Activation of a Mitogen-activated Protein Kinase (ERK2) by the 5-Hydroxytryptamine1A Receptor Is Sensitive Not Only to Inhibitors of Phosphatidylinositol 3-Kinase, but to an Inhibitor of Phosphatidylcholine Hydrolysis

Abstract

A variety of receptors coupled to GTP-binding regulatory proteins (G proteins) initiate signals that culminate in activation of the mitogen-activated protein kinases ERK1 and ERK2. We demonstrate here that the human 5-HT1A receptor expressed in Chinese hamster ovary cells similarly promotes activation of ERK1 and ERK2, but that the pathway used does not conform entirely to those proposed previously for G protein-coupled receptors. Activation of ERK2 by the 5-HT1A receptor-selective agonist 8-hydroxy-N,N-dipropyl-2-aminotetralin hydrobromide (8-OH-DPAT) was inhibited completely by pertussis toxin and substantially by prolonged treatment of cells with phorbol 12-myristate 13-acetate. The implied requirement for protein kinase C, however, was negated in studies with bisindolylmaleimide and Ro-31-8220, which, although completely inhibiting activation of ERK2 by phorbol ester, had no impact on activation by 8-OH-DPAT. The anticipated inhibition by the tyrosine kinase inhibitors genistein and herbimycin A, moreover, was marginal at best. As expected for a Gi-coupled receptor, the inhibitors of phosphatidylinositol 3-kinase wortmannin and LY294002 inhibited activation of ERK2, albeit only partly (70%). Of significance, an inhibitor of a phosphatidylcholine-specific phospholipase C, tricyclodecan-9-yl-xanthogenate (D609), caused a similar degree of inhibition. When the two types of inhibitors were combined, an almost complete inhibition was achieved. Our data suggest that phosphatidylinositol 3-kinase and phosphatidylcholine-specific phospholipase C represent components of different, but partly overlapping pathways that can account almost entirely for the activation of ERK2 by the 5-HT1A receptor.

Rapid activation of sodium–proton exchange and extracellular signal-regulated protein kinase in fibroblasts by G protein-coupled 5-HT1A receptor involves distinct signalling cascades

“…Although our results do not clarify which of those mechanisms is operant, they do provide further evidence for differential regulation of ERK and NHE by the 5-HT "A receptor. The current studies extend a previous study [28] showing that the 5-HT "A receptor activates ERK through a pathway that is partially sensitive to $ 200 µM D609, a putative specific inhibitor of PC-PLC [55], by showing that D609 also effectively blocks activation of NHE by the 5-HT "A receptor. Although the specificity of D609 is far from established, the remarkably different concentration-dependency and efficacy of D609 to inhibit both processes suggests that the pathways are not identical.…”

“…The relatively selective nature of the coupling of this receptor to G proteins would reduce potential ambiguities that might result from a more complex pattern of G protein coupling. This receptor has also been shown to stimulate both ERK [28,29] and NHE activities [30], and in the main, those data are consistent with a mutual regulatory pathway for NHE and ERK. However, it has been noted that rapid activation of ERK, but not of NHE, is sensitive to sequestration of G protein βγ-subunits, suggesting that ERK might not always be a proximal regulator of NHE in CHO cells over a short-time frame [29,30].…”

“…In particular, much work has focused upon the products of phospholipase C (PLC) and phosphoinositide-3h-kinase (PI-3K). Specifically for the 5-HT "A receptor expressed in CHO cells, evidence for the involvement of a phosphatidylcholine specific PLC (PC-PLC) [28] and a PI-3K [28,29] in the activation of ERK has been presented. Therefore, we studied the involvement of those enzymes in the activation of NHE activity.…”

Calcium/Calmodulin-dependent Protein Kinase II Binds to Raf-1 and Modulates Integrin-stimulated ERK Activation

“…Activation of PKC contributes to Raf/Erk-1/2 activation in response to cyclic pressure-induced strain in endothelial cells (36). Ras-mediated activation of Erk-1/2, either by insulin, insulin like-growth factor I, interleukin-8 and -2, platelet-derived growth factor, vasopressin, T cell receptor, and adrenergic receptors is increased by Raf-1 co-activation by PI3-K (37)(38)(39)(40)(41)(42)(43). Although a direct effect of CaM on Raf-1 activity has been proposed in response to epidermal growth factor receptor activation, a direct CaMKII/Raf-1 interaction has not yet been described (21,22,44).…”

Separation of integrin-dependent adhesion from morphological changes based on differential PLC specificities

“…Therefore, the involvement of PLC in morphological changes was investigated with the use of the putative PI-PLC inhibitor NEO and PC-PLC inhibitor D609 [21][22][23][24][25][26][27][28]. Figure 2 shows digital images of untreated ( Fig.…”