Multiple pathways for the dynamin-regulated internalization of muscarinic acetylcholine receptors

An important regulatory pathway of G-protein-coupled receptors (GPCRs) is the internalization of receptors into the cell interior. To unravel the molecular mechanisms by which GPCRs are internalized, we have studied the internalization of various members of the family of muscarinic acetylcholine receptors (mAChRs). Using the transient expression system of HEK-293 cells, we showed that the M₁, M₃ and M₄ mAChRs are internalized into clathrin-coated vesicles and recycle back to the plasma membrane. This internalization pathway is dependent on the concerted action of β-arrestin, c-Src and the GTPase dynamin, which ‘catalyses’ the budding of clathrin-coated vesicles (and other vesicles) from the plasma membrane. Internalization of the M₂ mAChR (which is highly structurally and functionally related to the M₄ receptor subtype) also requires dynamin, but proceeds in an apparent β-arrestin-, c-Src- and clathrin-independent manner. Internalized M₂ mAChRs also show virtually no receptor recycling, but are down-regulated. This demonstrates that GPCRs can be internalized by multiple dynamin-dependent pathways in a highly regulated manner.