Distinct Pathways of Gi- and Gq-mediated Mitogen-activated Protein Kinase Activation

Abstract

Receptors that couple to the heterotrimeric G proteins, Gi or Gq, can stimulate phosphoinositide (PI) hydrolysis and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC), which can stimulate increased MAPK activity. However, the relationship between PI hydrolysis and MAPK activation in Gi and Gq signaling has not been clearly defined and is the subject of this study. The effects of several signaling inhibitors are assessed including expression of a peptide derived from the carboxyl terminus of the beta adrenergic receptor kinase 1 (beta ARKct), which specifically blocks signaling mediated by the beta gamma subunits of G proteins (G beta gamma), expression of dominant negative mutants of p21ras (RasN17) and p74raf-1 (N delta Raf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC. The Gi-coupled alpha 2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of beta ARKct, RasN17, or N delta Raf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the Gq-coupled alpha 1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of beta ARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of N delta Raf or by PKC depletion. These data demonstrate that Gi- and Gq-coupled receptors stimulate MAPK activation via distinct signaling pathways. G beta gamma is responsible for mediating Gi-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, G alpha mediates Gq-coupled receptor-stimulated MAPK activation using a p21ras-independent mechanism employing PKC and p74raf. To define the role of G beta gamma in Gi-coupled receptor-mediated PI hydrolysis and MAPK activation, direct stimulation with G beta gamma was used. Expression of G beta gamma resulted in MAPK activation that was sensitive to inhibition by expression of beta ARKct, RasN17, or N delta Raf or by PTK inhibitors, but insensitive to PKC depletion. By comparison, G beta gamma-mediated PI hydrolysis was not affected by beta ARKct, RasN17, or N delta Raf expression or by PTK inhibitors. Together, these results demonstrate that G beta gamma mediates MAPK activation and PI hydrolysis via independent signaling pathways.

Regulation of cell proliferation by G proteins

“…The resultant activation of Ras can lead to the activation of ERK though Raf-MEK pathway (Rocca et al, 1997). This is consistent with the observation that the bg-activated ERK is not sensitive to PKC-inhibitors but sensitive to Ras, Raf and tyrosine kinase inhibitors (Hawes et al, 1995). Gbg-activation of PLC can also lead to an increase in DAGs and subsequently to the activation of PKC.…”

“…Results from these studies indicated that the expression of the Gb1g1, Gb1g2, Gb1g3, and Gb2g2-configurations increased the basal levels of ERK by 6 ± 10-folds while the con®gurations involving Gb3 or Gb4 failed to activate ERK. Similarly, only the Gb1g1, Gb1g2, Gb1g3, and Gb2g2 con®gurations stimulated 3 ± 4-fold increase in PI hydrolysis suggesting their activation of PI-PLC pathways (Hawes et al, 1995). Coexpression of a dominant negative mutant of Ras along with the Gbg completely inhibited the activation of ERK without having any e ect on PLC activity.…”

“…Lefkowitz and co-workers have characterized the ability of di erent con®gurations of bg-subunits to activate ERK in COS-7 cells (Hawes et al, 1995). Results from these studies indicated that the expression of the Gb1g1, Gb1g2, Gb1g3, and Gb2g2-configurations increased the basal levels of ERK by 6 ± 10-folds while the con®gurations involving Gb3 or Gb4 failed to activate ERK.…”

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).…”

“…Gi or Go) [90,91] or pertussis toxininsensitive (i.e. Gq) [92] G-protein-coupled pathways, which are dependent on the activation of either PKC [50,92] or Ras proteins [90,91,93]. 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.…”