title: Site-specific cleavage of G protein-coupled receptor-engaged beta-arrestin. Influence of the AT1 receptor conformation on scissile site selection.
abstract: The discovery of beta-arrestin-related approximately 46-kDa polypeptide in transfected cells and mouse hearts led us to examine angiotensin II type 1 receptor (AT(1)R)-dependent proteolytic cleavage of beta-arrestin(s). Receptor-ligand induced proteolysis of beta-arrestin(s) is novel, especially in the endocrine system, since proteolytic and/or splice variants of nonvisual arrestins are unknown. We used a strategy to retrieve AT(1)R-engaged isoforms of beta-arrestin 1 to confirm direct interaction of fragments with this G protein-coupled receptor and determine cleavage sites. Here we show that the angiotensin II-AT(1)R complex is associated with full-length and approximately 46-kDa beta-arrestin forms. Mass spectrometric analysis of the AT(1)R-associated short form suggested a scissile site located within the Arg(363)-Arg(393) region in the bovine beta-arrestin 1. Edman degradation analysis of a beta-arrestin 1 C-terminal fragment fused to enhanced green fluorescent protein confirmed the major cleavage to be after Phe(388) and a minor cleavage after Asn(375). Rather unexpectedly, the inverse agonist EXP3174-bound AT(1)R generated different fragmentation of bovine beta-arrestin 1, at Pro(276). The angiotensin II-induced cleavage is independent of inositol 1,4,5-trisphosphate- and Ca(2+)-mediated signaling pathways. The proteolysis of beta-arrestin 2 occurs, but the pattern is more complex. Our findings suggest that beta-arrestin cleavage upon AT(1)R stimulation is a part of the unraveling beta-arrestin-mediated G protein-coupled receptor signaling diversity.
authors.affiliation: Department of Molecular Cardiology and Molecular Biotechnology Core, Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
authors.forename: ChangWoo
authors.initials: C
authors.lastname: Lee
authors.forename: Sumantha
authors.initials: S
authors.lastname: Bhatt
authors.forename: Anita
authors.initials: A
authors.lastname: Shukla
authors.forename: Russell W
authors.initials: RW
authors.lastname: Desnoyer
authors.forename: Satya P
authors.initials: SP
authors.lastname: Yadav
authors.forename: Mijin
authors.initials: M
authors.lastname: Kim
authors.forename: Sei-Heon
authors.initials: SH
authors.lastname: Jang
authors.forename: Sadashiva S
authors.initials: SS
authors.lastname: Karnik
mesh_terms: D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000737:chemistry; D000818:Animals; D019390:Arrestins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002522:Chlorocebus aethiops; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000737:chemistry*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605255:Arrb1 protein, mouse; C000605256:Arrb1 protein, rat; C000605258:Arrb2 protein, mouse; C000605257:Arrb2 protein, rat; D019390:Arrestins; D044140:Receptor, Angiotensin, Type 1; D043562:Receptors, G-Protein-Coupled; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D000804:Angiotensin II; D002118:Calcium