Trafficking of green fluorescent protein-tagged muscarinic M4 receptors in NG108-15 cells.

The muscarinic M4 receptor for acetylcholine was tagged at its C terminus with green fluorescent protein (GFP) and expressed in NG108-15 cells, which normally express this receptor subtype. The binding affinity of the antagonist N-methylscopolamine was not significantly affected by the presence of the GFP tag, whereas the affinity of the receptor for the agonist carbachol was reduced by four-fold. Stimulation of the tagged receptor resulted in inhibition of adenylyl cyclase. Following agonist stimulation, the tagged receptor was slowly internalized, and became partially co-localized with the endosomal marker Texas Red-transferrin after 30 min. There was little co-localization with the lysosomal marker 1gp120 even after 60 min of internalization. Finally, the tagged receptor, unlike the endogenous receptor, failed to recycle to the plasma membrane on removal of the agonist. We conclude that the GFP-tagged muscarinic M4 receptor does not traffic normally in NG108-15 cells, most likely because of its gross overexpression.