Synucleins Are a Novel Class of Substrates for G Protein-coupled Receptor Kinases

Alexey N Pronin,Andrew J Morris,Andrei Surguchov,Jeffrey L Benovic

doi:10.1074/jbc.m003542200

Abstract

G protein-coupled receptor kinases (GRKs) specifically recognize and phosphorylate the agonist-occupied form of numerous G protein-coupled receptors (GPCRs), ultimately resulting in desensitization of receptor signaling. Until recently, GPCRs were considered to be the only natural substrates for GRKs. However, the recent discovery that GRKs also phosphorylate tubulin raised the possibility that additional GRK substrates exist and that the cellular role of GRKs may be much broader than just GPCR regulation. Here we report that synucleins are a novel class of GRK substrates. Synucleins (alpha, beta, gamma, and synoretin) are 14-kDa proteins that are highly expressed in brain but also found in numerous other tissues. alpha-Synuclein has been linked to the development of Alzheimer's and Parkinson's diseases. We found that all synucleins are GRK substrates, with GRK2 preferentially phosphorylating the alpha and beta isoforms, whereas GRK5 prefers alpha-synuclein as a substrate. GRK-mediated phosphorylation of synuclein is activated by factors that stimulate receptor phosphorylation, such as lipids (all GRKs) and Gbetagamma subunits (GRK2/3), suggesting that GPCR activation may regulate synuclein phosphorylation. GRKs phosphorylate synucleins at a single serine residue within the C-terminal domain. Although the function of synucleins remains largely unknown, recent studies have demonstrated that these proteins can interact with phospholipids and are potent inhibitors of phospholipase D2 (PLD2) in vitro. PLD2 regulates the breakdown of phosphatidylcholine and has been implicated in vesicular trafficking. We found that GRK-mediated phosphorylation inhibits synuclein's interaction with both phospholipids and PLD2. These findings suggest that GPCRs may be able to indirectly stimulate PLD2 activity via their ability to regulate GRK-promoted phosphorylation of synuclein.

Highlights

G protein-coupled receptor kinases (GRKs) recognize and phosphorylate the agonist-occupied form of numerous G protein-coupled receptors (GPCRs), resulting in desensitization of receptor signaling
The recent discovery that GRKs phosphorylate tubulin raised the possibility that additional GRK substrates exist and that the cellular role of GRKs may be much broader than just GPCR regulation
Common features shared by the GRKs include a centrally localized catalytic domain of ϳ270 amino acids, an N-terminal domain of ϳ190 amino acids that has been implicated in receptor interaction and GRK regulation, and a variable length C-terminal domain of 105–233 amino acids that is involved in phospholipid association

Summary

Introduction

G protein-coupled receptor kinases (GRKs) recognize and phosphorylate the agonist-occupied form of numerous G protein-coupled receptors (GPCRs), resulting in desensitization of receptor signaling. We identified the site phosphorylated by GRKs in ␣-synuclein and demonstrated that this phosphorylation inhibits the ability of ␣-synuclein to interact with phospholipids and results in reduced inhibition of phospholipase D2 (PLD2). The ability of ␣-synuclein to associate with phospholipids was analyzed by incubating 0.5 ␮M ␣-synuclein in the presence or absence of the indicated concentration of liposomes in 60 ␮l of 20 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol, 0.1 mg/ml ovalbumin on ice for 10 min.

Results

Conclusion