Abstract
G-protein-coupled receptors are thought to transmit extracellular signals to the cytoplasm from their position on the cell surface. Some receptors, including the metabotropic glutamate receptor 5 (mGluR5), are also highly expressed on intracellular membranes where they serve unknown functions. Here, we show that activation of cell surface versus intracellular mGluR5 results in unique Ca(2+) signatures leading to unique cellular responses. Specifically, activation of either cell surface or intracellular mGluR5 leads to JNK, Ca(2+)/calmodulin-dependent protein kinase (CaMK), and cyclic adenosine 3',5'-monophosphate-responsive element-binding protein phosphorylation, whereas activation of only intracellular mGluR5 leads to ERK1/2 and Elk-1 phosphorylation. Using pharmacological and genetic approaches, the present findings support a role for CaMK kinase in mediating mGluR5-dependent cyclic adenosine 3',5'-monophosphate-responsive element-binding protein phosphorylation, whereas CaMKII is upstream of intracellular mGluR5-mediated Elk-1 phosphorylation. Consistent with models showing Elk-1 regulating cascades of gene expression, the known Elk-1 targets c-fos and egr1 were up-regulated following intracellular mGluR5 activation, whereas a representative non-Elk-1 target, c-jun, was not. These findings emphasize that glutamate not only serves as a neurotransmitter for cell surface receptors but, when transported into the cell, can also activate intracellular receptors such as mGluR5. Glutamate activation of intracellular mGluR5 serves an important role in the regulation of nuclear Ca(2+), transcriptional activation, and gene expression necessary for physiological processes such as synaptic plasticity.
Highlights
An active role in neuronal excitability, synaptic transmission, as well as in various neurological and neurodevelopmental (Fragile X syndrome and autism) disorders, is found on endoplasmic reticulum and nuclear membranes [1,2,3]
What are the functional consequences of activating intracellular metabotropic glutamate receptor 5 (mGluR5)? In the nucleus, Ca2ϩ is generated by diffusion of cytosolic Ca2ϩ waves through nuclear pore complexes [6] or by release from the lumen of the nuclear envelope [7, 8]
Both pathways can lead to the phosphorylation of cyclic adenosine 3Ј,5Ј-monophosphate-responsive element-binding protein (CREB), which plays an important role in survival and plasticity
Summary
Materials—Drugs were as described previously [4, 5] or are listed in the supplemental material. Heterozygous mice were mated to produce wild type, heterozygous, and homozygous mGluR5-deficient pups that were individually genotyped by PCR using DNA prepared from cortical tissue. Transfected with vectors expressing dnCaMKK (K71A, T108A, and S458A), dnCaMKI (K49E, T177A, I286D/H287D/Q288E/S289E, and F307A), dnCaMKIV nuclear (T196A, K71E, and H305D/M306E/T308D), or dnCaMKII (K42M) fused to enhanced green fluorescent protein [14] or reporter gene constructs using. Tal neurons were fixed and stained as described previously [4]. Chromatin immunoprecipitation assays were performed largely as described previously [15] starting with ϳ5 ϫ 106 cells. Quantitative Reverse Transcriptase-PCR—Two-step quantitative reverse transcriptase-PCR was performed using procedures and sets of primers described in the supplemental material. Of target mRNAs were normalized to Gapdh mRNA and expressed as fold change compared with the untreated control
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