Surface clustering of metabotropic glutamate receptor 1 induced by long Homer proteins.
Animals; Carrier Proteins/genetics/*metabolism; Cell Membrane/metabolism; Confocal; Gene Expression; Green Fluorescent Proteins/genetics; Homer Scaffolding Proteins; In Vitro Techniques; Metabotropic Glutamate/genetics/*metabolism; Microscopy; Neurons/*metabolism; Rats; Receptor Aggregation/*physiology; Receptors; Superior Cervical Ganglion/cytology; Sympathetic Nervous System/cytology; Transfection; Wistar
BACKGROUND: Metabotropic glutamate receptors (mGluRs) regulate neuronal excitability and synaptic strength. The group I mGluRs, mGluR1 and 5, are widespread in the brain and localize to post-synaptic sites. The Homer protein family regulates group I mGluR function and distribution. Constitutively expressed 'long' Homer proteins (Homer 1b, 1c, 2 and 3) induce dendritic localization of group I mGluRs and receptor clustering, either internally or on the plasma membrane. Short Homer proteins (Homer 1a, Ania-3) exhibit regulated expression and act as dominant negatives, producing effects on mGluR distribution and function that oppose those of the long Homer proteins. There remains some controversy over whether long Homer proteins induce receptor internalization by inducing retention in the endoplasmic reticulum, or induce mGluR clustering on the plasma membrane. Further, an exhaustive study of the effects of each long Homer isoform on mGluR distribution has not been published. RESULTS: The distribution of a GFP-tagged group I mGluR, mGluR1-GFP, was examined in the absence of Homer proteins and in the presence of several Homer isoforms expressed in sympathetic neurons from the rat superior cervical ganglion (SCG) using total internal reflection fluorescence (TIRF-M) and confocal microscopy. Quantitative analysis of mGluR1-GFP fluorescence using TIRF-M revealed that expression of each long Homer isoform tested (Homer 1b, 1c, 2b and 3) induced a significant degree of surface clustering. Using confocal imaging, Homer-induced mGluR clusters were observed intra-cellularly as well as on the plasma membrane. Further, in approximately 40% of neurons co-expressing mGluR1-GFP and Homer 1b, intracellular inclusions were observed, but plasma membrane clusters were also documented in some Homer 1b coexpressing cells. CONCLUSION: All long Homer proteins examined (Homer 1b, 1c, 2b and 3) induced a significant degree of mGluR1-GFP clustering on the plasma membrane compared to cells expressing mGluR1-GFP alone. Clusters induced by long Homers appeared on the plasma membrane and intracellularly, suggesting that clusters form prior to plasma membrane insertion and/or persist after internalization. Finally, while Homer 1b induced surface clustering of mGluR1 in some cells, under some conditions intracellular retention may occur.
Kammermeier Paul J
BMC neuroscience
2006
2006-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1186/1471-2202-7-1" target="_blank" rel="noreferrer noopener">10.1186/1471-2202-7-1</a>
Activation of metabotropic glutamate receptor 1 dimers requires glutamate binding in both subunits.
Blotting; Calcium Channels/drug effects/metabolism; DNA/biosynthesis/genetics; Dose-Response Relationship; Drug; Fluorescent Antibody Technique; Genes; Glutamic Acid/*metabolism; Humans; Membrane Potentials/drug effects; Metabotropic Glutamate/genetics/*metabolism; myc/genetics; Patch-Clamp Techniques; Plasmids/genetics; Receptors; Signal Transduction/drug effects; Superior Cervical Ganglion/cytology/drug effects/metabolism; Sympathetic Nervous System/cytology/drug effects/metabolism; Western
Group I metabotropic glutamate receptors (mGluRs) form stable, disulfide-linked homodimers. Lack of a verifiably monomeric mGluR1 mutant has led to difficulty in assessing the role of dimerization in the molecular mechanism of mGluR1 activation. The related GABA(B) receptor exhibits striking intradimer cross talk (ligand binding at one subunit effectively produces G protein activation at the other), but it is unclear whether group I mGluRs exhibit analogous cross talk. Signaling of heterologously expressed mGluR1 was examined in isolated rat sympathetic neurons by measuring glutamate-mediated inhibition of native calcium currents. To examine mGluR1 activity when only one dimer subunit has access to glutamate ligand, wildtype mGluR1 was coexpressed with mGluR1 Y74A, a mutant with impaired glutamate binding, and the activity of the heterodimer (mutant/wild type) was examined. The mGluR1 Y74A mutant alone had a dose-response curve that was shifted by about 2 orders of magnitude. The half-maximal dose of glutamate shifted from 1.3 (wild-type mGluR1) to about 450 (mGluR1 Y74A) microM. However, the maximal effect was similar. Wild-type mGluR1 was expressed with excess Y74A mGluR1 to generate a receptor population consisting largely of mutant homodimers and mutant/wild-type heterodimers but without detectable wild-type homodimers. Under these conditions, no glutamate-mediated calcium current inhibition was observed below approximately 300 microM glutamate, although wild-type mGluR1 protein was detectable with immunofluorescence. These data suggest that mutant/wild-type heterodimeric receptors are inactive at ligand concentrations favoring glutamate association with receptor dimers at only one subunit.
Kammermeier Paul J; Yun June
The Journal of pharmacology and experimental therapeutics
2005
2005-02
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1124/jpet.104.073155" target="_blank" rel="noreferrer noopener">10.1124/jpet.104.073155</a>