Abstract

We investigated the functional role of a conserved motif, F(x)₆LL, in the membrane proximal C-tail of the human muscarinic M₁ (hM₁) receptor. By use of site-directed mutagenesis, several different point mutations were introduced into the C-tail sequence ⁴²³FRDTFRLLL⁴³¹. Wild-type and mutant hM₁ receptors were transiently expressed in Chinese hamster ovary cells, and the amount of plasma membrane-expressed receptor was determined by use of intact, whole-cell [³H]N-methylscopolamine binding assays. The plasma membrane expression of hM₁ receptors possessing either L430A or L431A or both point mutations was significantly reduced compared with the wild type. The hM₁ receptor possessing a L430A/L431A double-point mutation was retained in the endoplasmic reticulum (ER), and atropine treatment caused the redistribution of the mutant receptor from the ER to the plasma membrane. Atropine treatment also caused an increase in the maximal response and potency of carbachol-stimulated phosphoinositide hydrolysis elicited by the L430A/L431A mutant. The effect of atropine on the L430A/L431A receptor mutant suggests that L⁴³⁰ and L⁴³¹ play a role in folding hM₁ receptors, which is necessary for exit from the ER. Using site-directed mutagenesis, we also identified amino acid residues at the base of transmembrane-spanning domain 1 (TM1), V⁴⁶ and L⁴⁷, that, when mutated, reduce the plasma membrane expression of hM₁ receptors in an atropine-reversible manner. Overall, these mutagenesis data show that amino acid residues in the membrane-proximal C-tail and base of TM1 are necessary for hM₁ receptors to achieve a transport-competent state.