FIGURE 5.

Formation of disulfide cross-linked mutant M3R dimers after drastic reduction of receptor expression levels. To achieve relatively low receptor densities (∼0.2 pmol/mg of membrane protein), COS-7 cells were transfected with reduced amounts of plasmid DNA coding for the indicated receptor constructs (see ”Experimental Procedures“ for details). As a positive control, we included the L264C-M3′(3C)-Xa construct (16). Subsequently, membranes prepared from these cells were incubated with 100 μm CuPhen for 10 min at room temperature. Membrane extracts were then subjected to SDS-PAGE under non-reducing conditions, and receptor proteins were visualized via Western blotting using the polyclonal anti-M3R antibody. The blot shown is representative of three independent experiments. Asterisk, putative receptor monomers; arrows, putative cross-linked receptor dimers.