GPCRs represent the largest family of membrane signaling proteins and respond to a wide-array of stimuli. These seven transmembrane receptors couple to distinct classes of heterotrimeric G-proteins, leading to the activation or inhibition of a large number of protein targets3
. The diversity of signaling is vastly greater than can be accounted for by the four classes of G-proteins to which GPCRs couple. The additional diversity comes from several factors, including localization into specific subcellular compartments, corralling into signaling nanodomains with particular effectors, assembly of preformed GPCR-G protein-effector complexes, heteromultimerization into complexes with specialized properties, and unique profiles of interaction with regulatory proteins6,7
To elucidate GPCR function one needs a method that combines specific pharmacology with specificity for region, cell-type and subcellular compartment. At the same time one wants the approach to allow for the GPCR to be activated at physiological rates (i.e. the millisecond time scale) and to be reversible and reproducible to mimic physiological signaling and permit quantitative analysis. All this needs to be achieved on the full-length GPCR in order to maintain normal targeting and interaction with signaling partners and regulators. We overcame these obstacles by developing, via the rational design and synthesis of new PTLs called D-MAGs and a novel, simple and fast Monte Carlo simulation approach to select anchoring sites for these PTLs in order to generate photo-agonizing and photo-antagonizing versions of three of the eight mGluRs, representing two of the three mGluR groups. These approaches can readily be adapted to other target proteins and PTLs.
We most thoroughly characterized the photo-agonism with D-MAG-0 at position L300C of mGluR2 (LimGluR2). Unlike rhodopsin, which was the basis of most of the prior light-gated GPCRs, LimGluR2 can be actively toggled both on and off in less than one millisecond, enabling signaling to be controlled. on a synaptically-relevant timescale and providing for fast effector kinetics. Moreover, LimGluR2 permits repetitive stimulation at high rates without decline. Rhodopsin requires constant illumination to be activated, which increases the chance of tissue damage and can act as a confounding variable for behavioral studies, while LimGluR2 is bistable, eliminating the need for constant illumination. Most importantly, optical control of native GPCRs provides a unique opportunity to examine the specific synaptic and circuit functions of each receptor, which emerge from their restricted effector and regulatory profiles and cannot be deduced from widespread activation of the entire signaling pathway of the G-protein to which they couple.
We show that, despite their limited homology (66% identity between mGluR2 and mGluR3 and 44% identity between mGluR2 and mGluR6), photo-control can be generalized within the mGluR family from mGluR2 to the other Group II member mGluR3 and the Group III members mGluR6, with the same D stereoisomer linkage to the glutamate of MAG being required. Differences in photo-switching with a particular MAG at homologous sites of these three mGluRs reveals differences between their LBDs. This information may be useful for designing additional photoswitches or other pharmacological ligands as well as for probing the mechanism of clamshell closure.
The LimGluRs provide rapid, reversible, bistable, and highly reproducible control of excitability and synaptic transmission in dissociated cultured neurons as well as brain slice, two of the prime in vitro
systems where synaptic transmission and plasticity in general, and mGluR function in particular, are studied most extensively. Although the photo-agonism and photo-antagonism of LimGluR2 are not complete, the photo-agonism induces characteristic mGluR2-dependent modulation and the photo-antagonism prevents the induction of such changes by native glutamate release. The precise temporal control, which allows the agonist or antagonist to be toggled on and off in a time-coupled manner, repeatedly and reproducibly makes it possible to observe small effects that would be difficult to distinguish with classical drugs. In the case of LimGluR2block, the photoeffect in neurons is consistent with the behavior of most neurotransmitter-gated GPCRs, which tend to be localized outside of the synaptic cleft and experience sub-saturating concentrations of the neurotransmitter. The success of the D-MAG labeling and photo-control of mGluRs in brain slice suggests that the approach should also work in the mammalian brain in vivo
, as has been shown for a similar photoswitch directed to the ionotropic kainate receptor in the mouse retina in vivo48
. Indeed, we demonstrate that LimGluR2 works effectively in vivo
in zebrafish when D-MAG is simply added to the zebrafish larvae E3 salt water medium.
We used LimGluR2 to photo-manipulate mGluR2 signaling in the context of the zebrafish ASR, a widely-studied behavior that is intriguingly similar in architecture and pharmacological regulation to the mammalian acoustic startle response47,49
. In rodents, mGluRs have been implicated in various forms of the startle response, including regulation of paired-pulse inhibition by group II mGluRs, using pharmacological manipulation50
. Recently, it has been shown that group II mGluRs are expressed across all main subdivisions of the zebrafish brain51
. Indeed, we found that conventional agonism of group II mGluRs by L-CCG-1 lowers the zebrafish ASR threshold.
The ability to target light to a subregion of the nervous system allowed us to localize the mGluR2-mediated effect on the acoustic startle response to the spinal cord and hindbrain and to find that optical activation of LimGluR2 also reduces acoustic startle response threshold, but, unlike L-CCG-1, this effect can be shown to result from acute activation of mGluR2 and can be reversed and repeated, suggesting that mGluR2 signaling could dynamically modulate escape threshold. Such information regarding the temporal dynamics of the acoustic startle response would not be possible to obtain using pharmacological approaches that require complete wash-out of ligands or addition of compounds whose activities are constrained by the pharmacokinetics of intact animals.
As with other GPCRs, mGluRs that couple to the same G-protein often activate distinct effectors5
and are regulated distinctly3,7
. Photo-agonism and photo-antagonism of group II and III mGluRs should make it possible to determine the precise spatial (i.e. pre vs. post-synaptic; synaptic vs. peri-synaptic vs. astrocytic) and temporal properties of signaling by individual receptors to mediate lasting changes in synaptic strength. Furthermore, since LimGluR2 maintains close to native ligand sensitivity, knock-in mice with a single point mutation to introduce a single cysteine anchor should allow for high resolution, specific photo-agonism or photo-antagonism while maintaining the receptor’s native function. This would provide a new way to specifically probe the receptor’s function in synaptic plasticity and learning, but also in anxiety, depression, and schizophrenia, for which they are major drug targets33