GRIP1 controls dendrite morphogenesis by regulating EphB receptor trafficking

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Abstract

The function of the multi-PDZ domain scaffold protein GRIP1 (glutamate receptor interacting protein 1) in neurons is unclear. To explore the function of GRIP1 in hippocampal neurons, we used RNA interference (RNAi) to knock down the expression of GRIP1. Knockdown of GRIP1 by small interfering RNA (siRNA) in cultured hippocampal neurons caused a loss of dendrites, associated with mislocalization of the GRIP-interacting proteins GluR2 (AMPA receptor subunit), EphB2 (receptor tyrosine kinase) and KIF5 (also known as kinesin 1; microtubule motor). The loss of dendrites by GRIP1-siRNA was rescued by overexpression of the extracellular domain of EphB2, and was phenocopied by overexpression of the intracellular domain of EphB2 and extracellular application of ephrinB-Fc fusion proteins. Neurons from EphB1-EphB2-EphB3 triple knockout mice showed abnormal dendrite morphogenesis. Disruption of the KIF5-GRIP1 interaction inhibited EphB2 trafficking and strongly impaired dendritic growth. These results indicate an important role for GRIP1 in dendrite morphogenesis by serving as an adaptor protein for kinesin-dependent transport of EphB receptors to dendrites.

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Figure 1: RNAi knockdown of GRIP1 impairs dendrite morphogenesis of hippocampal neurons.
Figure 2: GRIP1 is essential for the maintenance of dendrites in mature hippocampal neurons.
Figure 3: GRIP1 constructs containing PDZ456 and L2 rescue the GRIP1-siRNA phenotype.
Figure 4: Effect of GRIP1 knockdown on subcellular distribution of GluR2 and EphB2.
Figure 5: Extracellular domain of EphB2 rescues GRIP1 siRNA phenotype.
Figure 6: Abnormal dendrite morphology in hippocampal neurons deficient for EphB1, EphB2 and EphB3.
Figure 7: EphB2 accumulates in the Golgi region in the absence of GRIP1.
Figure 8: KIF5/GRIP interaction required for surface expression of GluR2 and EphB2 and dendrite morphogenesis.

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Acknowledgements

We thank M. Greenberg for Eph receptor reagents, G. Banker for CD8, A. Hung for GRIP1(L732Y) and A. Akhmanova for pSuper-TRE. C.C.H. is a recipient of a long-term fellowship from Human Frontier Science Program Organization. I.M.E. and M.H. are supported by a National Institute of Mental Health grant and MH66332, respectively. M.S. is an Investigator of the Howard Hughes Medical Institute.

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Correspondence to Morgan Sheng.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

MAP2 staining in GRIP1-siRNA expressing hippocampal neurons. (PDF 1440 kb)

Supplementary Fig. 2

RNAi knock-down of GRIP1 impairs dendrite morphogenesis of pyramidal neurons in hippocampal slice cultures. (PDF 667 kb)

Supplementary Fig. 3

GRIP1 is essential for the formation and growth of dendrites but not axons in developing hippocampal neurons. (PDF 590 kb)

Supplementary Fig. 4

Reduced NMDA receptor clusters in GRIP1-knockdown neurons. (PDF 757 kb)

Supplementary Fig. 5

Brefeldin A treatment: EphB2 is trapped in the Golgi apparatus in GRIP1 siRNA expressing hippocampal neurons. (PDF 978 kb)

Supplementary Methods (PDF 47 kb)

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