Homer is concentrated at the postsynaptic density and does not redistribute after acute synaptic stimulation

• Long forms of Homer 1, 2, and 3 are concentrated at the PSD.
• Homer occupies domains in synapses different from those of type I mGluRs.
• Homer and Shank are co-localized at the PSD, ~30-100 nm from the postsynaptic membrane.
• Homer at the PSD does not redistribute upon acute (2 min) stimulation.
• Homer distribution at the PSD is not dependent on calcium concentration.

Homer is a postsynaptic density (PSD) scaffold protein that is involved in synaptic plasticity, calcium signaling and neurological disorders. Here, we use pre-embedding immunogold electron microscopy to illustrate the differential localization of three Homer gene products (Homer 1, 2, and 3) in different regions of the mouse brain. In cross-sectioned PSDs, Homer occupies a layer ∼30–100 nm from the postsynaptic membrane lying just beyond the dense material that defines the PSD core (∼30-nm-thick). Homer is evenly distributed within the PSD area along the lateral axis, but not at the peri-PSD locations within 60 nm from the edge of the PSD, where type I-metabotropic glutamate receptors (mGluR1 and 5) are concentrated. This distribution of Homer matches that of Shank, another major PSD scaffold protein, but differs from those of other two major binding partners of Homer, type I mGluR and IP3 receptors.Many PSD proteins rapidly redistribute upon acute (2 min) stimulation. To determine whether Homer distribution is affected by acute stimulation, we examined its distribution in dissociated hippocampal cultures under different conditions. Both the pattern and density of label for Homer 1, the isoform that is ubiquitous in hippocampus, remained unchanged under high K+ depolarization (90 mM for 2–5 min), N-methyl-d-asparic acid (NMDA) treatment (50 μM for 2 min), and calcium-free conditions (EGTA at 1 mM for 2 min). In contrast, Shank and calcium/calmodulin-dependent kinase II (CaMKII) accumulate at the PSD upon NMDA treatment, and CaMKII is excluded from the PSD complex under low calcium conditions.