High-resolution mass spectrometry for identifying phosphorylation sites of the metabotropic glutamate receptor mGlu3

Metabotropic glutamate receptors (mGlus) modulate synaptic transmission in the central nervous system, while β-arrestins (βarrs) mediate the desensitization and internalization of activated mGlus. We report two structures of the metabotropic glutamate receptor subtype 3 (mGlu3) coupled to βarr1 with stoichiometries of 2:1 and 2:2, respectively. Guided by the density map, we identified three phosphorylation sites (pSer857, pSer859 and pThr860), which follow the P-X-P-P type phosphorylation pattern. To verify the phosphorylation sites observed in the cryo-EM structure, we conducted mass spectrometry (MS) experiment to identify phosphorylation sites on mGlu3 mediated by GRK2. The results show that three phosphorylation sites (pSer857, pSer859 and pThr860) were identified in mass spectra, which is consistent with the findings from our cryo-EM density map.