New Modes of GPCR Signalling
New Modes of GPCR Signalling
New Modes of GPCR Signalling
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Quantitative Phosphoproteomics Analysis <strong>of</strong><br />
GABAB Receptor Signaling<br />
Meng FU1, Chanjuan XU2, Jianfeng LIU2,, Lin GUO1,<br />
1College <strong>of</strong> Life Science, Wuhan University, Wuhan, Hubei, P. R. China, 430072<br />
2College <strong>of</strong> Life Science and Technology, Huazhong University <strong>of</strong> Science and<br />
Technology, Wuhan, Hubei, P. R. China, 430074<br />
The G-protein-coupled receptors (<strong>GPCR</strong>s) play important role in cell-cell interaction.<br />
GABAB receptor, one <strong>of</strong> the best known <strong>GPCR</strong> family members, is activated by the<br />
neurotransmitter GABA and is expressed in most neurons. In the current study,<br />
primary cells from cerebellar granule neurons (CGNs) and cerebra cortex were cultured<br />
using SILAC (stable isotope labeling by amino acid in cell culture) method, with<br />
‘SILAC-Light’ as the control group, and ‘SILAC-Heavy’ treated with bacl<strong>of</strong>en, an<br />
agonist specific to mammalian GABAB receptors. After treatment, cell lysate were<br />
mixed 1:1 (protein ratio), and phosphopeptides were enriched by immobilized metal<br />
affinity chromatography (IMAC) method. A hydrophilic interaction chromatography<br />
(HILIC) fractionation step was used before phosphopeptide sequencing and<br />
quantification by liquid chromatography tandem mass spectrometry (LC-MSMS)<br />
analysis. In the preliminary data, we obtained quantitative information for 727<br />
phosphopeptides from cerebra cortex and 254 phosphopeptides from CGNs.<br />
Key wors: Phosphoproteomics, GABAB Receptor, Cell signaling,
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