Activating and inactivating mutations in N- and C-terminal i3 loop junctions of muscarinic acetylcholine Hm1 receptors.

The N- and C-terminal junctions of the third intracellular loop (i3) of G protein-coupled receptors play a role in the coupling process. We had previously constructed two triple point alanine mutants of the i3 junction of the muscarinic Hm1 receptor, W209A/I211A/Y212A and E360A/K362A/T366A, which are defective in mediating carbachol stimulation of phosphatidylinositol (PI) turnover (Moro, O., Lameh, J., Högger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 22273-22276). Each of the corresponding six single point mutations were constructed to determine residues crucial to receptor coupling. Mutants W209A and T366A were similar to or only slightly less effective than wild type Hm1 in stimulating PI turnover. In the N-terminal junction, I211A and Y212A were defective in coupling, and I211A was even more defective than the corresponding triple mutant. Therefore, the triple mutation compensated at least partially for the effect of these two single point mutations. In the C-terminal i3 loop junction, mutant K362A was again more strongly defective than the corresponding triple mutant. In contrast, mutation E360A was found to be activating, leading to elevated PI turnover in the absence of agonist and sensitization toward carbachol activation. Activating mutations in the C-terminal i3 loop junction have been reported previously for the adrenergic receptors, but E360A represents the first muscarinic receptor with substantial basal activity. The effects of the single point mutations observed in this study were not readily predictable from similar mutations from closely related G protein-coupled receptors despite sequence conservation in the i3 loop junctions. Our results caution against defining precise coupling domains in these regions by mutagenesis results.