Actinfilin: Linking synaptic proteins to the ubiquitin-proteasome pathway.

摘要

Actinfilin, a brain-specific protein characterized by an N-terminal BTB/POZ domain followed by six Kelch domain repeats, has previously been found to interact with the actin cytoskeleton. However, no defined role for actinfilin has been established in the brain. In the research presented here, we show that actinfilin, as a member of the BTB/POZ-Kelch protein family, links synaptic substrates with Cul3 and the ubiquitinproteasome pathway. We identify two such actinfilin substrates: GluR6 and Shank. GluR6, as a subunit of the kainate subtype of glutamate receptors, is a major mediator for neural excitation. The Kelch domain region of actinfilin binds GluR6 at a hydrophobic amino acid stretch proximal to its C-terminus. Upon binding, actinfilin targets GluR6 for degradation via Cul3 and the ubiquitin-proteasome pathway. GluR6 ubiquitination and degradation can be decreased by disrupting the ubiquitination pathway or the actinfilin/GluR6 interaction. Mice with lowered neuronal Cul3 expression have elevated levels of synaptic GluR6, but other subtypes of ionotropic glutamate receptors are not changed. Lowering actinfilin levels in dissociated hippocampal cultures with RNAi causes a significant increase in dendritic spine density, which may be due to an increase in the levels of Shank, a postsynaptic scaffolding protein. Actinfilin binds Shank in heterologous cell culture as well as in rat cerebellar extracts. Mice with lowered Cul3 expression have altered expression of many scaffolding proteins in their synapses, including elevated synaptic levels of Shank. These studies are the first to show that kainate receptors are ubiquitinated, and reveal a potential mechanism in regulation of the mammalian synapse.