DELETION ANALYSIS OF THE M4 MUSCARINIC ACETYLCHOLINE-RECEPTOR - MOLECULAR DETERMINANTS FOR ACTIVATION OF BUT NOT COUPLING TO THE G(I) GUANINE-NUCLEOTIDE-BINDING REGULATORY PROTEIN REGULATE RECEPTOR INTERNALIZATION

In order to investigate whether coupling to and/or activation of guani ne-nucleotide-binding proteins (G proteins) is involved in agonist-ind uced 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 hams ter ovary cells in essentially comparable amounts, exhibited identical antagonist-binding affinities. Consistent with the reported importanc e of the third cytoplasmic loop for G(i) protein activation, the des-( 264-394)mAChR showed a drastically reduced potential to mediate agonis t-induced inhibition of adenylyl cyclase. In contrast, the ability of the mutant receptor to couple to G(i) proteins was not impaired, as de monstrated by a similar guanine-nucleotide-sensitive and pertussis-tox in-sensitive high-affinity agonist-receptor binding for both mAChRs. I n contrast, des-(264-394)mAChR was hardly able to stimulate the GTPase activity of G proteins, suggesting impaired activation of G(i) protei ns 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, de s-(264-394)mAChR displayed a significantly reduced rate of receptor in ternalization. A similar attenuation of wild-type mAChR internalizatio n was obtained upon pertussis toxin treatment. In conclusion, our data provide evidence that the molecular determinants of the m4 mAChR invo lved in G(i)-protein coupling and activation are not identical and tha t activation of, but not coupling to, G(i) proteins regulates m4 mAChR internalization.