Pyk2 uncouples metabotropic glutamate receptor G protein signaling but facilitates ERK1/2 activation.

Group I metabotropic glutamate receptors (mGluRs) are coupled via Galphaq/11 to the activation of phospholipase Cbeta, which hydrolyzes membrane phospholipids to form inositol 1,4,5 trisphosphate and diacylglycerol. This results in the release of Ca2+ from intracellular stores and the activation of protein kinase C. The activation of Group I mGluRs also results in ERK1/2 phosphorylation. We show here, that the proline-rich tyrosine kinase 2 (Pyk2) interacts with both mGluR1 and mGluR5 and is precipitated with both receptors from rat brain. Pyk2 also interacts with GST-fusion proteins corresponding to the second intracellular loop and the distal carboxyl-terminal tail domains of mGluR1a. Pyk2 colocalizes with mGluR1a at the plasma membrane in human embryonic kidney (HEK293) cells and with endogenous mGluR5 in cortical neurons. Pyk2 overexpression in HEK293 results in attenuated basal and agonist-stimulated inositol phosphate formation in mGluR1 expressing cells and involves a mechanism whereby Pyk2 displaces Galphaq/11 from the receptor. The activation of endogenous mGluR1 in primary mouse cortical neuron stimulates ERK1/2 phosphorylation. Treatments that prevent Pyk2 phosphorylation in cortical neurons, and the overexpression of Pyk2 dominant-negative and catalytically inactive Pyk2 mutants in HEK293 cells, prevent ERK1/2 phosphorylation. The Pyk2 mediated activation of ERK1/2 phosphorylation is also Src-, calmodulin- and protein kinase C-dependent. Our data reveal that Pyk2 couples the activation mGluRs to the mitogen-activated protein kinase pathway even though it attenuates mGluR1-dependent G protein signaling.

Metabotropic glutamate receptor-mediated cell signaling pathways are altered in a mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (Htt). Group I metabotropic glutamate receptors (mGluRs) are coupled to G(alphaq) and play an important role in neuronal survival. We have previously demonstrated that mGluRs interact with Htt. Here we used striatal neuronal primary cultures and acute striatal slices to demonstrate that mGluR-mediated signaling pathways are altered in a presymptomatic mouse model of HD (Hdh(Q111/Q111)), as compared to those of control mice (Hdh(Q20/Q20)). mGluR1/5-mediated inositol phosphate (InsP) formation is desensitized in striatal slices from Hdh(Q111/Q111) mice and this desensitization is PKC-mediated. Despite of decreased InsP formation, (S)-3,5-dihydroxylphenylglycine (DHPG)-mediated Ca(2+) release is higher in Hdh(Q111/Q111) than in Hdh(Q20/Q20) neurons. Furthermore, mGluR1/5-stimulated AKT and extracellular signal-regulated kinase (ERK) activation is altered in Hdh(Q111/Q111) mice. Basal AKT activation is higher in Hdh(Q111/Q111) neurons and this increase is mGluR5 dependent. Moreover, mGluR5 activation leads to higher levels of ERK activation in Hdh(Q111/Q111) than in Hdh(Q20/Q20) striatum. PKC inhibition not only brings Hdh(Q111/Q111) DHPG-stimulated InsP formation to Hdh(Q20/Q20) levels, but also causes an increase in neuronal cell death in Hdh(Q111/Q111) neurons. However, PKC inhibition does not modify neuronal cell death in Hdh(Q20/Q20) neurons, suggesting that PKC-mediated desensitization of mGluR1/5 in Hdh(Q111/Q111) mice might be protective in HD. Together, these data indicate that group I mGluR-mediated signaling pathways are altered in HD and that these cell signaling adaptations could be important for striatal neurons survival.

Phosphorylation-independent regulation of metabotropic glutamate receptor 5 desensitization and internalization by G protein-coupled receptor kinase 2 in neurons.

The uncoupling of metabotropic glutamate receptors (mGluRs) from heterotrimeric G proteins represents an essential feedback mechanism that protects neurons against receptor overstimulation that may ultimately result in damage. The desensitization of mGluR signaling is mediated by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). Unlike mGluR1, the attenuation of mGluR5 signaling in HEK 293 cells is reported to be mediated by a phosphorylation-dependent mechanism. However, the mechanisms regulating mGluR5 signaling and endocytosis in neurons have not been investigated. Here we show that a 2-fold overexpression of GRK2 leads to the attenuation of endogenous mGluR5-mediated inositol phosphate (InsP) formation in striatal neurons and siRNA knockdown of GRK2 expression leads to enhanced mGluR5-mediated InsP formation. Expression of a catalytically inactive GRK2-K220R mutant also effectively attenuates mGluR5 signaling, but the expression of a GRK2-D110A mutant devoid in Galpha(q/11) binding increases mGluR5 signaling in response to agonist stimulation. Taken together, these results indicate that the attenuation of mGluR5 responses in striatal neurons is phosphorylation-independent. In addition, we find that mGluR5 does not internalize in response to agonist treatment in striatal neuron, but is efficiently internalized in cortical neurons that have higher levels of endogenous GRK2 protein expression. When overexpressed in striatal neurons, GRK2 promotes agonist-stimulated mGluR5 internalization. Moreover, GRK2-mediated promotion of mGluR5 endocytosis does not require GRK2 catalytic activity. Thus, we provide evidence that GRK2 mediates phosphorylation-independent mGluR5 desensitization and internalization in neurons.