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beta1-integrin mediates myelin-associated glycoprotein signaling in neuronal growth cones.

Goh EL, Young JK, Kuwako K, Tessier-Lavigne M, He Z, Griffin JW, Ming GL - Mol Brain (2008)

Bottom Line: Several myelin-associated factors that inhibit axon growth of mature neurons, including Nogo66, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), can associate with a common GPI-linked protein Nogo-66 receptor (NgR).In contrast, OMgp-induced repulsion is not affected by inhibition of b1-integrin function.These studies identify β1-integrin as a specific mediator for MAG in growth cone turning responses, acting through FAK activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Cell Engineering, The Johns Hopkins University School of Medicine, MD 21205, USA. egoh2@jhmi.edu

ABSTRACT
Several myelin-associated factors that inhibit axon growth of mature neurons, including Nogo66, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), can associate with a common GPI-linked protein Nogo-66 receptor (NgR). Accumulating evidence suggests that myelin inhibitors also signal through unknown NgR-independent mechanisms. Here we show that MAG, a RGD tri-peptide containing protein, forms a complex with β1-integrin to mediate axonal growth cone turning responses of several neuronal types. Mutations that alter the RGD motif in MAG or inhibition of β1-integrin function, but not removal of NgRs, abolish these MAG-dependent events. In contrast, OMgp-induced repulsion is not affected by inhibition of b1-integrin function. We further show that MAG stimulates tyrosine phosphorylation of focal adhesion kinase (FAK), which in turn is required for MAG-induced growth cone turning. These studies identify β1-integrin as a specific mediator for MAG in growth cone turning responses, acting through FAK activation.

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MAG-induced tyrosine phosphorylation of FAK is required for growth cone repulsion to MAG. A-C. MAG induces phosphorylation of FAK. Shown in (A) is the time course of FAK phosphorylation after MAG stimulation (2 μg/ml) of rat hippocampal neurons. Cell lysates were immunoprecipitated with anti-FAK antibodies and immunoblotted with the pY-20 antibody for phosphorylated tyrosine residues. Shown in (B) are experiments in the presence or absence of Ha2/5 (1.0 μg/ml) or echistatin (100 nM). Shown in (C) are experiments with the treatment of WT-MAG (RGD) or mutant MAG (KGE). D. MAG induces phosphorylation of FAK on residues Y397 and Y861. Cell lysates of hippocampal neurons after MAG stimulation were immunoprecipitated with anti-FAK antibodies and immunoblotted with tyrosine phosphorylation site-specific antibodies to FAK. E-G, Phosphorylation of FAK on residues Y397 and Y861 is required for MAG-induced growth cone repulsion. Hippocampal neurons were transfected with expression constructs for GFP, WT-FAK-GFP (E), FAK-Y397/861F-GFP (F), GFP and control shRNA, GFP and shRNAs against FAK. Growth cones of GFP+ neurons were examined in a gradient of MAG (150 μg/ml in the pipette). Sample images and traces were shown similarly as in Fig. 2 (A-C). Scale bar: 20 μm for microscopic images and 5 μm for traces. Shown in (G) is the summary of growth cone turning angles. Values represent mean ± s.e.m. Numbers associated with the bar graph indicate the number of growth cones analyzed. "*" indicates significant difference from the control (neurons transfected to express GFP alone; p < 0.01, ANOVA).
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Figure 7: MAG-induced tyrosine phosphorylation of FAK is required for growth cone repulsion to MAG. A-C. MAG induces phosphorylation of FAK. Shown in (A) is the time course of FAK phosphorylation after MAG stimulation (2 μg/ml) of rat hippocampal neurons. Cell lysates were immunoprecipitated with anti-FAK antibodies and immunoblotted with the pY-20 antibody for phosphorylated tyrosine residues. Shown in (B) are experiments in the presence or absence of Ha2/5 (1.0 μg/ml) or echistatin (100 nM). Shown in (C) are experiments with the treatment of WT-MAG (RGD) or mutant MAG (KGE). D. MAG induces phosphorylation of FAK on residues Y397 and Y861. Cell lysates of hippocampal neurons after MAG stimulation were immunoprecipitated with anti-FAK antibodies and immunoblotted with tyrosine phosphorylation site-specific antibodies to FAK. E-G, Phosphorylation of FAK on residues Y397 and Y861 is required for MAG-induced growth cone repulsion. Hippocampal neurons were transfected with expression constructs for GFP, WT-FAK-GFP (E), FAK-Y397/861F-GFP (F), GFP and control shRNA, GFP and shRNAs against FAK. Growth cones of GFP+ neurons were examined in a gradient of MAG (150 μg/ml in the pipette). Sample images and traces were shown similarly as in Fig. 2 (A-C). Scale bar: 20 μm for microscopic images and 5 μm for traces. Shown in (G) is the summary of growth cone turning angles. Values represent mean ± s.e.m. Numbers associated with the bar graph indicate the number of growth cones analyzed. "*" indicates significant difference from the control (neurons transfected to express GFP alone; p < 0.01, ANOVA).

Mentions: How does β1-integrin signaling transduce MAG-induced growth cone responses? Focal adhesion kinase (FAK) is a major mediator of integrin-dependent signaling in many contexts, including cell migration and axon guidance [26,50,51]. Interestingly, treatment of hippocampal neurons with MAG (2 μg/ml) induced tyrosine phosphorylation of FAK in a time-dependent manner (Fig. 7A). Such MAG-induced tyrosine phosphorylation of FAK was abolished in the presence of echistatin (100 nM) or Ha2/5 (2.0 μg/ml; Fig. 7B). In addition, mutant MAG-KGE failed to trigger tyrosine phosphorylation of FAK (Fig. 7C) while removing GPI-linked proteins following the PI-PLC treatment did not affect MAG-induced phosphorylation of FAK in these neurons [see Additional file 5]. Thus, MAG induces tyrosine phosphorylation of FAK in an integrin-dependent manner.


beta1-integrin mediates myelin-associated glycoprotein signaling in neuronal growth cones.

Goh EL, Young JK, Kuwako K, Tessier-Lavigne M, He Z, Griffin JW, Ming GL - Mol Brain (2008)

MAG-induced tyrosine phosphorylation of FAK is required for growth cone repulsion to MAG. A-C. MAG induces phosphorylation of FAK. Shown in (A) is the time course of FAK phosphorylation after MAG stimulation (2 μg/ml) of rat hippocampal neurons. Cell lysates were immunoprecipitated with anti-FAK antibodies and immunoblotted with the pY-20 antibody for phosphorylated tyrosine residues. Shown in (B) are experiments in the presence or absence of Ha2/5 (1.0 μg/ml) or echistatin (100 nM). Shown in (C) are experiments with the treatment of WT-MAG (RGD) or mutant MAG (KGE). D. MAG induces phosphorylation of FAK on residues Y397 and Y861. Cell lysates of hippocampal neurons after MAG stimulation were immunoprecipitated with anti-FAK antibodies and immunoblotted with tyrosine phosphorylation site-specific antibodies to FAK. E-G, Phosphorylation of FAK on residues Y397 and Y861 is required for MAG-induced growth cone repulsion. Hippocampal neurons were transfected with expression constructs for GFP, WT-FAK-GFP (E), FAK-Y397/861F-GFP (F), GFP and control shRNA, GFP and shRNAs against FAK. Growth cones of GFP+ neurons were examined in a gradient of MAG (150 μg/ml in the pipette). Sample images and traces were shown similarly as in Fig. 2 (A-C). Scale bar: 20 μm for microscopic images and 5 μm for traces. Shown in (G) is the summary of growth cone turning angles. Values represent mean ± s.e.m. Numbers associated with the bar graph indicate the number of growth cones analyzed. "*" indicates significant difference from the control (neurons transfected to express GFP alone; p < 0.01, ANOVA).
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Figure 7: MAG-induced tyrosine phosphorylation of FAK is required for growth cone repulsion to MAG. A-C. MAG induces phosphorylation of FAK. Shown in (A) is the time course of FAK phosphorylation after MAG stimulation (2 μg/ml) of rat hippocampal neurons. Cell lysates were immunoprecipitated with anti-FAK antibodies and immunoblotted with the pY-20 antibody for phosphorylated tyrosine residues. Shown in (B) are experiments in the presence or absence of Ha2/5 (1.0 μg/ml) or echistatin (100 nM). Shown in (C) are experiments with the treatment of WT-MAG (RGD) or mutant MAG (KGE). D. MAG induces phosphorylation of FAK on residues Y397 and Y861. Cell lysates of hippocampal neurons after MAG stimulation were immunoprecipitated with anti-FAK antibodies and immunoblotted with tyrosine phosphorylation site-specific antibodies to FAK. E-G, Phosphorylation of FAK on residues Y397 and Y861 is required for MAG-induced growth cone repulsion. Hippocampal neurons were transfected with expression constructs for GFP, WT-FAK-GFP (E), FAK-Y397/861F-GFP (F), GFP and control shRNA, GFP and shRNAs against FAK. Growth cones of GFP+ neurons were examined in a gradient of MAG (150 μg/ml in the pipette). Sample images and traces were shown similarly as in Fig. 2 (A-C). Scale bar: 20 μm for microscopic images and 5 μm for traces. Shown in (G) is the summary of growth cone turning angles. Values represent mean ± s.e.m. Numbers associated with the bar graph indicate the number of growth cones analyzed. "*" indicates significant difference from the control (neurons transfected to express GFP alone; p < 0.01, ANOVA).
Mentions: How does β1-integrin signaling transduce MAG-induced growth cone responses? Focal adhesion kinase (FAK) is a major mediator of integrin-dependent signaling in many contexts, including cell migration and axon guidance [26,50,51]. Interestingly, treatment of hippocampal neurons with MAG (2 μg/ml) induced tyrosine phosphorylation of FAK in a time-dependent manner (Fig. 7A). Such MAG-induced tyrosine phosphorylation of FAK was abolished in the presence of echistatin (100 nM) or Ha2/5 (2.0 μg/ml; Fig. 7B). In addition, mutant MAG-KGE failed to trigger tyrosine phosphorylation of FAK (Fig. 7C) while removing GPI-linked proteins following the PI-PLC treatment did not affect MAG-induced phosphorylation of FAK in these neurons [see Additional file 5]. Thus, MAG induces tyrosine phosphorylation of FAK in an integrin-dependent manner.

Bottom Line: Several myelin-associated factors that inhibit axon growth of mature neurons, including Nogo66, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), can associate with a common GPI-linked protein Nogo-66 receptor (NgR).In contrast, OMgp-induced repulsion is not affected by inhibition of b1-integrin function.These studies identify β1-integrin as a specific mediator for MAG in growth cone turning responses, acting through FAK activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Cell Engineering, The Johns Hopkins University School of Medicine, MD 21205, USA. egoh2@jhmi.edu

ABSTRACT
Several myelin-associated factors that inhibit axon growth of mature neurons, including Nogo66, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), can associate with a common GPI-linked protein Nogo-66 receptor (NgR). Accumulating evidence suggests that myelin inhibitors also signal through unknown NgR-independent mechanisms. Here we show that MAG, a RGD tri-peptide containing protein, forms a complex with β1-integrin to mediate axonal growth cone turning responses of several neuronal types. Mutations that alter the RGD motif in MAG or inhibition of β1-integrin function, but not removal of NgRs, abolish these MAG-dependent events. In contrast, OMgp-induced repulsion is not affected by inhibition of b1-integrin function. We further show that MAG stimulates tyrosine phosphorylation of focal adhesion kinase (FAK), which in turn is required for MAG-induced growth cone turning. These studies identify β1-integrin as a specific mediator for MAG in growth cone turning responses, acting through FAK activation.

Show MeSH
Related in: MedlinePlus