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. 2015 Sep 30;290(46):27972–27985. doi: 10.1074/jbc.M115.668491

FIGURE 5.

FIGURE 5.

Essential amino-terminal residues are required on the Nout copy of MRAP. Schematic representations of the presumed orientations of MRAP-MRAP-MC2R fusion proteins containing mutations in either the first copy of MRAP (mutations on the extracellular face) (A) or the second copy of MRAP (mutations on the intracellular face) (B) are shown. Mutated segments are shown in red. In A and B, cells were incubated for 4–5 h with ACTH, and the response was measured with the CRE-luciferase assay and normalized to the forskolin response. In C, cells were incubated with ACTH for 20 min in buffer containing 0.5 mm isobutylmethylxanthine, and cAMP mass was measured by ELISA. The data were normalized to surface HA-receptor measured in parallel cultures. Relative to the value in cells transfected with MRAP/MC2R, surface receptor levels were 58.3% (MRAP-MRAP-MC2R), 144% (MRAP-(18–21A)MRAP-MC2R), and 35.5% ((18–21A)MRAP-MRAP-MC2R). Color schemes and sequences are given in Figs. 1 and 4. Error bars show S.E.