RESIDUES SPECIFICALLY INVOLVED IN DOWN-REGULATION BUT NOT INTERNALIZATION OF THE M1 MUSCARINIC ACETYLCHOLINE-RECEPTOR

Human m1 muscarinic acetylcholine receptor mutants were screened to de termine receptor domains and cellular pathways relevant to down-regula tion. Mutations in the second intracellular loop and the junctions of the third intracellular loop of the receptor, where a role for recepto r activation or internalization had been previously demonstrated in HE K293 cells, were selected for this study. To assess receptor down-regu lation, the mi receptor mutants were transfected into Chinese hamster ovary cells. Because receptor internalization is expected to precede d own-regulation, mutants displaying intact internalization were selecte d to permit interpretation of mutational effects on down-regulation al one, Four mutations were identified that specifically impaired down-re gulation without altering receptor internalization: V127A, I211A, E360 A, and K362A. The results define new receptor domains in the second in tracellular loop and the junctions of the third intracellular loop tha t are involved in down-regulation. These same four mutants were also d efective in signaling via the phospholipase C and the adenylyl cyclase pathways and in G protein activation, as measured by [S-35]GTP gamma/ S binding, However, the level of second messenger stimulation correlat ed poorly with the extent of down-regulation. In summary, several muta tions of the mi receptor selectively affect down-regulation, demonstra ting that internalization and down-regulation represent distinct event s driven by different cellular mechanisms.