Cysteine pairs in the third intracellular loop of the muscarinic M ₁ acetylcholine receptor play a role in agonist-induced internalization

We determined the functional role of a small domain in the third intracellular loop of the human muscarinic M₁ (hM₁) receptor. Using site-directed mutagenesis, several mutant hM₁ receptors were made possessing either a deletion or point mutations within the third intracellular loop domain ²⁵²PETPPGRCCRCC²⁶³. Wild-type and mutant hM₁ receptors were transiently expressed in Chinese hamster ovary cells, and the effects of each mutation on radioligand binding, agonist-mediated phosphoinositide hydrolysis, and agonist-induced internalization were determined. The mutant receptors exhibited a modest reduction in affinity for [³H]N-methylscopolamine (pK_D = ∼9.0) and a moderately increased binding capacity relative to the wild-type receptor. This moderate increase in binding capacity was associated with small increases in the maximal response and potency of carbachol for eliciting phosphoinositide hydrolysis through the mutant receptors (pEC₅₀ = ∼5.5) relative to wild-type (pEC₅₀ = 5.35 ± 0.05). In contrast, carbachol-induced internalization of mutant hM₁ receptors possessing either C259A/C260A or C262A/C263A or both double point mutations was significantly reduced compared to the wild-type hM₁ receptor. Of the hM₁ receptor mutants tested, those possessing a C262D/C263D double point mutation had the least carbachol-induced internalization. The desensitization and down-regulation of receptors possessing either Cys/Ala or Cys/Asp double point mutations were similar to those observed for the wild-type hM₁ receptor. Collectively, these observations suggest that Cys pairs Cys259/Cys260 and Cys262/Cys263 play an important role in the agonist-induced internalization of hM₁ receptors.