Global phosphorylation analysis of beta-arrestin-mediated signaling downstream of a seven transmembrane receptor (7TMR).
beta-Arrestin-mediated signaling downstream of seven transmembrane receptors (7TMRs) is a relatively new paradigm for signaling by these receptors. We examined changes in protein phosphorylation occurring when HEK293 cells expressing the angiotensin II type 1A receptor (AT1aR) were stimulated with the beta-arrestin-biased ligand Sar(1), Ile(4), Ile(8)-angiotensin (SII), a ligand previously found to signal through beta-arrestin-dependent, G protein-independent mechanisms. Using a phospho-antibody array containing 46 antibodies against signaling molecules, we found that phosphorylation of 35 proteins increased upon SII stimulation. These SII-mediated phosphorylation events were abrogated after depletion of beta-arrestin 2 through siRNA-mediated knockdown. We also performed an MS-based quantitative phosphoproteome analysis after SII stimulation using a strategy of stable isotope labeling of amino acids in cell culture (SILAC). We identified 1,555 phosphoproteins (4,552 unique phosphopeptides), of which 171 proteins (222 phosphopeptides) showed increased phosphorylation, and 53 (66 phosphopeptides) showed decreased phosphorylation upon SII stimulation of the AT1aR. This study identified 38 protein kinases and three phosphatases whose phosphorylation status changed upon SII treatment. Using computational approaches, we performed system-based analyses examining the beta-arrestin-mediated phosphoproteome including construction of a kinase-substrate network for beta-arrestin-mediated AT1aR signaling. Our analysis demonstrates that beta-arrestin-dependent signaling processes are more diverse than previously appreciated. Notably, our analysis identifies an AT1aR-mediated cytoskeletal reorganization network whereby beta-arrestin regulates phosphorylation of several key proteins, including cofilin and slingshot. This study provides a system-based view of beta-arrestin-mediated phosphorylation events downstream of a 7TMR and opens avenues for research in a rapidly evolving area of 7TMR signaling.
Xiao
Kunhong
K
Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
Sun
Jinpeng
J
Kim
Jihee
J
Rajagopal
Sudarshan
S
Zhai
Bo
B
Villén
Judit
J
Haas
Wilhelm
W
Kovacs
Jeffrey J
JJ
Shukla
Arun K
AK
Hara
Makoto R
MR
Hernandez
Marylens
M
Lachmann
Alexander
A
Zhao
Shan
S
Lin
Yuan
Y
Cheng
Yishan
Y
Mizuno
Kensaku
K
Ma'ayan
Avi
A
Gygi
Steven P
SP
Lefkowitz
Robert J
RJ
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D019390:Arrestins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction; D049490:Systems Biology; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; D019390:Arrestins; D008024:Ligands; D010750:Phosphoproteins; D020543:Proteome; D034741:RNA, Small Interfering; D044140:Receptor, Angiotensin, Type 1; D043562:Receptors, G-Protein-Coupled; C502020:angiotensin II, Sar(1)-Ile(4)-Ile(8)-; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; C450520:seven-transmembrane G-protein-coupled receptor; D000804:Angiotensin II; D011494:Protein Kinases; D010744:Phosphoric Monoester Hydrolases