Figure 2. Phosphorylation of MOR is required for endocytosis of receptors, loss of surface receptors upon chronic activation, and the development of presynaptic tolerance.
(A) Representative images of axons of neurons marked with syp-mCh, expressing the endosomal marker VPS29-GFP andFLAG-tagged opioid receptors (SSF-MOR), surface labeled with a primary anti-FLAG antibody conjugated to Alexa 647. Neurons were imaged using oblique illumination at a frequency of 1 frame/min. Note the uniform distribution of the receptor before agonist addition (baseline) and the punctate distribution overlapping with a segmented mask of the endosomal marker after 25–30 min of incubation with DAMGO 10 μM. Scale bar is 5 μm. See also Figure 2—video 1. (B) Quantification of the enrichment of surface labeled SSF-MOR at VPS29-GFP marked structures along axons for cells treated with vehicle (n=5 acquisitions) or cells treated with DAMGO 10 μM (n=8 acquisitions). Right axis indicates p-values for unpaired t-test between the two conditions. (C) Same as A,B, for FLAG-tagged mutant opioid receptors where all serine and threonine residues of the C-terminal tail have been mutated to alanine (MOR S/T to A), for vehicle (n=5 acquisitions) or DAMGO 10 μM (n=7 acquisitions) treated cells. Note the diffused distribution of surface labeled MOR-S/T to A after 25–30 min of incubation with DAMGO 10 μM. (D) Quantification of presynaptic inhibition mediated by the MOR S/T to A mutant acutely (inset n=717 synapses, n=7 acquisitions) or after 18 hr of incubation with DAMGO 10 μM (inset n=2360 synapses, n=11 acquisitions). (E) Same as A,B, for FLAG-tagged mutant opioid receptors where serine and threonine residues of STANT motif on the C-terminal tail have been mutated to alanine (STANT), for vehicle (n=5 acquisitions) or DAMGO 10 μM treated cells (n=6 acquisitions). Note the diffused distribution of surface labeled STANT after 25–30 min of incubation with DAMGO 10 μM. (F) Quantification of presynaptic inhibition mediated by the STANT MOR mutant acutely (inset n=1,882 synapses, n=11 acquisitions) or after 18 hr of incubation with DAMGO 10 μM (inset n=2605 synapses, n=14 acquisitions). (G) Same as A,B, for SSF-MOR in neurons treated with Cmpd101 30 μM (n=8 acquisitions) or DMSO control (n=6 acquisitions) and incubated with DAMGO 10 μM. Note the difference in distribution between the two conditions after 25–30 min of incubation with DAMGO. (H) Quantification of presynaptic inhibition mediated by wild type MOR in cells incubated with Cmpd101 30 μM (inset n=2157 synapses, n=15 acquisitions) or DMSO control (inset n=2346 synapses, n=17 acquisitions) together with DAMGO 10 μM for 18 hr. (I) Cumulative frequency curves of the normalized fluorescence at individual synapses for SEP-MOR signal (left panel) and syp-mCh for cells incubated with DMSO only (n=2273 synapses), cells pretreated with DMSO +DAMGO 10 μM for 18 hr (n=2209 synapses) and cells treated with Cmpd101 30 μM+DAMGO 10 μM for 18 hr (n=2456 synapses). Note that the left shift for SEP-MOR fluorescence after pretreatment with DMSO control +DAMGO is blocked by Cmpd101 while syp-mCh control signal is stable across conditions. *, ** represent p<0.05, 0.01, respectively. See also Figure 2—source data 1.
