FIGURE 5.

CNX-hδOR-Cys²⁷ interaction in CST-treated cells. Stably transfected HEK293S cells constitutively expressing the WT hδOR-Cys²⁷ were labeled with [³⁵S]methionine/cysteine for 60 min and chased for the indicated periods of time. The glucosidase inhibitor CST (200 μg/ml) was added to the culture medium at the beginning of depletion and was maintained thereafter or, alternatively, was only added to the chase medium, as indicated. The opioid receptor antagonist NTX (10 μm) was added to the chase medium (J), and the proteasomal inhibitor LA (10 μm) was added to the depletion, pulse, and chase media (K). Receptors were purified from cellular lysates by sequential immunoprecipitation to recover the total (top panels of A, C, and E; K and J) and CNX-bound receptors (bottom panels of A, C, and E). Samples were analyzed by SDS-PAGE, fluorography, and densitometric scanning of the fluorograms. Precursor and mature receptor forms are indicated with open and closed symbols, respectively. The precursor carrying only one N-glycan is indicated with an arrow, and the degradation intermediate is indicated with an asterisk. The arrowhead in J points to an unknown protein co-purifying with the receptor in LA-treated cells. B, D, and F represent quantitative analysis of fluorograms in A, C, and E, respectively, showing time-dependent changes in the intensity of total and CNX-bound pools of precursors. G shows corresponding changes in the amount of mature receptors. The values in B, D, and F are normalized to the precursor of the 0-h chase control samples, and the values in G are normalized to the corresponding precursor at the end of the pulse. The data shown are representative of three independent experiments. H depicts changes in the ratio of precursor and mature receptor forms in the 4-h chase samples, and I shows the relative changes in the total receptor amount compared with the corresponding nontreated control samples. The values are means ± S.E. of six independent experiments. The data in H were analyzed with the Tukey's multiple comparison test after the two-way analysis of variance, and the data in I were analyzed with the one-sample t test. *, p < 0.05; ***, p < 0.001. C, chase; P, pulse; Ab, antibody; IP, immunoprecipitation.