Single amino acid substitutions in the pm2 muscarinic receptor alter receptor/G protein coupling without changing...

The amino terminus of the third cytoplasmic loop of the porcine m2 muscarinic receptor plays an important role in receptor/effector coupling. Although large changes in coupling properties are easily detected, subtle changes are often overlooked. Three mutant receptors were characterized after expression in Chinese hamster ovary cells, and two of these exhibited subtle changes in coupling properties. Substitution of amino acids 219-223 (KDKKE) with those conserved in the m1/m3/m5 receptor subtype family (ELAAL) had little effect on coupling to effector systems, indicating that altering the charge distribution in this region did not affect receptor/G protein interactions. Substitution of alanine with glutamate at amino acid position 212 (A212E) or lysine with alanine in position 214 (K214A) resulted in receptors with IC50 values for inhibition of adenylyl cyclase that resembled those of wild-type, although maximal percent inhibition was reduced. All mutants moderately decreased coupling to phosphatidylinositol metabolism, but mutant A212E caused oxotremorine-M to become a weak partial agonist compared with carbachol, suggesting that receptor conformation is agonist dependent even for ligands normally thought of as full agonists. K214A coupled to PI metabolism through both PTX-sensitive and PTX-insensitive G proteins. The results indicated that these mutants superficially possessed effector coupling characteristics similar to those of wild-type, but on more detailed examination G protein/receptor interactions were altered.