Abstract

5-Hydroxytryptamine (5-HT)_2A receptors have been implicated to play a role in both the treatment and pathophysiology of a number of psychiatric disorders. Therefore, the coupling of this receptor to signals, such as extracellular signal-regulated kinase (ERK), that elicit long-term neuronal changes may be relevant. In the present study we examined the coupling of the G_q-coupled receptor to ERK in PC12 cells, a cell line commonly used as a neuronal model system. Activation of ERK occurred through a pathway different than the protein kinase C-dependent pathways described previously in studies of non-neuronal cells. Activation of ERK, in PC12 cells, was inhibited by both chelation of extracellular Ca²⁺ and by depletion of intracellular Ca²⁺ stores. Surprisingly, activation was not inhibited, but actually potentiated, by a variety of protein kinase C inhibitors covering all known protein kinase C isoforms. In contrast, the coupling of receptor to activation of ERK was found to be sensitive to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7) andN-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide (W13), inhibitors of calmodulin, but not to 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-l-tyrosyl)-4-phenylpiperazine (KN62) and 2-[N-(2-hydroxyethyl)]-N-4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN93), inhibitors of calmodulin-dependent protein kinase. Additionally, the general tyrosine kinase inhibitor genistein, as well as the Src inhibitor PP1 and the epidermal growth factor receptor kinase inhibitor 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG 1478), inhibited receptor-mediated activation of ERK, suggesting a role for tyrosine kinases. In fact, 5-HT was found to stimulate tyrosine phosphorylation of a number of proteins, and this phosphorylation was inhibited by W7. 5-HT_2A receptor-activation of ERK through a protein kinase C-independent pathway requiring Ca²⁺/calmodulin/tyrosine kinases represents a pathway distinct from those described in studies of non-neuronal cells.