Glutamate serves as both the mammalian brain's primary excitatory neurotransmitter and as a key neuromodulator to control synapse and circuit function over a wide range of spatial and temporal scales. This functional diversity is decoded by two receptor families: ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). The challenges posed by the complexity and physiological importance of each of these subtypes has limited our appreciation and understanding of how these receptors work in concert. In this review, by comparing both receptor families with a focus on their crosstalk, we argue for a more holistic understanding of neural glutamate signaling.

Summary Calcium/calmodulin-dependent kinase II (CaMKII) forms a highly conserved dodecameric assembly that is sensitive to the frequency of calcium pulse trains. Neither the structure of the dodecameric assembly nor how it regulates CaMKII are known. We present the crystal structure of an autoinhibited full-length human CaMKII holoenzyme, revealing an unexpected compact arrangement of kinase domains docked against a central hub, with the calmodulin binding sites completely inaccessible. We show that this compact docking is important for the autoinhibition of the kinase domains and for setting the calcium response of the holoenzyme. Comparison of CaMKII isoforms, which differ in the length of the linker between the kinase domain and the hub, demonstrates that these interactions can be strengthened or weakened by changes in linker length. This equilibrium between autoinhibited states provides a simple mechanism for tuning the calcium response without changes in either the hub or the kinase domains.

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors in eukaryotes. Crystal structures have provided insight into GPCR interaction with ligands and G-proteins1,2, but our understanding of the conformational dynamics of activation is incomplete. Metabotropic glutamate receptors (mGluRs) are dimeric class C GPCRs that modulate neuronal excitability, synaptic plasticity, and serve as drug targets for neurological disorders3,4. A “clamshell” ligand-binding domain (LBD), which contains the ligand binding site, is coupled to the transmembrane domain (TMD) via a cysteine rich domain, and LBD closure appears to be the first step in activation5,6. Crystal structures of isolated mGluR LBD dimers led to the suggestion that activation also involves a reorientation of the dimer interface from a “relaxed” to an “active” state7,8, but the relationship between ligand binding, LBD closure and dimer interface rearrangement in activation remains unclear. We used single-molecule fluorescence resonance energy transfer (smFRET) to probe the activation mechanism of full-length mammalian group II mGluRs. We find that the LBDs interconvert between three conformations: resting, activated and a short-lived intermediate state. Orthosteric agonists induce transitions between these conformational states with efficacy determined by occupancy of the active conformation. Unlike mGluR2, mGluR3 displays basal dynamics, which are Ca2+ dependent and lead to basal protein activation. Our results support a general mechanism for the activation of mGluRs in which agonist binding induces closure of the LBDs followed by dimer interface reorientation. Our experimental strategy should be widely applicable to study conformational dynamics in GPCRs and other membrane proteins.

G-protein coupled receptors (GPCRs), the largest family of membrane signaling proteins, respond to neurotransmitters, hormones and small environmental molecules. The neuronal function of many GPCRs has been difficult to resolve because of an inability to gate them with subtype-specificity, spatial precision, speed and reversibility. To address this, we developed an approach for opto-chemical engineering native GPCRs. We applied this to the metabotropic glutamate receptors (mGluRs) to generate light-agonized and light-antagonized “LimGluRs”. The light-agonized “LimGluR2”, on which we focused, is fast, bistable, and supports multiple rounds of on/off switching. Light gates two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release. The light-antagonized “LimGluR2block” can be used to manipulate negative feedback of synaptically released glutamate on transmitter release. We generalize the optical control to two additional family members: mGluR3 and 6. The system works in rodent brain slice and in zebrafish in vivo, where we find that mGluR2 modulates the threshold for escape behavior. These light-gated mGluRs pave the way for determining the roles of mGluRs in synaptic plasticity, memory and disease.

Summary G protein-coupled receptors (GPCRs) mediate cellular responses to a wide variety of extracellular stimuli. GPCR dimerization may expand signaling diversity and tune functionality, but little is known about the mechanisms of subunit assembly and interaction or the signaling properties of heteromers. Using single molecule subunit counting on Class C metabotropic glutamate receptors (mGluRs), we map dimerization determinants and define a heterodimerization profile. Intersubunit fluorescence resonance energy transfer measurements reveal that interactions between ligand binding domains control the conformational rearrangements underlying receptor activation. Selective liganding with photoswitchable tethered agonists conjugated to one or both subunits of covalently linked mGluR2 homodimers reveals that receptor activation is highly cooperative. Strikingly, this cooperativity is asymmetric in mGluR2/mGluR3 heterodimers. Our results lead to a model of cooperative activation of mGluRs that provides a framework for understanding how class C GPCRs couple extracellular binding to dimer reorganization and G protein activation.