Extended Data Fig. 9: Oligomeric state characterization of the phosphorylation deficient constitutively active T279K mutant and verification of effectors on the wild-type µOR. | Nature Chemical Biology

Extended Data Fig. 9: Oligomeric state characterization of the phosphorylation deficient constitutively active T279K mutant and verification of effectors on the wild-type µOR.

From: Single-molecule analysis reveals agonist-specific dimer formation of µ-opioid receptors

Extended Data Fig. 9

a, Intensity histograms of the dimeric T279K mutant and its phosphorylation-deficient modification. The phosphorylation-deficient modified mutant shows a monomeric distribution of intensities. b, Bleaching experiments over 400 frames (each 40 ms) and high laser-power reveal an almost exactly doubled half-life time for the T279K mutant (8.9 ± 0.3 s) in contrast to its phosphorylation-deficient modification (4.5 ± 0.2 s). c, Oligomerization states of wild-type µOR, its phosphorylation-deficient modification (pd) as well as wild-type µOR treated with Pitstop2 (Ps2) before and after activation with 10 µM DAMGO. The phosphorylation-deficient modification results in a monomeric distribution of intensities. Pitstop2 had within our measurement time window no significant effect on the basal monomeric state of the µOR, nor on the DAMGO-induced formation of dimers. d, Individual datapoints and significance of the measurements shown in (c). Whiskers show SD, the box represents IQR of the data and the line indicates the median. [Data in (a) were generated from 347 (T279K-pd) and 287 (T279K) detected PSFs from 5 different cells tested on 3 experimental days. (b) shows a representative bleaching trace over 400 frames extracted from datasets acquired in (a). Data in c and d are from n = 8, 5, 5, 5, 4, and 6 different cells for “wt”, “wt+DAMGO”, “wt-pd”, “wt-pd+DAMGO”, “wt+Ps2” and “wt+Ps2+DAMGO”, respectively. Cells of each variant are from three independent experiments. One Datapoint in (d) corresponds to one PSF- intensity. P values were determined by one-way ANOVA and Tukey’s multiple comparison test].

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