Ras-Dependent Activation of Fibroblast Mitogen-Activated Protein Kinase by 5-HT_1A Receptor via a G Protein βγ-Subunit-Initiated Pathway

Abstract

Serotonin (5-HT) is a potent mitogen in many cells types, an action which is frequently mediated through pertussis toxin-sensitive G proteins. In the current study, we used pharmacological inhibitors and dominant negative signaling constructs to delineate elements which participate in the activation of MAPK, a growth-associated mitogen-activated protein kinase, by human G protein-coupled 5-HT1A receptor transfected into CHO-K1 cells in a stable manner. The activation pathway does not directly involve phorbol ester-sensitive protein kinase C types, but does require (i) pertussis toxin-sensitive G protein beta gamma-subunits, (ii) a staurosporine- and genistein-sensitive protein kinase, (iii) phosphoinositide-3'-kinase activity, (iv) activation of Sos in a multimolecular complex that contains p46Shc, and p52Shc, and Grb2, (v) the GTPase p21Ras, and (vi) the protein kinase p74Raf-1. These data demonstrate that the 5-HT1A receptor mediates MAPK activity by convergence upon a common activation pathway that is shared with receptor tyrosine kinases.

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

“…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].…”

“…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]. Thus, the current studies were undertaken to establish whether ERK, when activated by the 5-HT "A receptor, is a rapid upstream regulator of NHE in CHO fibroblast cells.…”

“…"A receptors ($ 1 pmol of receptor\mg of protein) were obtained as previously described [29]. Cell culture supplies were obtained from Gibco-BRL (Grand Island, NY), the Comprehensive Cancer Center at Duke University, or Corning Costar (Cambridge, MA).…”

“…We recently showed that ERK activation in CHO cells by the 5-HT "A receptor does not depend upon phorbol ester-sensitive PKC types [29]. Because PKC types are frequently implicated in NHE regulation [1,2], we tested their involvement in our model.…”

Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma

“…EMALA et al [94], confirming the observations of others [75,82], recently showed in human cultured airway smooth muscle cells that activation of Ras by ET was blocked by pertussis toxin, suggesting that the receptors for ET, which activate Ras, couple Gi but not Gq. Furthermore, in the same study, overexpression of a G protein bc-subunit scavenger failed to prevent activation of Ras, indicating that ET receptors most probably couple to a Gi that activates Ras via the Gia subunit [94] and not a Gibc (or Gqbc) subunit as proposed in other cell types [92,95] (fig. 3).…”