receptor internalization over a 30 min period in transiently transfected CHO-K1 cells, following stimulation with vehicle (HBSS, 1% DMSO), NT(8-13) (1 μmol/L), PP-001 (50 μmol/L), NP-001 (50 μmol/L) or PP-SCR-001 (50 μmol/L). (d, e) Kinetic assay monitoring NT(8-13) (1 μmol/L)-induced NTSR1 receptor internalization over a 30 min period, in transiently transfected CHO-hNTSR1 cells pre-stimulated (15 min) with PP-001 (50, 100 μmol/L), NP-001 (50 μmol/L), or PP-SCR-001 (50 μmol/L). Data represent mean ± SEM of four independent experiments, tested in duplicate. To determine a “ΔBRET2 change factor”, each condition was normalized according to itself and corrected to vehicle: baseline data for each condition was set as a value of 1 and subtracted from vehicle (or vehicle + HBSS)-treated condition. Schema was…

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  Figure 4. PP-001 promotes receptor-receptor interactions. (a) Principle of BRET2 homo- and heteromerization assay. Formation of multimeric donor (RlucII) and acceptor (GFP10)-tagged receptor units leads to an increase in BRET2 signal. (b) BRET2 titration experiments monitoring receptor-receptor interactions in transiently transfected CHO-K1 (NTSR1RlucII-NTSR1GFP10) and HEK293 (NTSR1RlucII-APJGFP10, APJRlucII-NTSR1GFP10) cells treated with vehicle (HBSS, 1% DMSO) or pepducin series PP-001, NP-001, and PP-SCR-001 (100 μmol/L). In the NTSR1RlucII-APJGFP10 experiments (middle panel), cells were treated with 50 μmol/L of pepducin series. In these titration experiments, cells were transfected with fixed amounts of 7TMRRlucII and increasing amounts of 7TMRGFP10; specific receptor-receptor interactions is evidenced by a saturation curve rather than a linear increase. Data represent mean ± SEM of three independent experiments, tested in duplicate. A one-way ANOVA followed by Dunnett’s multiple comparisons test was performed on the BRET50 and BRETmax parameters derived from each curve. The resulting values are provided in Table S4. Schemas was created with Biorender.com.

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  Figure 5. PP-001 induces a sustained drop in arterial blood pressure in rats. (a) Changes in mean arterial blood pressure (MABP) recorded in carotid-artery cannulated male Sprague‒Dawley rats over a 5 min-period, following injection into the jugular vein of vehicle (saline, 5% DMSO), NT(8-13), PP-001, NP-001, or PP-SCR-001 at 55 nmol/kg doses. In the SR48692 + PP-001 condition, rats received the NTSR1-selective non-peptide antagonist SR48692 (1 mg/kg) 10 min prior to PP-001 (55 nmol/kg). (b) Maximal arterial blood pressure decreases (ΔMABP) recorded for each condition. Data represent mean ± SEM, n = 4 (NP-001, SR48692 + PP-001), n = 5 (vehicle, NT(8-13), PP-SCR-001), n = 7 (PP-001) rats. Data were statistically analyzed by a One-way ANOVA followed by Dunnett’s multiple comparisons test, comparing each condition to PP-001-treated condition. ∗∗∗P < 0.001; ∗∗P < 0.01.

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  Figure 6. An alanine scan of PP-001 reveals critical residues for biological activity. (a) Color-coded representation of the effects of alanine substitutions on PP-001-induced biological activity. Alanine substitutions of the amino acids shown in red and orange resulted in a total or almost complete loss, respectively, of the pepducin-induced cellular response in an assay monitoring the dynamic mass redistribution (DMR) kinetics of CHO-hNTSR1 cells. For those shown in black, a response profile similar to that of PP-001 was observed; for those indicated in green, the response profile was altered. (b) DMR kinetics of CHO-K1 cells stably expressing NTSR1, treated with 10 μmol/L concentrations of PP-001, PP-SCR-001, or each of the PP-001 alanine scan pepducins. DMR was monitored by Electric Cell-substrate Impedance Sensing (ECIS) for 50 min post-stimulation. Data represent mean ± SEM, n = 3, tested in duplicate. Each condition was normalized to reflect a “normalized response” (or “change factor”) relative to baseline resistance values (ohms).

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  Figure 7. [Ala2]PP-001 exhibits reduced biological activity compared to PP-001, whereas [Ala13]PP-001 behaves similarly. (a) Table comparing the peptide sequences of PP-001, [Ala2]PP-001, and [Ala13]PP-001. Alanine substitutions are shown in red. (b) Displacement of 125I-radiolabeled NT following incubation of CHO-hNTSR1 membranes with increasing concentrations of NT(8-13), PP-001 (for reference), [Ala2]PP-001, and [Ala13]PP-001. n = 3, tested in triplicate. (c) Gα13 protein dissociation and βarr2 recruitment, monitored by BRET2, in CHO-K1 cells transiently expressing NTSR1, in response to increasing concentrations of NT(8-13), PP-001 (for reference), [Ala2]PP-001, and [Ala13]PP-001. n = 4, tested in triplicate. (d, e) NT(8-13)-induced Gα13 protein dissociation and βarr2 recruitment in CHO-K1 cells transiently expressing NTSR1, pre-treated (10 min) with fixed concentrations (0, 1, 10, 32, and 100 μmol/L) of [Ala2]PP-001 (d) and [Ala13]PP-001 (e), as monitored by BRET2. n = 4, tested in triplicate. (f) NTSR1 internalization over a 30-min period, in response to treatment with vehicle (HBSS, 1% DMSO), NT(8-13) (1 μmol/L), [Ala2]PP-001 (50 μmol/L), or [Ala13]PP-001 (50 μmol/L), in transiently…

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  Figure 8. PP-001 binds to NTSR1. Representative CPM thermodenaturation curves of purified (a) NTSR1-3 or (b) NTSR1-22 in the presence of ligands or vehicle. (c, d) First derivative of the CPM thermodenaturation curves presented in (a) and (b), respectively. (e) Table of the Tm values determined from vehicle, NT(8-13) and PP-001 conditions. The data are expressed as a mean ± SEM of 3 independent experiments in duplicate. Statistical significance is determined by a one-way ANOVA followed by a Dunnett’s post hoc analysis. ∗∗∗P-value < 0.001; and ∗P-value < 0.05.

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  Figure 9. Alanine substitutions in NTSR1 ICL domains impede PP-001-driven inhibition of NT binding. (a) Cell surface expression of the HA-hNTSR1 WT receptor and the derived ICL mutant constructs (Mutants 1–27) in transiently transfected HEK293 cells, as determined by ELISA. Data was normalized to WT (set as 100% expression) and mock (set as 0% expression) conditions. n = 3, tested in duplicate. (b) Displacement of 125I-radiolabeled NT by 10 μmol/L PP-001 in HEK293 cell membranes transiently transfected with the HA-hNTSR1 WT receptor (left) or the ICL mutant constructs (right). No reliable results could be generated with mutants 1, 4, 5, 8, and 18. Mutants represented in color (2, 16, 17, 19, 20) were found to retain greater binding of [125I]-NT. n = 2, tested in duplicate. (c) Snake plot of the human NTSR1 receptor, cropped to ICL domains. Substituted residues in Mutants 2, 16, 17, 19 and 20 are identified in color. (d‒f) Displacement of 125I-radiolabeled NT following incubation of transiently transfected HEK293 membranes with increasing concentrations of “cold” NT(8-13) (left) or PP-001 (right). Data for the WT condition are compared to mutants in the ICL1 (Mutant 2, d), ICL3 (Mutant 16,…

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  Figure 10. Docking of PP-001 to the intracellular domains of NTSR1. (a) Overall view of NTSR1 showing the lowest-energy docking pose of PP-001 (blue) and the positions of residues corresponding to mutants with altered PP-001 activity: Mutant 2 (pink), Mutant 16 (green), Mutant 17 (yellow), Mutant 19 (cyan), and Mutant 20 (red). (b) Close-up of the intracellular region of NTSR1 highlighting the proximity of PP-001 to the identified mutant sites. Ribbon colors represent structural domains: helix 8 (H8, green), intracellular loop 1 (ICL1, pink), intracellular loop 3 (ICL3, brown), and helix 7 C-terminal extension/H8 transition (orange). (c) Intracellular bottom view obtained after a 90° rotation, illustrating the spatial distribution of residues surrounding the PP-001 binding site.