Sequential inter- and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1.
The metabotropic glutamate receptor 1 (mGluR1), a class C member of the heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor family, is a constitutive dimer that regulates excitatory neurotransmission. We investigated the role of homodimer formation in mGluR1 activation by examining activation-dependent inter- and intrasubunit conformational changes by fluorescence resonance energy transfer (FRET). We inserted yellow and cyan fluorescent proteins in the second intracellular loop and at the carboxyl terminus of mGluR1 to act as FRET sensors and expressed these proteins in human embryonic kidney 293 cells. Agonist-dependent activation of these mGluR1 chimeras rapidly increased the intersubunit FRET, suggesting rapid movement of the subunits relative to each other. After intersubunit movement, the intrasubunit FRET decreased, reflecting conformational changes within a subunit. Cotransfection of chimeric receptor subunits that were capable or incapable of G protein coupling revealed that only a single subunit assumes an active state in an mGluR1 receptor dimer.
Institute of Pharmacology and Toxicology, University of Würzburg, 97078 Würzburg, Germany.
Veronika
V
Hlavackova
Ulrike
U
Zabel
Daniela
D
Frankova
Julia
J
Bätz
Carsten
C
Hoffmann
Laurent
L
Prezeau
Jean-Philippe
JP
Pin
Jaroslav
J
Blahos
Martin J
MJ
Lohse
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D020125:Mutation, Missense / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry*; D018094:Receptors, Metabotropic Glutamate / Q000737:chemistry* / Q000378:metabolism; D009435:Synaptic Transmission / Q000502:physiology*
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D017931:DNA Primers; D008164:Luminescent Proteins; D021122:Protein Subunits; D018094:Receptors, Metabotropic Glutamate; C104077:metabotropic glutamate receptor type 1; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D002118:Calcium