Muscarinic m4 receptor recycling requires a motif in the third intracellular loop

The present study was performed to identify sequence(s) in the third intracellular loop (i3) of the muscarinic acetylcholine receptor M4 subtype (M4 receptor) involved in its internalization and recycling. In transiently transfected human embryonic kidney 293-tsA201 cells, 40 to 50% of cell-surface M4 receptors are internalized in an agonist-dependent manner, and approximately 65% of internalized receptors are recycled back to the cell surface after removal of the agonist. We examined the internalization and recycling of M4 receptor mutants with partial deletion in i3 and found that various mutants (M4del-K-235-K-240, M4del-T-241-K-271, and M4del-W-339-N-372) showed internalization and cell-surface recycling in a similar manner to the M4 receptor. We also found that the mutant M4del-L-272-R-338 was internalized to only half the extent of the M4 receptor and was recycled after agonist removal, and the mutant M4del-V(373)A(393) was also internalized to half the extent of the wild type but was not recycled back to the cell surface after agonist removal. When the sequence corresponding to Val(373)-Ala(393) was grafted onto the i3 portion of a recycling-negative mutant of muscarinic M2 receptor with deletion of almost the whole of the i3 sequence, approximately 40% of the chimeric receptor on the cell surface was internalized, and more than 65% of the internalized receptors were recycled back to the cell surface. These results indicate that the regions including Leu(272)-Arg(338) and Val(373)-Ala(393) are involved in internalization of the M4 receptor, and the region including Val(373)-Ala(393) is indispensable for its recycling, whereas the other regions of i3 are dispensable for internalization and recycling.