Surface clustering of metabotropic glutamate receptor 1 induced by long Homer proteins

Abstract

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.

Figure 1

TIRF-M images of mGluR1-GFP fluorescence in SCG neurons expressing the receptor alone, "mGluR1-GFP", or with co-expression of the indicated Homer protein. Two representative neurons from each group are shown. The total number of neurons examined for each group were: mGluR1-GFP (19 cells), + Homer 1a (5), + Homer 1b (6), + Homer 1c (9), + Homer 2b (8), + Homer 3 (10).

Figure 2

A, Cumulative histograms showing average (error bars indicate SEM) fraction of clusters in cells as a function of cluster size. The data analyzed were from the neurons described in Figure 1. [Asterisks indicate a statistically significant change from control for 1 or more long Homer protein groups.] B, An illustration of the particle analysis for a Homer 2b co-expressing cell. Left shows the image of the region of interest for this cell. Right shows the areas exceeding 1.5× the average fluorescence of the region of interest. 'Clusters' were defined as contiguous regions above this threshold. Two examples of clusters of various sizes are shown. Differences were seen in cluster size ranging from 10 to 50 pixels². Asterisks indicate bins at which all long Homer groups differed from control and + Homer 1a cells. Scale bar is 5 μm.

Figure 3

Confocal images of mGluR1-GFP fluorescence in SCG neurons expressing the receptor alone, "mGluR1-GFP", or with co-expression of the indicated Homer protein. Two representative neurons from each group are shown. The total number of neurons examined for each group were: mGluR1-GFP (26 cells), + Homer 1a (9), + Homer 1b (12), + Homer 1c (12), + Homer 2b (34), + Homer 3 (7).

Figure 4

Differential cluster formation in SCG neurons expressing mGluR1-GFP with Homer 1b. A, Examples of Homer 1b and 1c co-expressing cells with surface clustering (TIRF; left), and apparent intracellular aggregations of mGluR1-GFP (Epifluorescence; right). B, Expanded views of a confocal image showing intracellular aggregations of receptor clusters taken from a Homer 1b co-expressing cell. C, Anomalous mGluR1-GFP distribution induced by Homer proteins indicated in a small number of TIRF-M images.