Rapid internalization and surface expression of a functional, fluorescently tagged G-protein-coupled glutamate receptor.
l-Glutamate is the principal excitatory neurotransmitter in the vertebrate central nervous system, where it mediates many of its actions via G-protein-coupled metabotropic glutamate (mGlu) receptors. Since little is known about the dynamics of mGlu receptors at the plasma membrane, we have constructed a fusion protein comprising the mGlu receptor subtype 1alpha (mGlu1alpha) and green fluorescent protein (GFP). Using imaging of Ca2+ release from intracellular stores as a functional assay, the agonist pharmacology of this fluorescently tagged receptor was found to be similar to that of the wild-type receptor when expressed in HEK-293 cells. Receptor movement and function were measured simultaneously by combined imaging of Ca2+, using fura-red, and GFP fluorescence in single cells. Exposure to agonist induced a rapid loss of up to 30% of membrane-associated fluorescence, with a corresponding decrease in the functional response. Following removal of the agonist there was recovery of both the membrane fluorescence and the functional response. These data suggest that the surface expression of G-protein-coupled glutamate receptors might be rapidly regulated in response to agonist activation.
MRC Centre for Synaptic Plasticity, Department of Anatomy, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, U.K.
A J
AJ
Doherty
V
V
Coutinho
G L
GL
Collingridge
J M
JM
Henley
D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism* / Q000648:ultrastructure; D015398:Signal Transduction
D008164:Luminescent Proteins; D018094:Receptors, Metabotropic Glutamate; D049452:Green Fluorescent Proteins; D002118:Calcium