Abstract
Metabotropic glutamate receptors (mGluRs), members of family 3 G protein-coupled receptors, play pivotal roles in the modulation of synaptic transmission and are important drug targets for various neurological diseases. The structures of the extracellular ligand-binding domain (ECD) of mGluRs and its changes upon ligand binding have been well studied by various techniques, including X-ray crystallography. In contrast, little is known about the structure and structural changes of the seven-transmembrane domain (TMD). Here we searched for constitutively active mutation (CAM) sites in the TMD of mGluR8 to get insight into the epicenter of the structural changes in TMD, a potential target for allosteric ligands. Mutational analyses based on the knowledge of activating mutations of calcium-sensing receptor showed the presence of several CAM sites in the TMD of mGluR8. Among them, the site at position 764 in helix V, where threonine is present, is unique in that any substitution resulted in elevation of the basal activity, and some substitutions caused a loss of responsiveness to agonist. Further comprehensive mutational analyses indicated that the additional mutation of the CAM site at position 663 in helix III, where a tyrosine residue is present, generated a revertant phenotype. Mutations at these sites also increased the agonist binding affinity, although these sites are far from the ECD. These results indicated that the specific pair of amino acids at these CAM sites forms an activation switch that stabilizes the inactive state of mGluR8 and mediates the signal flow from the ECD to the cytoplasmic G protein-interacting site.
Footnotes
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This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan [Grants-in-Aid for Scientific Research 19045018, 19770130; the Global Center of Excellence Program “Formation of a Strategic Base for Biodiversity and Evolutionary Research: from Genome to Ecosystem” (Program A06)]; and by a Japan Society for the Promotion of Science Research Fellowship for Young Scientists [Grant 08J01259].
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ABBREVIATIONS: mGluR, metabotropic glutamate receptor; GPCR, G protein-coupled receptor; ECD, extracellular ligand-binding domain; TMD, transmembrane domain; CAM, constitutively active mutation; LY341495, (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid; l-AP4, l-(+)-2-amino-4-phosphonobutyric acid; GTPγS, guanosine 5′-3-O-(thio)triphosphate; DM, dodecylmaltoside; CaSR, calcium-sensing receptor.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Accepted April 27, 2009.
- Received March 26, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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