G protein-coupled receptors (GPCRs) are major players in cell communication. Although they form functional monomers, increasing evidence indicates that GPCR dimerization has a critical role in cooperative phenomena that are important for cell signal integration. However, the structural bases of these phenomena remain elusive. Here, using well-characterized receptor dimers, the metabotropic glutamate receptors (mGluRs), we show that structural changes at the dimer interface are linked to receptor activation. We demonstrate that the main dimer interface is formed by transmembrane α helix 4 (TM4) and TM5 in the inactive state and by TM6 in the active state. This major change in the dimer interface is required for receptor activity because locking the TM4-TM5 interface prevents activation by agonist, whereas locking the TM6 interface leads to a constitutively active receptor. These data provide important information on the activation mechanism of mGluRs and improve our understanding of the structural basis of the negative cooperativity observed in these GPCR dimers.

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G protein-coupled receptors (GPCRs) can integrate extracellular signals via allosteric interactions within dimers and higher-order oligomers. However, the structural bases of these interactions remain unclear. Here, we use the GABA B receptor heterodimer as a model as it forms large complexes in the brain. It is subjected to genetic mutations mainly affecting transmembrane 6 (TM6) and involved in human diseases. By cross-linking, we identify the transmembrane interfaces involved in GABA B1 -GABA B2 , as well as GABA B1 -GABA B1 interactions. Our data are consistent with an oligomer made of a row of GABA B1 . We bring evidence that agonist activation induces a concerted rearrangement of the various interfaces. While the GB1-GB2 interface is proposed to involve TM5 in the inactive state, cross-linking of TM6s lead to constitutive activity. These data bring insight for our understanding of the allosteric interaction between GPCRs within oligomers.

We propose a novel single-cell chemical proteomics (SCCP) strategy to profile low-abundance membrane proteins in single cells. In this approach, the membrane protein GB1 and its splicing variants were targeted on cultured cell lines and primary neurons using a specifically designed activity-based probe. The functionally labeled single cells were encapsulated in individual buffer droplets on a PDMS microwell array, and were further picked up one at a time and loaded into a capillary electrophoresis system for cell lysis, separation, and laser-induced fluorescence detection of the targeted proteins. The results revealed the expression of GB1 splicing variants in HEK and MEF cells, which was previously only suggested at the transcriptional level. We further applied this method to investigate single primary cells and observed significant heterogeneity among individual mouse cerebellar granule neurons. Interference experiments with GB1 antagonist and agonist validated this observation.

Many GPCRs (G-protein-coupled receptors) can activate RTKs (receptor tyrosine kinases) in the absence of RTK ligands, a phenomenon called transactivation. However, the underlying molecular mechanisms remain undefined. In the present study we investigate the molecular basis of GABA(B) (γ-aminobutyric acid B) receptor-mediated transactivation of IGF-1R (insulin-like growth factor type I receptor) in primary neurons. We take a chemical biology approach by developing an activity-based probe targeting the GABA(B) receptor. This probe enables us first to lock the GABA(B) receptor in an inactive state and then activate it with a positive allosteric modulator, thereby permitting monitoring of the dynamic of the protein complex associated with IGF-1R transactivation. We find that activation of the GABA(B) receptor induces a dynamic assembly and disassembly of a protein complex, including both receptors and their downstream effectors. FAK (focal adhesion kinase), a non-RTK, plays a key role in co-ordinating this dynamic process. Importantly, this dynamic of the GABA(B) receptor-associated complex is critical for transactivation and transactivation-dependent neuronal survival. The present study has identified an important mechanism underlying GPCR transactivation of RTKs, which was enabled by a new chemical biology tool generally applicable for dissecting GPCR signalling.