Mechanisms of activation and modulation of homomeric and heteromeric metabotropic glutamate receptors

Synaptic plasticity underlies all learning and memory and is a cornerstone of neurological function. The adaptive and flexible nature of these processes makes them complex and challenging to define with the precision needed to develop quantitative models or effective therapeutics. The main excitatory neurotransmitter in the mammalian nervous system is glutamate which is also critical in setting synaptic strength and plasticity. Glutamate induces synaptic changes through two classes of receptors, ionotropic glutamate receptors and metabotropic glutamate receptors. Ionotropic glutamate receptors are glutamate gated calcium channels which can alter synaptic strength in the time scale of tens of milliseconds to 1 second. Metabotropic glutamate receptors (mGluRs) are Class C G-protein coupled receptors (GPCRs) which induce synaptic changes on the time scale of tens of seconds to minutes, hours and even days. This makes mGluRs critical to understanding the molecular induction of synaptic plasticity and very attractive therapeutic targets. Despite much effort put towards developing therapeutics that target mGluRs none have been approved by the FDA. A deeper understanding of mGluR activation will aid in designing more effective therapeutics as well as provide a more precise role of glutamate on synaptic changes.mGluRs are obligate dimers with large N-terminal domains. Each subunit contains a clamshell like glutamate binding domain (LBD), a rigid cysteine rich domain (CRD) and the canonical 7-pass transmembrane domain that defines all GPCRs. Numerous studies of GPCRs have pointed to the fact that these receptors are highly dynamic and that ligand binding changes the time a receptor spends in several distinct conformational states. The last ten years has seen an explosion in the number of high-resolution GPCRs structures. Unfortunately, these only provide a snapshot of a subset of potential receptor conformations and the amount of knowledge on the conformational dynamics of GPCRs has lagged behind. One very promising technique to address the questions surrounding GPCR conformational dynamics is single molecule fluorescence resonance energy transfer (smFRET). smFRET allows for very precise measurement of receptor motions and compliments the wealth of structural information to create a much more complete understanding of GPCRs. To this end I used smFRET to measure and study the conformational motions associated with activation of mGluR homodimers and heterodimers. First I examined the effects of different regions on mGluR2 homodimer activation and mGluR2/3 heterodimer activation which demonstrated a functional role of a LBD interface and the cysteine loop. Next I examined mGluR7, which has an incredibly low glutamate affinity, and found that while mGluR7 homodimers are virtually insensitive to glutamate, mGluR2/7 heterodimers are present in native tissue and are super sensitive to glutamate due to an apo state rotation demonstrating a potential biologic and modulatory role of heterodimerization. Lastly, I examined regions of mGluR7 and find that the lower dimer interface is responsible for very low glutamate sensitivity and that the cysteine bridge flexibility is responsible for the mGluR2/7 super sensitivity to glutamate.

Advisor: Isacoff, Ehud Y