A functional enhanced green fluorescent protein (EGFP)-tagged angiotensin II at(1a) receptor recruits the endogenous Galphaq/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 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 Galphaq/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 Galphaq/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 Galphaq/11 protein intracellular translocation, which presents a similar kinetic pattern but occurs predominantly in a different compartment; and 3) This Galphaq/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 (D(74)N and Delta329) for which the coupling and internalization functions are modified.
Miserey-Lenkei
S
S
Institut national de la santé et de la recherche médicale U36, Collège de France, 3 rue d'Ulm, 75005 Paris, France.
Lenkei
Z
Z
Parnot
C
C
Corvol
P
P
Clauser
E
E
D000804:Angiotensin II / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000648:ultrastructure; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics*; D018613:Microscopy, Confocal; D016296:Mutagenesis; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D008164:Luminescent Proteins; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D011993:Recombinant Fusion Proteins; D000804:Angiotensin II; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins