Author response: Conformational dynamics between transmembrane domains and allosteric modulation of a metabotropic glutamate receptor

Differences in interactions between transmembrane domains tune the activation of metabotropic glutamate receptors

“…Next, we wondered whether the 3xTM4 residues also influence allosteric activation. Our work and others’ have demonstrated that PAMs can function as direct agonists of mGluR2 (Gutzeit et al, 2019, El Moustaine et al, 2012, Goudet et al, 2004, O’Brien et al, 2018). We tested the mGluR2 PAM LY487379 (LY48) (Johnson et al, 2003) with mGluR2 and mGluR2-3xTM4.…”

“…Motivated by our recent finding that isolated mGluR TMDs are able to dimerize to variable levels in the absence of extracellular domains (Gutzeit et al, 2019), we first sought to further understand the molecular basis of mGluR TMD dimerization using single molecule pulldown (SiMPull). In this technique, detergent-solubilized receptor complexes are isolated from fresh cell lysate and sparsely immobilized via antibodies on a glass coverslip.…”

“…Consistent with previous work, SNAP-mGluR2-TMD molecules photobleached primarily in one-step (∼55%) or two-step (∼40%) events with a small population of > 3 steps (∼4 %) (Figure 1C). This represents a population of ∼80% dimers when normalized to the obligatory dimerization of SNAP-mGluR2 (Figure 1D; see Materials and methods) (Gutzeit et al, 2019).…”

“…Structural interpretation of such studies has been limited but the consistently observed FRET efficiency increase seen with intersubunit sensors points to a rearrangement that leads to increased inter-TMD interaction upon LBD activation. Notably, we recently developed an isolated TMD FRET sensor which showed that TMD-targeting allosteric drugs are able to produce intersubunit re-arrangements autonomously without allosteric input from the LBDs (Gutzeit et al, 2019), pointing to a multi-state model of inter-TMD interaction and raising questions about TMD rearrangement in response to allosteric versus orthosteric activation. In line with this, a prior cross-linking study of full-length mGluR2 provided evidence for close proximity of TM4 and TM5 helices in inactive states and TM6 helices in active states (Xue et al, 2014).…”

“…This raises the possibility that such variability between subtypes also exists at the TMDs and can be used as a lens to dissect the TMD activation mechanism. Intriguingly, we recently used a single molecule imaging approach to determine that while all isolated mGluR TMDs tested show a clear propensity for dimerization, the mGluR2 subtype shows substantially higher dimerization propensity than other subtypes (Gutzeit et al, 2019), providing a jumping off point for this study.…”