Lm. Tolbert et J. Lameh, HUMAN MUSCARINIC CHOLINERGIC RECEPTOR HM1 INTERNALIZES VIA CLATHRIN-COATED VESICLES, The Journal of biological chemistry, 271(29), 1996, pp. 17335-17342
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.