FIGURE 10.

Orientation and responses of MRAPs and MRAP-MRAP-receptor fusion constructs in CHO cells. A, CHO cells were transfected with either V5-MRAP-3xFLAG or tandem MRAP-V5-MRAP-3xFLAG proteins with mutations as shown. Relative expression of V5 and FLAG epitopes on the cell surface and in permeabilized cells was determined as described under “Experimental Procedures” and expressed relative to total for each epitope. B, CHO cells were transfected with wild-type or mutant MRAP-MRAP-MC2R fusion proteins. Total and surface HA and V5 epitopes were measured, and results were expressed relative (rel.) to total for each epitope. Although relative expression of an epitope can be compared for different mutants, surface expression of proteins cannot be compared directly with total because of differences in assay conditions. C, CHO cells were transfected to express MRAP plus MC2 receptor or the MRAP-MRAP-MC2 receptor constructs shown, and responses to ACTH were determined. Error bars show S.E.