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Ca(2+)/calmodulin-dependent protein kinase II interacts with group I metabotropic glutamate and facilitates receptor endocytosis and ERK1/2 signaling: role of β-amyloid.

Raka F, Di Sebastiano AR, Kulhawy SC, Ribeiro FM, Godin CM, Caetano FA, Angers S, Ferguson SS - Mol Brain (2015)

Bottom Line: The expression of both mGluR5a and PrP(C) together, but not alone resulted in the agonist-stimulated subcellular distribution of CaMKIIα into cytoplasmic puncta.Taken together these results indicate that CaMKIIα selectively regulates mGluR1a and mGluR5a ERK1/2 signaling.As mGluR5 and CaMKIIα are involved in learning and memory and Aβ and mGluR5 are implicated in Alzheimer's disease, results of these studies could provide insight into potential pharmacological targets for treatment of Alzheimer's disease.

View Article: PubMed Central - PubMed

Affiliation: J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute, and the Department of Physiology and Pharmacology, University of Western Ontario, 100 Perth Dr. London, Ontario, N6A 5K8, Canada. fitoreraka@gmail.com.

ABSTRACT

Background: Agonist stimulation of Group I metabotropic glutamate receptors (mGluRs) initiates their coupling to the heterotrimeric G protein, Gαq/11, resulting in the activation of phospholipase C, the release of Ca(2+) from intracellular stores and the subsequent activation of protein kinase C. However, it is now recognized that mGluR5a also functions as a receptor for cellular prion protein (PrP(C)) and β-amyloid peptide (Aβ42) oligomers to facilitate intracellular signaling via the resulting protein complex. Intracellular mGluR5a signaling is also regulated by its association with a wide variety of intracellular regulation proteins.

Results: In the present study, we utilized mass spectroscopy to identify calmodulin kinase IIα (CaMKIIα) as a protein that interacts with the second intracellular loop domain of mGluR5. We show that CaMKIIα interacts with both mGluR1a and mGluR5a in an agonist-independent manner and is co-immunoprecipitated with mGluR5a from hippocampal mouse brain. CaMKIIα positively regulates both mGluR1a and mGluR5a endocytosis, but selectively attenuates mGluR5a but not mGluR1a-stimulated ERK1/2 phosphorylation in a kinase activity-dependent manner. We also find that Aβ42 oligomers stimulate the association of CaMKIIα with mGluR5a and activate ERK1/2 in an mGluR5a-dependent manner. However, Aβ42 oligomer-stimulated ERK1/2 phosphorylation is not regulated by mGluR5a/CaMKIIα interactions suggesting that agonist and Aβ42 oligomers stabilize distinct mGluR5a activation states that are differentially regulated by CaMKIIα. The expression of both mGluR5a and PrP(C) together, but not alone resulted in the agonist-stimulated subcellular distribution of CaMKIIα into cytoplasmic puncta.

Conclusions: Taken together these results indicate that CaMKIIα selectively regulates mGluR1a and mGluR5a ERK1/2 signaling. As mGluR5 and CaMKIIα are involved in learning and memory and Aβ and mGluR5 are implicated in Alzheimer's disease, results of these studies could provide insight into potential pharmacological targets for treatment of Alzheimer's disease.

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Effect of CaMKIIα overexpression on mGluR1a- and mGluR5a- -stimulated ERK1/2 phosphorylation. A) Shown are representative immunoblots for FL-mGluR1a expression, p-ERK1/2 activity and total-ERK1/2 expression in HEK 293 cells transiently transfected with 3 μg of pcDNA3.1 encoding FLAG-mGluR1a along with 0.5 μg of plasmid cDNA encoding either GFP, GFP-CaMKIIα or GFP-CaMKIIα-T286A in response to 50 μM quisqualate treatment for 0, 5 and 10 min. Bar graph shows the densitometric analysis of ERK1/2 phosphorylation normalized to both basal and total ERK1/2 protein expression. Data represents the mean ± SD of five independent experiments. *P < 0.05 versus GFP transfected control cells. B) Shown are representative immunoblots for, FL-mGluR5a expression, p-ERK1/2 activity and total-ERK1/2 expression in HEK 293 cells transiently transfected with 3 μg of pcDNA3.1 encoding, FL-mGluR5a along with 0.5 μg of plasmid cDNA encoding either GFP, GFP-CaMKIIα or GFP-CaMKIIα-T286A in response to 50 μM quisqualate treatment for 0, 5 and 10 min. Bar graph shows the densitometric analysis of ERK1/2 phosphorylation normalized to both basal total ERK1/2 protein expression. Data represents the mean ± SD of five independent experiments. *P < 0.05 versus GFP transfected control cells.
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Fig4: Effect of CaMKIIα overexpression on mGluR1a- and mGluR5a- -stimulated ERK1/2 phosphorylation. A) Shown are representative immunoblots for FL-mGluR1a expression, p-ERK1/2 activity and total-ERK1/2 expression in HEK 293 cells transiently transfected with 3 μg of pcDNA3.1 encoding FLAG-mGluR1a along with 0.5 μg of plasmid cDNA encoding either GFP, GFP-CaMKIIα or GFP-CaMKIIα-T286A in response to 50 μM quisqualate treatment for 0, 5 and 10 min. Bar graph shows the densitometric analysis of ERK1/2 phosphorylation normalized to both basal and total ERK1/2 protein expression. Data represents the mean ± SD of five independent experiments. *P < 0.05 versus GFP transfected control cells. B) Shown are representative immunoblots for, FL-mGluR5a expression, p-ERK1/2 activity and total-ERK1/2 expression in HEK 293 cells transiently transfected with 3 μg of pcDNA3.1 encoding, FL-mGluR5a along with 0.5 μg of plasmid cDNA encoding either GFP, GFP-CaMKIIα or GFP-CaMKIIα-T286A in response to 50 μM quisqualate treatment for 0, 5 and 10 min. Bar graph shows the densitometric analysis of ERK1/2 phosphorylation normalized to both basal total ERK1/2 protein expression. Data represents the mean ± SD of five independent experiments. *P < 0.05 versus GFP transfected control cells.

Mentions: Group I mGluRs couple to the activation of ERK1/2 phosphorylation via a number of different mechanisms, that include Gαq/11-mediated activation of Ca2+ signaling leading to the activation of both protein kinase C and Src, as well as their interaction with both Pyk2 and Homer [22,40]. Therefore, we examined whether over-expression of either CaMKIIα or an autophosphorylation incompetent CaMKIIα-T286A mutant altered either FL-mGluR1a- or FL-mGluR5a-stimulated ERK1/2 phosphorylation. We found that neither GFP-CaMKIIα nor CaMKIIα-T286A over-expression affected FL-mGluR1a-stimulated ERK1/2 phosphorylation in response to 50 μM quisqualate stimulation for either 5 or 10 min (Figure 4A). In contrast, CaMKIIα over-expression significantly attenuated FL-mGluR5a stimulated ERK1/2 phosphorylation in response to 5 and 10 min treatment with 50 μM quisqualate, whereas the expression of CaMKIIα-T286A mutant had no effect on quisqualate stimulated ERK1/2 phosphorylation in FL-mGluR5a expressing cells (Figure 4B).Figure 4


Ca(2+)/calmodulin-dependent protein kinase II interacts with group I metabotropic glutamate and facilitates receptor endocytosis and ERK1/2 signaling: role of β-amyloid.

Raka F, Di Sebastiano AR, Kulhawy SC, Ribeiro FM, Godin CM, Caetano FA, Angers S, Ferguson SS - Mol Brain (2015)

Effect of CaMKIIα overexpression on mGluR1a- and mGluR5a- -stimulated ERK1/2 phosphorylation. A) Shown are representative immunoblots for FL-mGluR1a expression, p-ERK1/2 activity and total-ERK1/2 expression in HEK 293 cells transiently transfected with 3 μg of pcDNA3.1 encoding FLAG-mGluR1a along with 0.5 μg of plasmid cDNA encoding either GFP, GFP-CaMKIIα or GFP-CaMKIIα-T286A in response to 50 μM quisqualate treatment for 0, 5 and 10 min. Bar graph shows the densitometric analysis of ERK1/2 phosphorylation normalized to both basal and total ERK1/2 protein expression. Data represents the mean ± SD of five independent experiments. *P < 0.05 versus GFP transfected control cells. B) Shown are representative immunoblots for, FL-mGluR5a expression, p-ERK1/2 activity and total-ERK1/2 expression in HEK 293 cells transiently transfected with 3 μg of pcDNA3.1 encoding, FL-mGluR5a along with 0.5 μg of plasmid cDNA encoding either GFP, GFP-CaMKIIα or GFP-CaMKIIα-T286A in response to 50 μM quisqualate treatment for 0, 5 and 10 min. Bar graph shows the densitometric analysis of ERK1/2 phosphorylation normalized to both basal total ERK1/2 protein expression. Data represents the mean ± SD of five independent experiments. *P < 0.05 versus GFP transfected control cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Fig4: Effect of CaMKIIα overexpression on mGluR1a- and mGluR5a- -stimulated ERK1/2 phosphorylation. A) Shown are representative immunoblots for FL-mGluR1a expression, p-ERK1/2 activity and total-ERK1/2 expression in HEK 293 cells transiently transfected with 3 μg of pcDNA3.1 encoding FLAG-mGluR1a along with 0.5 μg of plasmid cDNA encoding either GFP, GFP-CaMKIIα or GFP-CaMKIIα-T286A in response to 50 μM quisqualate treatment for 0, 5 and 10 min. Bar graph shows the densitometric analysis of ERK1/2 phosphorylation normalized to both basal and total ERK1/2 protein expression. Data represents the mean ± SD of five independent experiments. *P < 0.05 versus GFP transfected control cells. B) Shown are representative immunoblots for, FL-mGluR5a expression, p-ERK1/2 activity and total-ERK1/2 expression in HEK 293 cells transiently transfected with 3 μg of pcDNA3.1 encoding, FL-mGluR5a along with 0.5 μg of plasmid cDNA encoding either GFP, GFP-CaMKIIα or GFP-CaMKIIα-T286A in response to 50 μM quisqualate treatment for 0, 5 and 10 min. Bar graph shows the densitometric analysis of ERK1/2 phosphorylation normalized to both basal total ERK1/2 protein expression. Data represents the mean ± SD of five independent experiments. *P < 0.05 versus GFP transfected control cells.
Mentions: Group I mGluRs couple to the activation of ERK1/2 phosphorylation via a number of different mechanisms, that include Gαq/11-mediated activation of Ca2+ signaling leading to the activation of both protein kinase C and Src, as well as their interaction with both Pyk2 and Homer [22,40]. Therefore, we examined whether over-expression of either CaMKIIα or an autophosphorylation incompetent CaMKIIα-T286A mutant altered either FL-mGluR1a- or FL-mGluR5a-stimulated ERK1/2 phosphorylation. We found that neither GFP-CaMKIIα nor CaMKIIα-T286A over-expression affected FL-mGluR1a-stimulated ERK1/2 phosphorylation in response to 50 μM quisqualate stimulation for either 5 or 10 min (Figure 4A). In contrast, CaMKIIα over-expression significantly attenuated FL-mGluR5a stimulated ERK1/2 phosphorylation in response to 5 and 10 min treatment with 50 μM quisqualate, whereas the expression of CaMKIIα-T286A mutant had no effect on quisqualate stimulated ERK1/2 phosphorylation in FL-mGluR5a expressing cells (Figure 4B).Figure 4

Bottom Line: The expression of both mGluR5a and PrP(C) together, but not alone resulted in the agonist-stimulated subcellular distribution of CaMKIIα into cytoplasmic puncta.Taken together these results indicate that CaMKIIα selectively regulates mGluR1a and mGluR5a ERK1/2 signaling.As mGluR5 and CaMKIIα are involved in learning and memory and Aβ and mGluR5 are implicated in Alzheimer's disease, results of these studies could provide insight into potential pharmacological targets for treatment of Alzheimer's disease.

View Article: PubMed Central - PubMed

Affiliation: J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute, and the Department of Physiology and Pharmacology, University of Western Ontario, 100 Perth Dr. London, Ontario, N6A 5K8, Canada. fitoreraka@gmail.com.

ABSTRACT

Background: Agonist stimulation of Group I metabotropic glutamate receptors (mGluRs) initiates their coupling to the heterotrimeric G protein, Gαq/11, resulting in the activation of phospholipase C, the release of Ca(2+) from intracellular stores and the subsequent activation of protein kinase C. However, it is now recognized that mGluR5a also functions as a receptor for cellular prion protein (PrP(C)) and β-amyloid peptide (Aβ42) oligomers to facilitate intracellular signaling via the resulting protein complex. Intracellular mGluR5a signaling is also regulated by its association with a wide variety of intracellular regulation proteins.

Results: In the present study, we utilized mass spectroscopy to identify calmodulin kinase IIα (CaMKIIα) as a protein that interacts with the second intracellular loop domain of mGluR5. We show that CaMKIIα interacts with both mGluR1a and mGluR5a in an agonist-independent manner and is co-immunoprecipitated with mGluR5a from hippocampal mouse brain. CaMKIIα positively regulates both mGluR1a and mGluR5a endocytosis, but selectively attenuates mGluR5a but not mGluR1a-stimulated ERK1/2 phosphorylation in a kinase activity-dependent manner. We also find that Aβ42 oligomers stimulate the association of CaMKIIα with mGluR5a and activate ERK1/2 in an mGluR5a-dependent manner. However, Aβ42 oligomer-stimulated ERK1/2 phosphorylation is not regulated by mGluR5a/CaMKIIα interactions suggesting that agonist and Aβ42 oligomers stabilize distinct mGluR5a activation states that are differentially regulated by CaMKIIα. The expression of both mGluR5a and PrP(C) together, but not alone resulted in the agonist-stimulated subcellular distribution of CaMKIIα into cytoplasmic puncta.

Conclusions: Taken together these results indicate that CaMKIIα selectively regulates mGluR1a and mGluR5a ERK1/2 signaling. As mGluR5 and CaMKIIα are involved in learning and memory and Aβ and mGluR5 are implicated in Alzheimer's disease, results of these studies could provide insight into potential pharmacological targets for treatment of Alzheimer's disease.

Show MeSH
Related in: MedlinePlus