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(Received for publication, July 6, 1995, and in revised form, April 22, 1996)
From the Departments of Biopharmaceutical Sciences and
Pharmaceutical Chemistry, University of California, San Francisco,
California 94143-0446
The mechanism by which muscarinic receptors
internalize upon agonist exposure is poorly understood. To determine
the endocytic pathways responsible for muscarinic receptor
internalization, we have stably transfected human embryonic kidney (HEK
293) cells with the Hm1 (human muscarinic subtype 1) receptor tagged at
the amino terminus with the epitope EYMPME. The subcellular location of
the receptor was visualized by immunofluorescence confocal microscopy
and quantified with the use of binding studies. The receptor
redistributed into intracellular compartments following agonist
treatment. This process was reversible upon removal of agonist and
inhibited by antagonist. Acid treatment of the cells, which disrupts
internalization via clathrin-coated vesicles, inhibited
carbachol-stimulated internalization. Phorbol 12-myristate 13-acetate,
on the other hand, which inhibits caveolae-mediated endocytosis, had no
effect on carbachol-induced endocytosis. Double-labeling confocal
microscopy was used to characterize the intracellular vesicles
containing Hm1 receptor following agonist treatment. The Hm1 receptor
was shown to be colocalized with clathrin and
Volume 271, Number 29,
Issue of July 19, 1996
pp. 17335-17342
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-adaptin, a subunit of
the AP2 adaptor protein which links endocytosed proteins with clathrin
in the intracellular vesicles. In addition, endosomes containing Hm1
also contained the transferrin receptor, which internalizes via
clathrin-coated vesicles. In contrast, caveolin, the protein that
comprises caveolae, did not colocalize with Hm1 in intracellular
vesicles following agonist treatment, indicating that caveolae are not
involved in the agonist-induced internalization of Hm1. These results
indicate that agonist-induced internalization of the Hm1 receptor
occurs via clathrin-coated vesicles in HEK cells.
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