Depolarization-induced, glutamate receptor-mediated, and transactivation-dependent extracellular-signal regulated kinase phosphorylation in cultured cerebellar granule neurons.

Depolarization of 7-8-day-old mouse cerebellar granule neurons in primary cultures, a glutamatergic preparation, by elevation of the extracellular potassium ion concentration ([K+]e) to 45 mM induces an increase of phosphorylation of extracellular-signal regulated kinase 1 and 2 (ERK1/2) at two time periods: 20 min and 60 min after the [K+]e increase. This effect can be mimicked by 5 min of exposure to 50 microM glutamate, suggesting that ERK1/2 phosphorylation in response to the depolarization is brought about by the resulting glutamate release. This concept is supported by the observation that the K+ -mediated stimulation of phosphorylation at both times is inhibited by MK-801, an NMDA antagonist, and by CNQX, an AMPA/kainate antagonist. These antagonists also inhibit the response to glutamate. Both increases in ERK1/2 phosphorylation are also inhibited by GM 6001 (a metalloproteinase inhibitor, preventing 'shedding' of growth factors), by AG 1478 (a receptor tyrosine kinase inhibitor, preventing epidermal growth factor [EGF] receptor activation), and also partly by heparin (inactivating heparin-binding epidermal growth factor [HB-EGF]), suggesting transactivation of epidermal growth factor receptors (EGFR). Transactivation is an intracellular/extracellular signal transduction pathway in which release from receptor- or depolarization-stimulated cells of EGFR ligand(s) (including HB-EGF), catalyzed by a metalloproteinase, stimulates receptor tyrosine kinases on the same (an autocrine effect) or adjacent (a paracrine effect) cells. The expression of HB-EGF as well as of transforming growth factor-alpha (TGF-alpha), two of the EGFR ligands, in the cells was confirmed by reverse transcription polymerase chain reaction, and the only partial inhibition by heparin suggests that both of these EGFR agonists are involved. Such a transactivation may play a major role in glutamate-mediated signaling and plasticity.