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

Daniel S Cowen,Rebecca S Sowers,David R Manning

doi:10.1074/jbc.271.37.22297

Daniel S Cowen, Rebecca S Sowers + Show 1 more

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https://doi.org/10.1074/jbc.271.37.22297

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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.

Highlights

Perhaps the most detailed models of activation of mitogenactivated protein kinases (MAP kinases)1 such as ERK1 and ERK2 by receptors coupled to GTP-binding regulatory proteins (G proteins) are those provided by Lefkowitz and colleagues, in which the scheme of events depends on the G protein utilized
Conflicts exist as to whether inhibitors of tyrosine kinases necessarily inhibit activation of Ras and/or MAP kinases achieved through Gi [5], an issue that may devolve to the nature of the cells and agonists employed
Daub et al [10], for example, provide evidence for a scheme wherein the stimulation of MAP kinases in fibroblasts by endothelin, lysophosphatidic acid, and thrombin occurs through G protein-coupled receptors, but with epidermal growth factor (EGF) receptors used as an intermediate in the activation cascade

Summary

Introduction

Perhaps the most detailed models of activation of mitogenactivated protein kinases (MAP kinases)1 such as ERK1 and ERK2 by receptors coupled to GTP-binding regulatory proteins (G proteins) are those provided by Lefkowitz and colleagues, in which the scheme of events depends on the G protein utilized. Conflicts exist as to whether inhibitors of tyrosine kinases necessarily inhibit activation of Ras and/or MAP kinases achieved through Gi [5], an issue that may devolve to the nature of the cells and agonists employed. In the studies described here, the human 5-hydroxytryptophan1A (5-HT1A) receptor was employed to investigate further the pathways leading to activation of MAP kinases by G protein-coupled receptors.

Results

Conclusion