HUMAN MUSCARINIC CHOLINERGIC RECEPTOR HM1 INTERNALIZES VIA CLATHRIN-COATED VESICLES

The mechanism by which muscarinic receptors internalize upon agonist e xposure is poorly understood. To determine the endocytic pathways resp onsible for muscarinic receptor internalization, we have stably transf ected human embryonic kidney (HEK 293) cells with the Hm1 (human musca rinic subtype 1) receptor tagged at the amino terminus with the epitop e EYMPME. The subcellular location of the receptor was visualized by i mmunofluorescence confocal microscopy and quantified with the use of b inding studies. The receptor redistributed into intracellular compartm ents following agonist treatment, This process was reversible upon rem oval of agonist and inhibited by antagonist, Acid treatment of the cel ls, which disrupts internalization via clathrin coated vesicles, inhib ited carbachol-stimulated internalization. Phorbol 12-myristate 13-ace tate, on the other hand, which inhibits caveolae-mediated endocytosis, had no effect on carbachol-induced endocytosis, Double-labeling confo cal microscopy was used to characterize the intracellular vesicles con taining Hm1 receptor following agonist treatment. The Hm1 receptor was shown to be colocalized with clathrin and alpha-adaptin, a subunit of the AP2 adaptor protein which links endocytosed proteins with clathri n in the intracellular vesicles, In addition, endosomes containing Hm1 also contained the transferrin receptor, which internalizes via clath rin-coated vesicles, In contrast, caveolin, the protein that comprises caveolae, did not colocalize with Hml in intracellular vesicles follo wing agonist treatment, indicating that caveolae are not involved in t he agonist-induced internalization of Hm1. These results indicate that agonist-induced internalization of the Hm1 receptor occurs via clathr in-coated vesicles in HEK cells.