FIGURE 6.

Ubiquitination in the first cytoplasmic loop is also required for pharmacological down-regulation of the MOR1B isoform. A, HEK293 cells stably expressing FLAG-tagged MOR1B (F-MOR1B) or FLAG-tagged MOR1B in which only the lysine residues present in the first cytoplasmic loop were mutated to arginine (F-MOR1B-K94R,K96R) and treated with 10 μm DADLE for the indicated time period before assessing receptor down-regulation by radioligand binding using [³H]DPN. Points represent mean determinations from independent experiments, with each time point analyzed in triplicate tubes in each experiment. Error bars represent the S.E. calculated across the experiments (n = 4). B, shown is a down-regulation assay comparing the MOR1 and the MOR1B splice variants. Data are replotted from Figs. 1B and 6A to reveal that, in the present experiments, there was no detectable difference in pharmacological down-regulation of the wild type versions of MOR1 (F-MOR) compared with MOR1B (F-MOR1B) isoforms. C, shown is a diagram describing the proposed sequential sorting operations in the hierarchical sorting model. The C-tail (containing the previously described MRS) determines the overall trafficking itinerary of internalized MORs between recycling and lysosomal routes and does not require MOR ubiquitination. Ubiquitination of the first cytoplasmic loop (1st icl) specifically promotes redistribution of receptors from the limiting membrane to lumen of late endosomes/multivesicular bodies. This intra-multivesicular body “topological” sorting operation does not dictate the overall trafficking itinerary of internalized receptors but is required for efficient destruction of the transmembrane helical bundle containing the diprenorphine binding site. This step is effectively rate-limiting for pharmacological down-regulation of both the MOR1 and MOR1B isoforms. Therefore, traditional down-regulation assays based on loss of radioligand binding sites may not be sensitive to functionally significant differences in the regulated endocytic trafficking itinerary of naturally occurring MOR isoforms.