FIGURE 4.

Expression and binding studies of the MC3R/GHSR heterodimer. A, total receptor expression was determined with N-terminally HA (NHA) and C-terminally FLAG-tagged (CFLAG) constructs. N-terminally HA-tagged MC3R served as a negative control. Three days after transfection, cells were lysed, and total receptor expression was determined as described under “Experimental Procedures.” B, for determination of cell surface expression, COS-7 cells were cotransfected with equal amounts of plasmid DNA coding for N-terminally HA-tagged MC3R or GHSR and the C-terminally FLAG-tagged control (thyrotropin receptor). ELISA measurements were carried out with intact COS-7 cells in 48-well plates as described under “Experimental Procedures.” Values are given as -fold over GFP expression. C, to investigate the binding properties of MC3R and MC3R/GHSR, COS-7 cells were transiently transfected, and after 48 h, [¹²⁵I-Nle⁴,d-Phe⁷]α-MSH (¹²⁵-NDP-α-MSH) displacement binding was performed as described under “Experimental Procedures.” Comparison of the binding properties of MC3R coexpressed with GHSR or with GFP to keep DNA amounts equal implicated no changes in ligand binding between MC3R and MC3R/GHSR. GHSRs were used as a negative control. Displacement with unlabeled α-MSH resulted in IC₅₀ values of 53 nm for the MC3R homodimer and 39 nm for the MC3R/GHSR heterodimer. IC₅₀ values were obtained using GraphPad Prism software.