Journal: Nature Communications
Article Title: Myosin VI drives arrestin-independent internalization and signaling of GPCRs
doi: 10.1038/s41467-024-55053-9
Figure Lengend Snippet: a Schematic representation of intramolecular FRET sensor for detection of a conformational change in myosin VI (M6). Activation of M6 is indicated by an open conformation (lower FRET). b FRET ratio for intramolecular M6 sensor with GIPC (2 μM) and D2R C-tail (100 μM) (Supplementary Fig. ). c Schematic diagram of actin gliding assay to assess M6 motility. d Motility speed for M6 with GIPC (2 μM) and D2R C-tail (100 μM). e PBM sequences for GPCR C-tails used in motility assay, color-coded based on PBM class. Refer to Supplementary Table for full sequence of GPCR C-tails used in motility assay. f Motility speed of M6 with GIPC and different GPCR C-tails (100 μM). b , d , f Mean +/- SD is reported for three biological replicates, with statistical significance computed by one-way ANOVA, followed by Dunnett’s post hoc test. ns, not significant ( p > 0.05). b , F = 14.28, d.f. = 9; d , f , F = 18.98, d.f. = 29. g Schematic for ELISA assay for biotinylated PDZ1 domain of GIPC binding to mCitrine (mCit)-GPCR C-tails. h Binding of GPCR C-tails (D2R, LPA1R, and V2R normalized to VIPR1) to PDZ1 at matching concentration (100 nM). Mean +/- SD is reported for three biological replicates, with statistical significance computed by one-way ANOVA, followed by Dunnett’s post hoc test. F = 160.4, d.f. = 9. i Motile speeds for M6 in the presence of GIPC and indicated GPCR C-tails plotted against mCit counts from the ELISA assay. Mean +/- SD is reported for three biological replicates. Simple linear regression was fit to the data to obtain the R 2 value. j M6 motility with GIPC and chimeric GPCR C-tails and affinity optimized KKETAV. Mean +/- SD is reported for three biological replicates, with statistical significance computed by one-way ANOVA, followed by Dunnett’s post hoc test. ns, not significant ( p > 0.05). F = 59.10, d.f. = 20. k Representative images of DOR, LPA1R, VIPR1, and V2R internalization in HEK293 WT cells upon stimulation with respective agonists (1 μM DPDPE, 10 μM LPA, 500 nM VIP, and 1 μM AVP) for 15 min (VIPR1 and V2R) or 30 min (DOR and LPA1R). Myosin VI activity was inhibited by TIP treatment (100 μM). l , m Number of puncta per cell l or cytosol-to-cell surface intensity m under basal, agonist, and agonist+TIP conditions. Mean +/- SD is reported for three biological replicates (n > 10 cells analyzed for each replicate), with statistical significance assessed by one-way ANOVA, followed by Tukey’s post hoc test. ns, not significant ( p > 0.05). l , F = 186.9, d.f. = 35; m , F = 35.38, d.f. = 35.
Article Snippet: DOR and LPA1R expressing cells were stimulated with DPDPE (Tocris; 1 μM) and LPA (Cayman; 10 μM), respectively, for 30 min. VIPR1 was stimulated with VIP (Tocris; 500 nM) for 15 min. For NCAMp, dyngo4a, or TIP treatment, cells were treated 15 minutes prior to agonist addition with NCAMp (30 μM), dyngo (Abcam; 30 μM), or TIP (Sigma; 100 μM) for 15 min. NCAMp contained a cell penetrating TAT sequence (CYGRKKRRQRRRC) attached to the D2R binding sequence of NCAM (VNLCGKAGPGAKGKDMEEG) with a GSG sequence in between.
Techniques: Activation Assay, Gliding Assay, Motility Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Activity Assay
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