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Identification of GPCR-interacting cytosolic proteins using HDL particles and mass spectrometry-based proteomic approach.

ABSTRACT: G protein-coupled receptors (GPCRs) have critical roles in various physiological and pathophysiological processes, and more than 40% of marketed drugs target GPCRs. Although the canonical downstream target of an agonist-activated GPCR is a G protein heterotrimer; there is a growing body of evidence suggesting that other signaling molecules interact, directly or indirectly, with GPCRs. However, due to the low abundance in the intact cell system and poor solubility of GPCRs, identification of these GPCR-interacting molecules remains challenging. Here, we establish a strategy to overcome these difficulties by using high-density lipoprotein (HDL) particles. We used the ?(2)-adrenergic receptor (?(2)AR), a GPCR involved in regulating cardiovascular physiology, as a model system. We reconstituted purified ?(2)AR in HDL particles, to mimic the plasma membrane environment, and used the reconstituted receptor as bait to pull-down binding partners from rat heart cytosol. A total of 293 proteins were identified in the full agonist-activated ?(2)AR pull-down, 242 proteins in the inverse agonist-activated ?(2)AR pull-down, and 210 proteins were commonly identified in both pull-downs. A small subset of the ?(2)AR-interacting proteins isolated was confirmed by Western blot; three known ?(2)AR-interacting proteins (Gs?, NHERF-2, and Grb2) and 3 newly identified known ?(2)AR-interacting proteins (AMPK?, acetyl-CoA carboxylase, and UBC-13). Profiling of the identified proteins showed a clear bias toward intracellular signal transduction pathways, which is consistent with the role of ?(2)AR as a cell signaling molecule. This study suggests that HDL particle-reconstituted GPCRs can provide an effective platform method for the identification of GPCR binding partners coupled with a mass spectrometry-based proteomic analysis.

SUBMITTER: Chung KY

PROVIDER: S-EPMC3556083 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Identification of GPCR-interacting cytosolic proteins using HDL particles and mass spectrometry-based proteomic approach.

Chung Ka Young KY Day Peter W PW Vélez-Ruiz Gisselle G Sunahara Roger K RK Kobilka Brian K BK

PloS one 20130125 1

G protein-coupled receptors (GPCRs) have critical roles in various physiological and pathophysiological processes, and more than 40% of marketed drugs target GPCRs. Although the canonical downstream target of an agonist-activated GPCR is a G protein heterotrimer; there is a growing body of evidence suggesting that other signaling molecules interact, directly or indirectly, with GPCRs. However, due to the low abundance in the intact cell system and poor solubility of GPCRs, identification of thes ...[more]

PMID: 23372797

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Project description:In the brain, the Homer protein family modulates excitatory signal transduction and receptor plasticity through interactions with other proteins in dendritic spines. Homer proteins are implicated in a variety of psychiatric disorders such as schizophrenia and addiction. Since long Homers serve as scaffolding proteins, identifying their interacting partners is an important first step in understanding their biological function and could help to guide the design of new therapeutic strategies. The present study set out to document Homer2-interacting proteins in the mouse brain using a co-immunoprecipitation-based mass spectrometry approach where Homer2 knockout samples were used to filter out non-specific interactors. We found that in the mouse brain, Homer2 interacts with a limited subset of its previously reported interacting partners (3 out of 31). Importantly, we detected an additional 15 novel Homer2-interacting proteins, most of which are part of the N-methyl-D-aspartate receptor signaling pathway. These results corroborate the central role Homer2 plays in glutamatergic transmission and expand the network of proteins potentially contributing to the behavioral abnormalities associated with altered Homer2 expression.SignificanceLong Homer proteins are scaffolding proteins that regulate signal transduction in neurons. Identifying their interacting partners is key to understanding their function. We used co-immunoprecipitation in combination with mass spectrometry to establish the first comprehensive list of Homer2-interacting partners in the mouse brain. The specificity of interactions was evaluated using Homer2 knockout brain tissue as a negative control. The set of proteins that we identified minimally overlaps with previously reported interacting partners of Homer2; however, we identified novel interactors that are part of a signaling cascade activated by glutamatergic transmission, which improves our mechanistic understanding of the role of Homer2 in behavior.

Dataset Information

Identification of GPCR-interacting cytosolic proteins using HDL particles and mass spectrometry-based proteomic approach.

Publications

Identification of GPCR-interacting cytosolic proteins using HDL particles and mass spectrometry-based proteomic approach.

Similar Datasets

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