title: Heteromerization of angiotensin receptors changes trafficking and arrestin recruitment profiles.
abstract: The cardiovascular hormone angiotensin II (AngII) exerts its actions via two G protein-coupled receptor (GPCR) subtypes, AT(1) and AT(2), which often display antagonistic functions. Methodological constraints have so far precluded detailed analyses of the ligand-dependency, cellular localization, and functional relevance of AngII receptor interactions in live cells. In this study, we utilize a protein-fragment complementation assay (PCA) and GPCR-Heteromer Identification Technology (GPCR-HIT) to provide the first detailed investigation of the ligand-dependency and cellular localization of AngII receptor interactions in human embryonic kidney 293 cells. Fluorescent-tagged receptor constructs for PCA and GPCR-HIT displayed normal affinity and selectivity for AngII (AT(1): IC(50)=1.0-1.6nM; AT(2): IC(50)=2.0-3.0nM). Well-characterized angiotensin receptor interactions were used as positive and negative controls to demonstrate the sensitivity and specificity of these fluorescence-based assays. We report that AT(1)-AT(2) receptor heteromers form constitutively, are localized to the plasma membrane and perinuclear compartments, and do not internalize following AngII stimulation despite arrestin being recruited specifically to the heteromer. Our findings using novel fluorescence-based technologies reveal a previously unrecognized mechanism of angiotensin receptor cross-talk involving cross-inhibition of AT(1) receptor internalization through heteromerization with the AT(2) receptor subtype.
authors: Department of Physiology, The University of Melbourne, Victoria 3010, Australia. enzo.porrello@utsouthwestern.edu
authors: Enzo R
authors: ER
authors: Porrello
authors: Kevin D G
authors: KD
authors: Pfleger
authors: Ruth M
authors: RM
authors: Seeber
authors: Hongwei
authors: H
authors: Qian
authors: Cristina
authors: C
authors: Oro
authors: Fe
authors: F
authors: Abogadie
authors: Lea M D
authors: LM
authors: Delbridge
authors: Walter G
authors: WG
authors: Thomas
mesh_terms: D000804:Angiotensin II / Q000378:metabolism* / Q000494:pharmacology; D019390:Arrestins / Q000378:metabolism* / Q000494:pharmacology; D001681:Biological Assay; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D018613:Microscopy, Confocal; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids; D058105:Polymerization; D011485:Protein Binding; D020239:Receptor Cross-Talk / Q000187:drug effects*; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*; D044139:Receptor, Angiotensin, Type 2 / Q000235:genetics / Q000378:metabolism*; D012084:Renin-Angiotensin System / Q000502:physiology*; D012680:Sensitivity and Specificity; D014162:Transfection
chemical_list: D019390:Arrestins; D008024:Ligands; D010446:Peptide Fragments; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D000804:Angiotensin II