Functional specialization of beta-arrestin interactions revealed by proteomic analysis - PubMed (original) (raw)
Functional specialization of beta-arrestin interactions revealed by proteomic analysis
Kunhong Xiao et al. Proc Natl Acad Sci U S A. 2007.
Abstract
Beta-arrestins are cytosolic proteins that form complexes with seven-transmembrane receptors after agonist stimulation and phosphorylation by the G protein-coupled receptor kinases. They play an essential role in receptor desensitization and endocytosis, and they also serve as receptor-regulated signaling scaffolds and adaptors. Moreover, in the past decade, a growing list of protein-protein interactions of beta-arrestins pertinent to these functions has been documented. The discovery of several novel functions of beta-arrestins stimulated us to perform a global proteomics analysis of beta-arrestin-interacting proteins (interactome) as modulated by a model seven-transmembrane receptor, the angiotensin II type 1a receptor, in an attempt to assess the full range of functions of these versatile molecules. As determined by LC tandem MS, 71 proteins interacted with beta-arrestin 1, 164 interacted with beta-arrestin 2, and 102 interacted with both beta-arrestins. Some proteins bound only after agonist stimulation, whereas others dissociated. Bioinformatics analysis of the data indicates that proteins involved in cellular signaling, organization, and nucleic acid binding are the most highly represented in the beta-arrestin interactome. Surprisingly, both S-arrestin (visual arrestin) and X-arrestin (cone arrestin) were also found in heteromeric complex with beta-arrestins. The beta-arrestin interactors distribute not only in the cytoplasm, but also in the nucleus as well as other subcellular compartments. The binding of 16 randomly selected newly identified beta-arrestin partners was validated by coimmunoprecipitation assays in HEK293 cells. This study provides a comprehensive analysis of proteins that bind beta-arrestin isoforms and underscores their potentially broad regulatory roles in mammalian cellular physiology.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Confirmation of β-arrestin-interacting proteins by coimmunoprecipitation. Shown are three examples (PP2Cα, PP2Cβ, and 14-3-3τ) of the coimmunoprecipitation assays used to validate the β-arrestin-interacting proteins identified by mass spectrometry. The cDNAs of 16 randomly selected proteins from the β-arrestin interactome were subcloned into a p3xFLAG-CMV-10 vector and transfected together with pcDNA3–β-arrestin 1 or 2 into HEK293 cells stably overexpressing HA-AT1aR. The 3xFLAG-tagged protein complexes were coimmunoprecipitated with anti-FLAG M2 affinity agarose beads and analyzed by Western blot using anti-β-arrestin antibody A1CT or A2CT. These data show representative blots from one of the three independent experiments. The detailed coimmunoprecipitation assay data are summarized in
SI Table 8
. Ang II, angiotensin II (100 nM).
Fig. 2.
Subcellular and functional distribution of β-arrestin interactome. (A) Subcellular distribution of β-arrestin-interacting proteins. The subcellular distribution information of proteins was obtained from the human International Protein Index database (
) and the Human Protein Reference Database (
). (B–D) Functional distributions of the whole human genome (B), β-arrestin-interacting proteins (C), and 14-3-3-interacting proteins (D). The functional distribution information of the whole human genome and 14-3-3-interacting proteins was taken from previously published reports (7, 10).
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