A protein cross-linking assay for measuring cell surface expression of glutamate receptor subunits in the rodent brain after in vivo treatments

Abstract

Trafficking of neurotransmitter receptors between intracellular and cell surface compartments is important for regulating neurotransmission. We developed a method for determining if an in vivo treatment has altered receptor distribution in a particular region of rodent brain. After the treatment, brain slices are rapidly prepared from the region of interest. Then, cell surface-expressed proteins are covalently cross-linked using the membrane-impermeable, bifunctional cross-linker bis(sulfosuccinimidyl)suberate (BS(3)). This increases the apparent molecular weight of surface receptors, while intracellular receptors are not modified. Thus, surface and intracellular receptor pools can be separated and quantified using SDS-PAGE and immunoblotting. This method is particularly useful for analyzing AMPA receptor subunits, offering advantages in accuracy, efficiency, and cost compared to biotinylation. A disadvantage is that some antibodies no longer recognize their target protein after cross-linking. We have used this method to quantify changes in receptor distribution after acute and chronic exposure to psychomotor stimulants.

Fig. 1

BS³ crosslinking enables the measurement of surface and intracellular pools of GluR1. A) The nucleus accumbens was dissected from a naive rat. Tissue from one hemisphere was cross-linked, whereas the other was not, generating paired samples that were then subjected to SDS-PAGE and immunoblotting for GluR1. Both high (surface-expressed) and predicted (intracellular) molecular weight bands are detected in tissue from the crosslinked side (Xlinked), whereas non-crosslinked (Non-Xlinked) tissue yields only a band corresponding to the predicted weight of GluR1. B) In crosslinked nucleus accumbens tissue, only proteins found both on the surface and inside the cell (GluR1, left) result in both high and predicted molecular weight bands. Proteins that are exclusively intracellular [tyrosine hydroxylase (TH); right] result in a predicted molecular weight band only, confirming that the crosslinker does not cross cell membranes. Reprinted from Boudreau and Wolf (2005) with permission from The Journal of Neuroscience.

Fig. 2

Time course of GluR1 crosslinking in nucleus accumbens tissue incubated with BS³ at two temperatures, 4°C and room temperature (RT) (*p<0.05, RT vs. 4°C). The full time course is shown in the top panel, with early times expanded in the bottom panels. Crosslinking at both temperatures occurred in three apparent phases indicated by boxed numbers 1-3 that correspond to bracketed numbers in the text.