A functional enhanced green fluorescent protein (EGFP)-tagged angiotensin II AT(1A) receptor recruits the endogenous G alpha q/11 protein to the membrane and induces its specific internalization independently of receptor-G protein coupling in HEK-293 cells

The angiotensin II (Ang II) AT(1A) receptor was tagged at its C terminus wi th the enhanced green fluorescent protein (EGFP), and the corresponding chi meric cDNA was expressed in HEK-293 cells, This tagged receptor presents wi ld-type pharmacological and signaling properties and can be immunodetected by Western blotting and immunoprecipitation using EGFP antibodies, Therefor e, this EGFP-tagged AT(1A) receptor is the perfect tool for analyzing in pa rallel the subcellular distributions of the receptor and its interacting G protein and their trafficking using confocal microscopy. Morphological obse rvation of both the fluorescent receptor and its cognate G alphaq/11 protei n, identified by indirect immunofluorescence, and the development of a spec ific software for digital image analysis together allow examination and qua ntification 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 recrui tment of the cytosolic G alphaq/11 protein at the membrane; 2) Internalizat ion of the EGFP-tagged AT(1A) induced by peptide ligands but not nonpeptide ligands is accompanied by a G alphaq/11 protein intracellular translocatio n, which presents a similar kinetic pattern but occurs predominantly in a d ifferent compartment; and 3) This G alphaq/11 protein cellular translocatio n is dependent on receptor internalization process, but not G protein coupl ing 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 internalizati on functions are modified.