Deletion analysis of the m4 muscarinic acetylcholine receptor

In order to investigate whether coupling to and/or activation of guanine‐nucleotide‐binding proteins (G proteins) is involved in agonist‐induced internalization of m4 muscarinic acetylcholine receptors (mAChRs), a deletion mutant [des‐(264–394)mAChR] was constructed that lacks a substantial portion of the putative third intracellular loop. The wild‐type receptor and des‐(264–394)mAChR stably expressed in Chinese hamster ovary cells in essentially comparable amounts, exhibited identical antagonist‐binding affinities. Consistent with the reported importance of the third cytoplasmic loop for G i protein activation, the des‐(264–394)mAChR showed a drastically reduced potential to mediate agonist‐induced inhibition of adenylyl cyclase. In contrast, the ability of the mutant receptor to couple to G i proteins was not impaired, as demonstrated by a similar guanine‐nucleotide‐sensitive and pertussis‐toxin‐sensitive high‐affinity agonist‐receptor binding for both mAChRs. In contrast, des‐(264–394)mAChR was hardly able to stimulate the GTPase activity of G proteins, suggesting impaired activation of G i proteins rather than ineffective coupling to G i proteins. Internalization of wild‐type receptor and des‐(264–394)mAChR was observed with similar agonist concentrations and showed similar maximal values. However, des‐(264–394)mAChR displayed a significantly reduced rate of receptor internalization. A similar attenuation of wild‐type mAChR internalization was obtained upon pertussis toxin treatment. In conclusion, our data provide evidence that the molecular determinants of the m4 mAChR involved in G i ‐protein coupling and activation are not identical and that activation of, but not coupling to, G i proteins regulates m4 mAChR internalization.