Abstract
The extracellular domain of the metabotropic glutamate receptor 1α (mGluR1α) forms a dimer and the ligand, glutamate, induces a structural rearrangement in this domain. However, the conformational change in the cytoplasmic domain, which is critical for mGluR1α′s interaction with G proteins, remains unclear. Here we investigated the ligand-induced conformational changes in the cytoplasmic domain by fluorescence resonance energy transfer (FRET) analysis of mGluR1α labeled with fluorescent protein(s) under total internal reflection field microscopy. Upon ligand binding, the intersubunit FRET efficiency between the second loops increased, whereas that between first loops decreased. In contrast, the intrasubunit FRET did not change clearly. These results show that ligand binding does not change the structure of each subunit, but does change the dimeric allocation of the cytoplasmic regions, which may underlie downstream signaling.
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References
Aiba, A. et al. Reduced hippocampal long-term potentiation and context-specific deficit in associative learning in mGluR1 mutant mice. Cell 79, 365–375 (1994).
Aiba, A. et al. Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice. Cell 79, 377–388 (1994).
Conquet, F. et al. Motor deficit and impairment of synaptic plasticity in mice lacking mGluR1. Nature 372, 237–243 (1994).
Shigemoto, R., Abe, T., Nomura, S., Nakanishi, S. & Hirano, T. Antibodies inactivating mGluR1 metabotropic glutamate receptor block long-term depression in cultured Purkinje cells. Neuron 12, 1245–1255 (1994).
Hermans, E. & Challiss, R.A. Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C G-protein-coupled receptors. Biochem. J. 359, 465–484 (2001).
Farrens, D.L., Altenbach, C., Yang, K., Hubbell, W.L. & Khorana, H.G. Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science 274, 768–770 (1996).
Ghanouni, P., Steenhuis, J.J., Farrens, D.L. & Kobilka, B.K. Agonist-induced conformational changes in the G-protein-coupling domain of the beta 2 adrenergic receptor. Proc. Natl. Acad. Sci. USA 98, 5997–6002 (2001).
Vilardaga, J.P., Bunemann, M., Krasel, C., Castro, M. & Lohse, M.J. Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nat. Biotechnol. 21, 807–812 (2003).
Kunishima, N. et al. Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 407, 971–977 (2000).
Hammerland, L.G. et al. Domains determining ligand specificity for Ca2+ receptors. Mol. Pharmacol. 55, 642–648 (1999).
Litschig, S. et al. CPCCOEt, a noncompetitive metabotropic glutamate receptor 1 antagonist, inhibits receptor signaling without affecting glutamate binding. Mol Pharmacol 55, 453–461 (1999).
Tsuchiya, D., Kunishima, N., Kamiya, N., Jingami, H. & Morikawa, K. Structural views of the ligand-binding cores of a metabotropic glutamate receptor complexed with an antagonist and both glutamate and Gd3+. Proc. Natl. Acad. Sci. USA 99, 2660–2665 (2002).
Remy, I., Wilson, I.A. & Michnick, S.W. Erythropoietin receptor activation by a ligand-induced conformation change. Science 283, 990–993 (1999).
Sato, T., Shimada, Y., Nagasawa, N., Nakanishi, S. & Jingami, H. Amino acid mutagenesis of the ligand binding site and the dimer interface of the metabotropic glutamate receptor 1. Identification of crucial residues for setting the activated state. J. Biol. Chem. 278, 4314–4321 (2003).
Abe, H., Tateyama, M. & Kubo, Y. Functional identification of Gd3+ binding site of metabotropic glutamate receptor 1α. FEBS Lett. 545, 233–238 (2003).
Francesconi, A. & Duvoisin, R.M. Role of the second and third intracellular loops of metabotropic glutamate receptors in mediating dual signal transduction activation. J. Biol. Chem. 273, 5615–5624 (1998).
Gomeza, J. et al. The second intracellular loop of metabotropic glutamate receptor 1 cooperates with the other intracellular domains to control coupling to G-proteins. J. Biol. Chem. 271, 2199–2205 (1996).
Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).
Tsuboi, T., Lippiat, J.D., Ashcroft, F.M. & Rutter, G.A. ATP-dependent interaction of the cytosolic domains of the inwardly rectifying K+ channel Kir6.2 revealed by fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 101, 76–81 (2004).
Riven, I., Kalmanzon, E., Segev, L. & Reuveny, E. Conformational rearrangements associated with the gating of the G protein-coupled potassium channel revealed by FRET microscopy. Neuron 38, 225–235 (2003).
Bunemann, M., Frank, M. & Lohse, M.J. Gi protein activation in intact cells involves subunit rearrangement rather than dissociation. Proc. Natl. Acad. Sci. USA 100, 16077–16082 (2003).
Miyawaki, A. Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell 4, 295–305 (2003).
Steyer, J.A. & Almers, W. A real-time view of life within 100 nm of the plasma membrane. Nat. Rev. Mol. Cell Biol. 2, 268–275 (2001).
Xia, Z. & Liu, Y. Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys. J. 81, 2395–2402 (2001).
Jensen, A.A., Hansen, J.L., Sheikh, S.P. & Brauner-Osborne, H. Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET). Eur. J. Biochem. 269, 5076–5087 (2002).
Miyashita, T. & Kubo, Y. Extracellular Ca2+ sensitivity of mGluR1α induces an increase in the basal cAMP level by direct coupling with Gs protein in transfected CHO cells. Receptors Channels 7, 77–91 (2000).
Yang, F., Moss, L.G. & Phillips, G.N. Jr. The molecular structure of green fluorescent protein. Nat. Biotechnol. 14, 1246–1251 (1996).
Mary, S., Gomeza, J., Prezeau, L., Bockaert, J. & Pin, J.P. A cluster of basic residues in the carboxyl-terminal tail of the short metabotropic glutamate receptor 1 variants impairs their coupling to phospholipase C. J. Biol. Chem. 273, 425–432 (1998).
Gasparini, F., Kuhn, R. & Pin, J.P. Allosteric modulators of group I metabotropic glutamate receptors: novel subtype-selective ligands and therapeutic perspectives. Curr. Opin. Pharmacol. 2, 43–49 (2002).
Pagano, A. et al. The non-competitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors. J. Biol. Chem. 275, 33750–33758 (2000).
Kubo, Y., Miyashita, T. & Murata, Y. Structural basis for a Ca2+-sensing function of the metabotropic glutamate receptors. Science 279, 1722–1725 (1998).
Saunders, R., Nahorski, S.R. & Challiss, R.A. A modulatory effect of extracellular Ca2+ on type 1α metabotropic glutamate receptor-mediated signalling. Neuropharmacology 37, 273–276 (1998).
Palczewski, K. et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745 (2000).
Bhave, G. et al. Membrane topology of a metabotropic glutamate receptor. J. Biol. Chem. 278, 30294–30301 (2003).
Tu, J.C. et al. Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors. Neuron 21, 717–726 (1998).
Abe, H., Misaka, T., Tateyama, M. & Kubo, Y. Effects of coexpression with Homer isoforms on the function of metabotropic glutamate receptor 1α. Mol. Cell Neurosci. 23, 157–168 (2003).
Acknowledgements
We thank J. Miyazaki for pCXN2 expression vector and A. Miyawaki for suggesting FRET. We are also grateful to T. Misaka for advice and discussion and to R. Watanabe for technical support. This work was supported partly by research grants from the Ministry of Education, Science, Sports, Culture and Technology of Japan and from foundations of Naitoh, Takeda and Yamada.
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Supplementary Fig. 1
Insertion of fluorescent proteins into the third loop of mGluR1α disturbed the fluorescence and membrane trafficking of the mGluR1α. (PDF 60 kb)
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Tateyama, M., Abe, H., Nakata, H. et al. Ligand-induced rearrangement of the dimeric metabotropic glutamate receptor 1α. Nat Struct Mol Biol 11, 637–642 (2004). https://doi.org/10.1038/nsmb770
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DOI: https://doi.org/10.1038/nsmb770
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