Figure 4.

Increased surface levels and decreased internalization rate of MPR-FFWYLL-A. (A) Schematic illustration of the cytoplasmic tails of the CD-MPR constructs. The amino acids are shown in single-letter code. The internalization signals that are mutated to alanines in the mutant construct and the palmitoylated cysteines are indicated by bold letters. (B) Internalization rates. Cell surface proteins of mouse L cells stably expressing MPR wt (▪), MPR-FFWYLL-A (•), and MPR-CC-A (▴) were derivatized at 4°C by using sulfo-NHS-SS-biotin. The cells were then incubated at 37°C for the indicated time and subsequently chilled on ice. The biotin groups remaining at the cell surface were removed by incubation in a reducing glutathione solution. The cells were lysed, and the wild-type and mutant forms of MPR were immunoprecipitated. Immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblotting by using a streptavidin-horseradish peroxidase conjugate. The immunoblots were quantitated for each construct, and the values were expressed as their percentage of the sample that was kept at 4°C and not treated with glutathione. The values are averages of three individual experiments. (C) Surface levels. Mouse L cells stably expressing MPR wt and MPR-FFWYLL-A were incubated with iodinated antibodies against CD-MPR for 2 h on ice either without saponin for the surface levels or with 0.1% saponin to determine the total CD-MPR levels. The cells were lysed and the cell-associated radioactivity was determined with a gamma-counter. The bars represent the percentage of wt and mutant CD-MPR that were present at the cell surface at steady state. The values are expressed as mean ± SEM from four separate experiments.