Actinfilin is a Cul3 substrate adaptor, linking GluR6 kainate receptor subunits to the ubiquitin-proteasome pathway.
Kainate receptors have been implicated in excitotoxic neuronal death induced by diseases such as epilepsy and stroke. Actinfilin, a synaptic member of the BTB-Kelch protein family, is known to bind to the actin cytoskeleton. However, little is understood about its function at the synapse. Here, we report that actinfilin is able to bind to GluR6, a kainate-type glutamate receptor subunit, and target GluR6 for degradation. Like many members of its protein family, actinfilin acts as a substrate adaptor, binding Cullin 3 (Cul3) and linking GluR6 to the E3 ubiquitin-ligase complex. We map this interaction to the Kelch repeat domain of actinfilin and the GluR6 C terminus. Co-immunoprecipitation and immunofluorescence studies show that GluR6 is ubiquitinated, and that GluR6 levels are decreased by actinfilin overexpression but increased when actinfilin levels are reduced by specific RNA interference. Furthermore, actinfilin-Cul3 interactions appear to be important for regulating surface GluR6 expression. Synaptic GluR6 levels are elevated in mice with lowered neuronal Cul3 expression and when dominant-negative forms of Cul3 are transfected into hippocampal neurons. Together our data demonstrate that actinfilin acts as a scaffold, linking GluR6 to the Cul3 ubiquitin ligase to provide a novel mechanism for kainate receptor degradation.
Salinas
Gregory D
GD
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island 02912, USA.
Blair
Leslie A C
LA
Needleman
Leigh A
LA
Gonzales
Justina D
JD
Chen
Ying
Y
Li
Min
M
Singer
Jeffrey D
JD
Marshall
John
J
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D044842:Cullin Proteins / Q000737:chemistry / Q000502:physiology*; D015536:Down-Regulation / Q000502:physiology; D006624:Hippocampus / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000502:physiology*; D009474:Neurons / Q000201:enzymology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000502:physiology*; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018092:Kainic Acid Receptors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013379:Substrate Specificity / Q000502:physiology; D020798:Two-Hybrid System Techniques; D000097806:GluK2 Kainate Receptor
D044842:Cullin Proteins; D008840:Microfilament Proteins; D046988:Proteasome Endopeptidase Complex; D021122:Protein Subunits; D018092:Kainic Acid Receptors; D000097806:GluK2 Kainate Receptor; C516956:Klhl17 protein, rat