A functional enhanced green fluorescent protein (EGFP)-tagged angiotensin II AT1A receptor recruits the endogenous Gαq/11 protein to the membrane and induces its specific internalization independently of receptor-G protein coupling in HEK-293 cells

被引:29
作者
Miserey-Lenkei, S [1 ]
Lenkei, Z [1 ]
Parnot, C [1 ]
Corvol, P [1 ]
Clauser, E [1 ]
机构
[1] Coll France, INSERM U36, F-75005 Paris, France
关键词
D O I
10.1210/me.15.2.294
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
The angiotensin II (Ang II) AT(1A) receptor was tagged at its C terminus with the enhanced green fluorescent protein (EGFP), and the corresponding chimeric cDNA was expressed in HEK-293 cells, This tagged receptor presents wild-type pharmacological and signaling properties and can be immunodetected by Western blotting and immunoprecipitation using EGFP antibodies, Therefore, this EGFP-tagged AT(1A) receptor is the perfect tool for analyzing in parallel the subcellular distributions of the receptor and its interacting G protein and their trafficking using confocal microscopy. Morphological observation of both the fluorescent receptor and its cognate G alphaq/11 protein, identified by indirect immunofluorescence, and the development of a specific software for digital image analysis together allow examination and quantification of the cellular distribution of these proteins before and after the binding of different agonist or antagonist ligands, These observations result in several conclusions: 1) Expression of increasing amounts of the AT(1A) receptor at the cell surface is associated with a progressive recruitment of the cytosolic G alphaq/11 protein at the membrane; 2) Internalization of the EGFP-tagged AT(1A) induced by peptide ligands but not nonpeptide ligands is accompanied by a G alphaq/11 protein intracellular translocation, which presents a similar kinetic pattern but occurs predominantly in a different compartment; and 3) This G alphaq/11 protein cellular translocation is dependent on receptor internalization process, but not G protein coupling and signal transduction mechanisms, as assessed by pharmacological data using agonists and antagonists and the characterization of AT(1A) receptor mutants ((DN)-N-74 and Delta 329) for which the coupling and internalization functions are modified.
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页码:294 / 307
页数:14
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