Human muscarinic cholinergic receptor Hm1 internalizes via clathrin-coated vesicles

被引:73
|
作者
Tolbert, LM
Lameh, J
机构
[1] UNIV CALIF SAN FRANCISCO,DEPT BIOPHARMACEUT SCI,SAN FRANCISCO,CA 94143
[2] UNIV CALIF SAN FRANCISCO,DEPT PHARMACEUT CHEM,SAN FRANCISCO,CA 94143
关键词
D O I
10.1074/jbc.271.29.17335
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 alpha-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 Hml 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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页码:17335 / 17342
页数:8
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