Fig 1: DAMGO and morphine differentially assemble protein-interaction networks to control MOR spatiotemporal signaling. A, DAMGO activation of the MOR leads to rapid recruitment of GRK2 and ß-arrestins and facilitates a redistribution of the MOR at the plasma membrane (66). The DAMGO-stimulated receptor is in close proximity to IQGAP1, a large scaffolding protein that is required for the activation of Rac1 and nuclear ERK. IQGAP1 has been previously shown to bind both ERK and Rac1 (38). The DAMGO-stimulated MOR also associates with the adaptor protein, CRKL, which is essential for the activation of both Rac1 and nuclear ERK. The activation of Rac1 and nuclear ERK (10 min) occurs prior to receptor internalization (30–60 min (8)). B, in contrast, morphine activation of the MOR stimulates a Gai/o–Gß?–PKCa pathway, which increases the proximity of MOR to desmosomal proteins. Desmosomes are formed by two transmembrane proteins, DSC and DSG, which in turn interact with the intracellular proteins plakoglobin/JUP, plakophilin (PKP) and desmoplakin (DSP) (15). The formation of stabilized desmosomal-like plasma membrane domains likely contributes to the inability of the morphine-stimulated MOR to redistribute and therefore controls the sustained increase in cytosolic ERK activity. Knockdown of DSC1 (green) or JUP (cyan) facilitated a transient increase in nuclear ERK in response to morphine, indicative of a redistribution of the receptor at the plasma membrane (8).
Fig 2: DAMGO, but not morphine, activates Rac1 and its spatiotemporal signaling profile depends on the scaffolding proteins IQGAP1 and CRKL. A–C, stimulation of MOR–APEX2 with DAMGO, but not morphine, for 60-min enriched proteins involved in actin cytoskeleton signaling, signaling by Rho family GTPases, and integrin signaling (n = 3). A, heat map showing proteins from these three pathways that were increased (orange) or decreased (purple) following treatment with DAMGO or morphine compared with a vehicle control. Data are expressed as the average log2 change in protein abundance compared with vehicle. B, interaction networks for the proteins that were differentially affected by DAMGO (left panel) versus morphine (right panel) were determined by IPA. Red indicates increased abundance, and green indicates a decrease. Data are expressed as the average log2 change in protein abundance compared with a vehicle control. C, proteins from B, with data expressed as the log2 change in protein abundance compared with vehicle control. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus vehicle control, two-way ANOVA with Dunnett's multiple comparison test. D–J, analysis of the activation of Rac1 in single HEK293 cells transiently expressing MOR and RaichuEV–Rac1 and stimulated with vehicle, 1 µm DAMGO, or 1 µm morphine. D, time course of Rac1 activity (n = 3). E, AUC of data from D, F, G, and I. ***, p < 0.001 versus vehicle control, two-way ANOVA with Dunnett's multiple comparison test. F, time course of Rac1 activity in cells co-transfected with scrambled siRNA (n = 3). G, time course of Rac1 activity in cells co-transfected with IQGAP1 siRNA (n = 3). H, confirmation of knockdown of IQGAP1 protein by immunoblotting. Left panel is a representative blot, and right panel is the grouped intensity measurements from four independent experiments. **, p < 0.01 versus pcDNA transfected control, one-way ANOVA with Dunnett's multiple comparisons test. I, time course of Rac1 activity in cells co-transfected with CRKL siRNA (n = 3). J, confirmation of knockdown of CRKL protein by immunoblotting. Left panel is a representative blot, and right panel is the grouped intensity measurements from three independent experiments. *, p < 0.05 versus pcDNA transfected control, one-way ANOVA with Dunnett's multiple comparisons test. K, AUC of nuclear ERK measured in single HEK293 cells transiently expressing MOR and nucEKAR, and stimulated with vehicle, 1 µm DAMGO, or 1 µm morphine, under control conditions or following co-transfection with scrambled siRNA, IQGAP1 siRNA, or CRKL siRNA (n = 3). AUC was calculated from the time courses in Fig. S6. *, p < 0.05 versus vehicle control, two-way ANOVA with Dunnett's multiple comparison test. Scatter plots show individual data points; symbols/bars represent means, and error bars indicate standard deviation of the mean from n experiments as stated.
Supplier Page from Abcam for Anti-CrkL antibody