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. 2007 Nov 28;27(48):13273-8.
doi: 10.1523/JNEUROSCI.3334-07.2007.

Characterization of the role of microtubule-associated protein 1B in metabotropic glutamate receptor-mediated endocytosis of AMPA receptors in hippocampus

Genoveva Davidkova  1 , Reed C Carroll
Affiliations

Characterization of the role of microtubule-associated protein 1B in metabotropic glutamate receptor-mediated endocytosis of AMPA receptors in hippocampus

Genoveva Davidkova et al. J Neurosci. .

Abstract

The mGluR-dependent endocytosis of AMPA receptors (AMPARs) in the CA1 region is protein synthesis dependent. However, why this form of trafficking, and not that mediated by NMDA receptor activation, is dependent on protein translation is unclear. Here we have studied the contribution of the cytoskeletal microtubule-associated protein 1B (MAP1B) to the pathway-specific internalization of AMPARs. Treatments of cultured neurons with 3,4-dihydroxyphenylglycol (DHPG) or NMDA, both of which drive AMPAR endocytosis, caused a translation-dependent increase in the dendritic levels of MAP1B protein. Although interfering with protein synthesis using short interfering RNA (siRNA) to eEF2 kinase (eukaryotic elongation factor 2 kinase) blocked the dendritic MAP1B increase by both pathways, it selectively blocked the DHPG- and not the NMDA-induced AMPAR endocytosis. In support of MAP1B synthesis contributing to metabotropic glutamate receptor (mGluR)-mediated AMPAR endocytosis, siRNA against MAP1B in CA1 cultured neurons specifically blocked the DHPG-induced AMPAR internalization. Previous studies suggest a direct interaction between MAP1B and the AMPAR-binding protein GRIP1. Biochemical studies establish that MAP1B associates with GRIP1 and forms a complex with GluR2 in vivo in rat hippocampus. Furthermore, the interaction between MAP1B and GRIP1 increased significantly in acute slices after treatment with DHPG and not NMDA. Together, these findings suggest that MAP1B plays a selective role in the DHPG-induced endocytosis of AMPARs, perhaps through its interaction with GRIP1.

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Figures

Figure 1.
Figure 1.
Immunocytochemical expression of MAP1B in dendrites of cultured CA1 pyramidal neurons. A, Colocalization (yellow) between MAP1B (red) and PSD95 (green) in hippocampal neurons. MAP1B, PSD-95, and merged labeling in the inset is enlarged on the right. B–E, NMDA or DHPG treatments, which induce AMPAR endocytosis, increase the dendritic levels and synthesis of MAP1B protein. B, MAP1B immunostaining without and with anisomycin (Anis.) pretreatment in control and DHPG- or NMDA-treated cells. C, Quantitation of MAP1B immunostaining after DHPG in the presence and absence of anisomycin and rapamycin (Rap) (Con, DHPG n = 8; anisomycin n = 5; rapamycin n = 5; *p ≤ 0.05). D, Surface GluR1 immunostaining in neurons labeled live with N-terminal-specific GluR1 antibody in NMDA- or DHPG-treated neurons in the presence or absence of anisomycin. E, Quantitation of surface GluR1 after DHPG treatment in the presence and absence of anisomycin or rapamycin (Con, DHPG n = 8, anisomycin, rapamycin n = 4; *p ≤ 0.05). Scale bars, 10 μm.
Figure 2.
Figure 2.
Acute knockdown of eEF2 kinase blocks DHPG- and NMDA-induced increases in dendritic MAP1B protein and DHPG-induced AMPAR endocytosis. A, eEF2 kinase and MAP1B immunostaining in neurons transfected with scrambled (Scr.) control or eEF2 kinase siRNA. B, Quantitation of dendritic MAP1B immunostaining. n = 4 transfections; *p < 0.01. C, Surface GluR2 immunostaining in transfected neurons labeled live with N-terminal-specific GluR2 antibody. D, Quantitation of surface GluR2 immunostaining. n = 5 (NMDA) and 4 (DHPG) transfections; *p < 0.05. Scale bars, 10 μm. si, siRNA; NS, not significantly different.
Figure 3.
Figure 3.
MAP1B siRNA blocks DHPG-induced AMPAR endocytosis. A, Surface GluR2 immunostaining in neurons transfected with scrambled or MAP1B siRNAs (si). After labeling of GluR2 receptors in live neurons, cells were treated with NMDA or DHPG. Scale bar, 10 μm. B, Graph depicts effects of MAP1B siRNA on surface GluR2 as depicted in A. *p ≤ 0.05. C, MAP1B siRNA does not have effects on number of primary dendrites (# Primary Dend.) and total dendritic length (Dend. Length).
Figure 4.
Figure 4.
MAP1B-GRIP1 interaction in vivo in hippocampus. A, MAP1B colocalizes with GRIP1 in dendritic shafts of hippocampal pyramidal neurons. Scale bar, 10 μm. B, GRIP or GluR2 coimmunoprecipitation demonstrates a complex between MAP1B, GRIP, and GluR2 but not PSD95. C, Western blot of biotinylated proteins showing that NMDA or DHPG treatments induce AMPAR endocytosis. n = 3 experiments; *p < 0.05. Total AMPAR (Tot) and isolated biotin-labeled surface GluR2 protein (Surf) from NMDA- or DHPG-treated hippocampal lysates were analyzed by Western blot. D, GRIP coimmunoprecipitation showing that DHPG treatment is associated with increased binding of MAP1B to GRIP1. E, Quantitation of the MAP1B/GRIP1 ratio; NMDA n = 4, DHPG n = 6; *p < 0.05. F, Coimmunoprecipitation experiment demonstrates that rapamycin (Rap.) pretreatment blocks the DHPG-dependent increase in GRIP–MAP1B association. C, Control; N, NMDA; D, DHPG.
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