Site-specific Cleavage of G Protein-coupled Receptor-engaged β-Arrestin

Abstract

The discovery of ␤-arrestin-related ϳ46-kDa polypeptide in transfected cells and mouse hearts led us to examine angiotensin II type 1 receptor (AT 1 R)-dependent proteolytic cleavage of ␤-arrestin(s). Receptor-ligand induced proteolysis of ␤-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 ␤-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 ϳ46-kDa ␤-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 ␤-arrestin 1. Edman degradation analysis of a ␤-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 ␤-arrestin 1, at Pro 276 . The angiotensin II-induced cleavage is independent of inositol 1,4,5-trisphosphateand Ca 2؉ -mediated signaling pathways. The proteolysis of ␤-arrestin 2 occurs, but the pattern is more complex. Our findings suggest that ␤-arrestin cleavage upon AT 1 R stimulation is a part of the unraveling ␤-arrestin-mediated G protein-coupled receptor signaling diversity.