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A Functional Enhanced Green Fluorescent Protein (EGFP)-Tagged Angiotensin II AT_1A Receptor Recruits the Endogenous Gq/11 Protein to the Membrane and Induces Its Specific Internalization Independently of Receptor- G Protein Coupling in HEK-293 Cells

Institut national de la santé et de la recherche médicale U36 Collège de France 75005 Paris, France

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 Gq/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 Gq/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 Gq/11 protein intracellular translocation, which presents a similar kinetic pattern but occurs predominantly in a different compartment; and 3) This Gq/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 (D⁷⁴N and 329) for which the coupling and internalization functions are modified.

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