Received for publication April 2, 2007.
Revised May 29, 2007.
Accepted for publication May 30, 2007.

Cysteine Pairs in the Third Intracellular Loop of the Muscarinic M₁ Acetylcholine Receptor Play a Role in Agonist-Induced Internalization

Abstract

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 252-PETPPGRCCRCC-263. Wild-type and mutant hM₁ receptors were transiently expressed in CHO 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 (pK_D = 9.57 ± 0.07). 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 CC259-260AA or CC262-263AA, or both, double point mutations was significantly reduced when compared to the wild-type hM₁ receptor. Of the hM₁ receptor mutants tested, those possessing a CC262-263AA double point mutation had the least extensive carbachol-induced internalization. The desensitization and downregulation of receptors possessing this Cys/Ala double point mutation was similar to that observed for the wild-type hM₁ receptor. Collectively, these observations suggest that Cys pairs 259-CC-260 and 262-CC-263 play an important role in the agonist-induced internalization of hM₁ receptors.

Key words: Cysteines, Desensitization, Internalization, Muscarinic, Mutagenesis, Receptors