FIGURE 6.

Formation of disulfide cross-links between mutant M3Rs containing Cys substitutions within the i2 loop is agonist-independent. Membranes prepared from COS-7 cells expressing the indicated mutant M3Rs containing Cys substitutions within the C-terminal segment of the i2 loop were incubated for 10 min with 100 μm CuPhen (at room temperature) in the absence (−) or presence (+) of 1 mm carbachol (CCh). As a positive control, we included the L264C-M3′(3C)-Xa construct (16). Subsequently, membrane extracts were 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; arrow, putative cross-linked receptor dimers.