Figure 6. The endocytic sequences are differentially involved in the postendocytic trafficking of M2 and M4 mAChRs.

(A) Representative immunofluorescences of EGFP-tagged M2, M2/M4(R³⁸⁶-A³⁹³), M2(del K³⁷⁴-S³⁸⁰), M4 or M4/M2(K³⁷⁴-S³⁸⁰) with respect to the late endocytic and lysosomal marker LAMP-1 in response to CCh (120 min; 100 μM). (B) Representative images showing the EGFP-tagged M2, M2/M4(R³⁸⁶-A³⁹³), M4 or M4/M2(K³⁷⁴-S³⁸⁰) in the absence or presence of CCh (60 min; 100 μM), or followed by 60 min of washout after CCh stimulation. (C) The levels of colocalization of various mAChR mutants with LAMP-1 depicted in A were determined by Manders coefficient as described in ′′Methods′′. (D) The recycling of various mAChR mutants depicted in B was quantified, as described in ′′Methods′′. Scale bars, 10 μm. Mean ± SEM of at least 15 cells from at least three independent experiments are shown. *** p < 0.001.