FIGURE 2.

Lack of N-glycosylation destabilizes hδOR variants at the cell surface, but only the Cys²⁷ variant shows compromised ligand binding ability. Stably transfected HEK293_i cells were induced for 24 h to express WT receptor variants and their non-N-glycosylated mutants, and samples were subjected to flow cytometry (A) or cell surface biotinylation chase (B) assays. For A, cells were incubated with c-Myc (9E10) antibody followed by phycoerythrin-conjugated secondary antibody to detect the total receptor pool at the cell surface (black bars) or with the FITC-conjugated NTX (gray bars) to detect the ligand binding competent receptors. The GeoMean values were normalized to those obtained for the WT Cys²⁷ variant. The data shown are from three to five independent experiments performed in triplicate or quadruplicate. In B, cells were labeled with sulfo-NHS-biotin and chased for the indicated periods of time at 37 °C. Receptors were immunoprecipitated from cellular lysates with FLAG M2 antibody and analyzed by Western blotting with HRP-streptavidin. The data from densitometric analysis from five independent experiments are shown. The values are means ± S.E., are normalized to the 0-h chase samples, and were fitted to the one-phase decay equation. The data were analyzed with the two-way analysis of variance and the Bonferroni's multiple comparison test. **, p < 0.01; ***, p < 0.001. Ab, antibody; IP, immunoprecipitation; WB, Western blot.