In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 110, No. 15 ( 2013-04-09)
Abstract:
Most importantly, our approach will drive the development of cell-surface receptor biosensors compatible with high-throughput screening, which has not yet been achieved with conventional FRET technologies. This approach might prove useful in drug development and could help researchers better understand the function of mGluRs and other related receptors, as well as membrane receptors in general. Our study reveals important information about the activation and allosteric properties of mGluRs in living cells and about how drugs regulate mGluRs activity. Our data reveal how agonist binding in the VFTs causes a scissoring movement between the VFTs that activates the G protein-activating transmembrane domain. This mechanism explains why mGluRs are dimers and provides opportunities to develop drugs capable of modulating their activity. These findings certainly will create possibilities for the development of innovative modulators. Eventually, we find that mGluRs couple to G proteins with properties similar to those of other GPCRs, despite important structural differences (e.g., dimeric organization and the presence of a large extracellular domain). GPCRs activate intracellular G proteins, which in turn influence the receptor conformation and stabilize the active state of the receptor. Our findings confirm that this activation also holds true for mGluRs, because the active state of VFTs is further stabilized in the receptor–G protein complex. This observation explains why the agonist affects the FRET signal at higher concentrations than that required to generate a response in cells. Indeed, although all receptors can be detected in the FRET assay, only those responsible for signaling can be detected using a cellular functional assay. Thus, our findings strongly suggest that the receptor–G protein complex alone, representing a small fraction of the cell surface receptors, is responsible for the functional response. How can such compounds, acting in the transmembrane domain, regulate mGluR activity so precisely? Using the FRET assay, we show that PAMs and NAMs allosterically control the relative movement of the extracellular domains ( Fig. P1 D ). These findings reveal a clear interaction between the dimeric VFT and the transmembrane domains, with the conformation of one influencing the conformation of the other. Our method of observing the VFT states directly through FRET measurement could help researchers better understand how various drugs regulate mGluRs. We found that some agonists induce a smaller change in FRET compared with glutamate, and we confirmed through functional studies that these molecules are partial agonists (agonists that induce partial receptor activation) ( Fig. P1 C ). With regard to mGluRs, many synthetic molecules were found to either facilitate or limit the action of agonists. These compounds, called “positive” and “negative” allosteric modulators (NAMs and PAMs, respectively) interact in the transmembrane domain and are considered promising drugs for the treatment of various brain diseases. In the present study, we analyzed the movement of subunits within mGluR dimers at the level of the VFTs. In particular, we examined how these movements are related to receptor activation. To accomplish this goal, we used innovative technologies that labeled cell-surface proteins with molecules that fluoresce and therefore can be studied using a technique known as “fluorescence resonance energy transfer” (FRET) ( 4 , 5 ). This method measures FRET efficiency, which reflects the distance between two fluorophores. We show that glutamate induces a large and rapid decrease in the FRET efficiency ( Fig. P1 B ), indicating an important movement of the VFTs upon receptor activation. We show that this movement is both necessary and sufficient for receptor activation using two types of receptors carrying mutations in their extracellular domain. These findings demonstrate that glutamate activates these prototypical dimeric receptors by allowing or stabilizing a new orientation of the extracellular domains. A better understanding of how changes in protein shape alter the function of mGluRs would enable the development of improved drugs to modulate their activities. The mGluRs are strict dimers (i.e., are composed of two subunits). Each subunit is made of a large extracellular domain that binds glutamate and is associated with a seven-helix transmembrane domain that mediates G-protein activation ( Fig. P1 A ). Agonists (compounds that activate the receptor, as glutamate does) bind to a region within the extracellular domain known as the “Venus flytrap bilobate domain” (VFT). The VFT oscillates between two major states: an open state that is stabilized by antagonists (compounds that bind to the receptor and prevent activation) and a closed state that is stabilized by agonists and is required for receptor activation. However, it remains unclear how VFT closure leads to transmembrane domain-mediated activation of G protein, despite the publication of crystal structures showing the dimeric VFTs with bound agonists and antagonists ( 3 ). mGluRs are part of the large family of G protein-coupled receptors (GPCRs). These proteins sense glutamate molecules outside the cell and trigger intracellular pathways that help regulate synaptic transmission in the nervous system ( 1 ). As such, the mGluRs represent potential therapeutic targets for the treatment of several neurologic and psychiatric disorders, such as anxiety, depression, pain, schizophrenia, and Parkinson disease. More generally, the mGluRs are part of a group of GPCRs known as “class C,” which also contains structurally related receptors for sweet and umami taste, calcium, basic amino acids, and the inhibitory neurotransmitter GABA ( 2 ). The neurotransmitter known as “glutamate” can activate two types of receptors: ionotropic and metabotropic. Ionotropic receptors are glutamate-gated channels responsible for fast excitatory neurotransmission; metabotropic glutamate receptors (mGluRs) activate intracellular pathways through heterotrimeric (i.e., containing three different subunits) G proteins. Cell-surface membrane proteins play key roles in cell physiology and cell–cell communication and as such represent major therapeutic targets. Many of these proteins serve as cell-surface receptors, which are activated by extracellular messengers such as light, small molecules, peptides, or proteins. Many cell-surface membrane proteins are composed of multiple domains and polypeptide chains. Upon binding of the messenger to the protein’s extracellular domain, structural changes in the transmembrane domain activate the receptor and lead to intracellular signaling events.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1215615110
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2013
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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