Figure 5.

Most of mGlu₅ is intracellular in striatal, hippocampal, and cortical cultures. (A) mGlu₅ receptors are expressed on dendrites, within cell bodies, and on nuclear membranes in neuronal cultures. DIV 14 striatal, hippocampal, or cortical cultures showing colocalization of mGlu₅ receptor immunoreactivity (red) together with the inner nuclear membrane marker Lamin B2 (green). (B) Western blots of DIV 14 striatal lysates prepared from knockout (−/−) and wildtype (+/+) littermates generated by mating mGlu5 (+/−) heterozygote mice. (C) Western blots of neuronal culture lysates probed with the antibody against mGlu₅. (D) Comparison of mGlu₅ levels in hippocampus or cortex to striatal levels following normalization to β-actin. Data shown represent the mean of three experiments (hippocampal/striatal = 112.96 ± 9.17%; cortical/striatal = 101.06 ± 9.09%). (E) Cell surface proteins from DIV 14 striatal, hippocampal, and cortical cultures were labeled with cell-membrane-impermeable EZ-Link Sulfo-NHS-LC-Biotin for 30 min prior to quenching. Cells were lysed, and an aliquot representing 25% of the total protein was removed. The remaining 75% was incubated with NeutrAvidin agarose, washed with RIPA buffer, and bound proteins were resuspended in SDS sample buffer and heated at 60 °C. An amount of 10 μg of total protein (tot) was run on a gel next to the biotinylated proteins (bio) derived from 100 μg of the equivalent neuronal lysate. The Na⁺-K⁺ ATPase was used as a plasma membrane positive control, whereas β-actin served as a loading control. (F) Glycosylation analysis of mGlu₅ in cortical cultures. Biotinylated cortical cell lysate was either treated or not with PNGaseF and then run on SDS-PAGE along with 10 μg of total cell lysate followed by Western blotting with anti-mGlu₅. The biotinylated surface mGlu₅ was sensitive to the PNGase F treatment. (G) Quantitative analysis of Western blotting results in (E) showing that only a small percentage of total mGlu₅ is on the cell surface in striatal, hippocampal, and cortical cultures: 1.81 ± 0.84% for striatal, 5.58 ± 1.52% for hippocampal, and 6.37 ± 1.38% for cortical cultures. These values were derived following densitometric analyses using the ChemiDoc MP system (Bio-Rad) together with associated Image Lab software. The percentage of the cell surface receptor = (the background-subtracted density of the biotinylated band from 100 μg of total lysate/10 × the background-subtracted density of 10 μg of total lysate) × 100. Bars represent the mean of three independent experiments ± SE. *Denotes statistical significance compared with control with Student’s t test (p < 0.05).