Activity dependent dissociation of the Homer1 interactome

Abstract

Neurons encode information by rapidly modifying synaptic protein complexes, which changes the strength of specific synaptic connections. Homer1 is abundantly expressed at glutamatergic synapses, and is known to alter its binding to metabotropic glutamate receptor 5 (mGlu5) in response to synaptic activity. However, Homer participates in many additional known interactions whose activity-dependence is unclear. Here, we used co-immunoprecipitation and label-free quantitative mass spectrometry to characterize activity-dependent interactions in the cerebral cortex of wildtype and Homer1 knockout mice. We identified a small, high-confidence protein network consisting of mGlu5, Shank2 and 3, and Homer1–3, of which only mGlu5 and Shank3 were significantly reduced following neuronal depolarization. We identified several other proteins that reduced their co-association in an activity-dependent manner, likely mediated by Shank proteins. We conclude that Homer1 dissociates from mGlu5 and Shank3 following depolarization, but our data suggest that direct Homer1 interactions in the cortex may be more limited than expected.

Elaboration of the Homer1 Recognition Landscape Reveals Incomplete Divergence of Paralogous EVH1 Domains

“…For this reason, Homer-mediated interactions may evade standard techniques for identifying native protein-protein interactions, and this may explain the low recovery of annotated binding partners in various Homer co-IP mass spectrometry studies. 50,51 Limitations of these experiments also stem from their tendency to focus on a single tissue type, despite the wider expression of Homer. 26…”

“…76 In the cerebral cortex of mice, Homer1 dissociates from a complex containing the Arp2/3 members ARPC2, 4, and 5 in an activity-dependent manner. 50…”

“…For this reason, Homer-mediated interactions may evade standard techniques for identifying native protein-protein interactions, and this may explain the low recovery of annotated binding partners in various Homer co-IP mass spectrometry studies. 50,51 Limitations of these experiments also stem from their tendency to focus on a single tissue type, despite the wider expression of Homer. 26 Most Homer binding partners have been identified in low-throughput, candidate-driven studies by virtue of the inclusion of a canonical PPxxF motif, first identified in metabotropic glutamate receptors.…”

“…76 In the cerebral cortex of mice, Homer1 dissociates from a complex containing the Arp2/3 members ARPC2, 4, and 5 in an activitydependent manner. 50 The small number of interface residues that constitute SLiM-mediated interactions makes them highly amenable to regulation by post-translational modification. Motif phosphorylation is a well-documented switch for SLiM binding (e.g phosphorylation of the NPxY motif in the integrin β3 subunit shifts binding from talin to Dok1).…”

“…Homer3 knockdown in neutrophils impairs the recruitment of the WAVE2 complex to the leading edge of motile cells [76]. In the cerebral cortex of mice, Homer1 dissociates from a complex containing the Arp2/3 members ARPC2, 4, and 5 in an activity-dependant manner [49].…”