Investigation of the Role of an Amino Acid Triplet Repeat in Differentiating Drug-Receptor Interaction at ml and m2 Muscarinic Receptors

Abstract

The first putative extracellular domains of both ml and ml muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y), and threonine (T). This triplet is repeated as LYTLYT in m2 receptors. However, it is repeated in a transposed fashion (LYTTYL) in the sequence of ml receptors. In this work we employed site-directed mutagenesis to investigate the possible significance of this unique sequence diversity in determining the distinct differential drug-receptor interaction at the two receptor subtypes. Mutation of the LYTTYL sequence of ml receptors to the corresponding m2 receptor LYTLYT sequence, however, did not significantly change the binding affinity of the agonist carbachol or the affinity of the majority of a series of receptor antagonists which are able to discriminate between wild-type ml and m2 receptors. The reverse mutation at the m2 receptor also did not modify agonist affinity, but altered affinity of several receptor subtype-selective antagonists. The magnitude of affinity changes, however, was small, and the direction of these changes was opposite to what would be expected if the m2 receptor LYTLYT sequence were important for determining the binding profile of m2-receptor-selective antagonists. Our data suggest that the LYTTYL-LYTLYT sequence differences between ml and m2 muscarinic receptors are not important for determining receptor pharmacology.

M₂ and M₄ Muscarinic Receptor Subtypes Couple to Activation of Endothelial Nitric Oxide Synthase

“…Gene amplification, purification, subcloning and establishing stably transfected cell lines were carried out according to established procedures [11,12]. Chinese hamster ovary (CHO) cells already transfected with human M 2 or M 4 muscarinic acetylcholine receptors were co-transfected with the human eNOS gene using the PREP4 vector which encodes hygromycin resistance.…”