Stable interaction between β-arrestin 2 and angiotensin type 1A receptor is required for β-arrestin 2-mediated activation of extracellular signal-regulated kinases 1 and 2

Binding of β-arrestins to seven-membrane-spanning receptors (7MSRs) not only leads to receptor desensitization and endocytosis but also elicits additional signaling processes. We recently proposed that stimulation of the angiotensin type 1A (AT_1A) receptor results in independent β-arrestin 2- and G protein-mediated extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation. Here we utilize two AT_1A mutant receptors to study these independent pathways, one truncated at residue 324, thus removing all potential carboxyl-terminal phosphorylation sites, and the other bearing four mutations in the serine/threonine-rich clusters in the carboxyl terminus. As assessed by confocal microscopy, the two mutant receptors interacted with β-arrestin 2-green fluorescent protein with much lower affinity than did the wild-type receptor. In addition, the mutant receptors more robustly stimulated G protein-mediated inositol phosphate production. Approximately one-half of the wild-type AT_1A receptor-stimulated ERK1/2 activation was via a β-arrestin 2-dependent pathway (suppressed by β-arrestin 2 small interfering RNA), whereas the rest was mediated by a G protein-dependent pathway (suppressed by protein kinase C inhibitor). ERK1/2 activation by the mutant receptors was insensitive to β-arrestin 2 small interfering RNA but was reduced more than 80% by a protein kinase C inhibitor. The biochemical consequences of ERK activation by the G protein and β-arrestin 2-dependent pathways were also distinct. G-protein-mediated ERK activation enhanced the transcription of early growth response 1, whereas β-arrestin 2-dependent ERK activation did not. In addition, stimulation of the truncated AT_1A mutant receptor caused significantly greater early growth response 1 transcription than did the wild-type receptor. These findings demonstrate how the ability of receptors to interact with β-arrestins determines both the mechanism of ERK activation as well as the physiological consequences of this activation.