Metabotropic glutamate receptor 5-induced phosphorylation of extracellular signal-regulated kinase in astrocytes depends on transactivation of the epidermal growth factor receptor.

G-protein-coupled receptors (GPCRs) induce the phosphorylation of mitogen-activated protein (MAP) kinase by actions on any of a number of signal transduction systems. Previous studies have revealed that activation of the G(q)-coupled metabotropic glutamate receptor 5 (mGluR5) induces phosphorylation of the MAP kinase extracellular signal-regulated kinase 2 (ERK2) in cultured rat cortical astrocytes. We performed a series of studies to determine the mechanisms underlying mGluR5-induced phosphorylation of MAP kinase in these cells. Interestingly, our studies suggest that mGluR5-mediated ERK2 phosphorylation is dependent on the activation of G(alphaq) but is not mediated by the activation of phospholipase Cbeta1, activation of protein kinase C, or increases in intracellular calcium. Studies with peptide inhibitors suggest that this response is not dependent on G(betagamma) subunits. However, the activation of ERK2 was dependent on activation of the epidermal growth factor (EGF) receptor and activation of a Src family tyrosine kinase. Furthermore, activation of mGluR5 induced an association of this receptor and the EGF receptor, suggesting the formation of a signaling complex involved in the activation of ERK2. These data suggest that mGluR5 increases ERK2 phosphorylation in astrocytes by a novel mechanism involving the activation of G(alphaq) and both receptor and nonreceptor tyrosine kinases but that is independent of the activation of phospholipase Cbeta1.

Phosphorylation of mitogen-activated protein kinase in cultured rat cortical glia by stimulation of metabotropic glutamate receptors.

Activation of metabotropic glutamate receptors (mGluRs) in glia results in significant physiological effects for both the glia and the neighboring neurons; but in many cases, the mGluR subtypes and signal transduction mechanisms mediating these effects have not been determined. In this study, we report that mGluR activation in primary cultures of rat cortical glia results in tyrosine phosphorylation of several proteins, including p44/p42 mitogen-activated protein kinases, also referred to as extracellular signal-regulated kinases (ERK1/2). Incubation of glial cultures with the general mGluR agonist 1-aminocyclopentane-1S,3R-dicarboxylate and the mGluR group I-selective agonists (RS)-3,5-dihydroxyphenylglycine (DHPG) and L-quisqualate resulted in increased tyrosine phosphorylation of ERK1/2. The group II-selective agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine and group III-selective agonist L(+)-2-amino-4-phosphonobutyric acid had no effect on tyrosine phosphorylation. DHPG-induced ERK1/2 phosphorylation could be inhibited by an antagonist that acts at group I or group II mGluRs but not by antagonists for group II and group III mGluRs. Protein kinase C (PKC) activators also induced ERK1/2 phosphorylation, but the PKC inhibitor bisindolylmaleimide I did not inhibit DHPG-induced ERK1/2 phosphorylation at a concentration that inhibited the response to phorbol 12,13-dibutyrate. These data suggest that mGluR activation of ERK1/2 in cultured glia is mediated by group I mGluRs and that this effect is independent of PKC activation. Furthermore, immunoblots with antibodies against various mGluR subtypes show expression of mGluR5, but no other mGluRs in our cultures. Taken together, these results suggest that mGluR5 stimulation results in tyrosine phosphorylation of ERK1/2 and other glial proteins.

Coupling efficiencies of human alpha 1-adrenergic receptor subtypes: titration of receptor density and responsiveness with inducible and repressible expression vectors.

We compared the efficiencies with which human alpha 1-adrenergic receptor (AR) subtypes activate inositol phosphate (InsP) formation and increase intracellular Ca2+ in transfected cell lines. Expression of human alpha 1a-, alpha 1b-, and alpha 1d-AR cDNAs under the repressible control of anhydrotetracycline in human embryonic kidney (HEK) 293 cells, which normally express no alpha 1-ARs, was used to compare responses to norepinephrine (NE) at different receptor densities. Maximal NE-stimulated InsP formation was found to increase with increasing density of each subtype, whereas basal levels and responses to sodium fluoride did not change. A comparison of multiple subclones over equivalent ranges of receptor expression showed that activation of each subtype resulted in different maximal responses (alpha 1a > alpha 1b > alpha 1d) in HEK 293 cells. Analogous studies were carried out in human SK-N-MC cells, which normally express low levels of all three alpha 1-AR subtypes, using an isopropyl-beta-D-thiogalactoside-inducible expression system. Induction with isopropyl-beta-D-thiogalactoside increased the density of individual alpha 1-AR subtypes by 4-6-fold over the level of endogenous expression. Increased expression of each of these subtypes in SK-N-MC cells did not alter the EC50 value for NE in stimulating InsP formation or releasing [Ca2+]i but did increase maximal responses to NE. Similar to our findings in HEK 293 cells, a comparison of responses at similar expression levels in SK-N-MC cells showed different maximal responses stimulated by each subtype, for both InsP (alpha 1a > alpha 1b > or = alpha 1d) and [Ca2+]i (alpha 1a > alpha 1b > alpha 1d) responses. These studies show that agonist-occupied human alpha 1-AR subtypes have different efficiencies in activating phospholipase C in human cell lines. In both HEK 293 and SK-N-MC cells, alpha 1a-ARs couple most efficiently, whereas alpha 1d-ARs couple very poorly.