Gi2-mediated activation of the MAP kinase cascade

Area of Science:

• Cellular signaling pathways
• G protein-coupled receptors
• Oncogenesis

Background:

• Heterotrimeric G proteins of the Gi class transmit signals from receptors.
• The alpha subunit of Gi2 (alpha i2) is oncogenic when constitutively active (gip2).
• Gi2's role in cellular proliferation is linked to the MAPK signaling cascade.

Purpose of the Study:

• Investigate the mechanism of Gi2 activation of the MAPK cascade.
• Utilize a pertussis toxin (PTX)-resistant alpha i2 mutant to specifically study Gi2 signaling.
• Determine the role of Ras and Gi2 subunits in MAPK activation.

Main Methods:

• Transient expression of PTX-resistant alpha i2 in Chinese hamster ovary (CHO) cells.
• PTX treatment to isolate Gi2 signaling.
• Analysis of MAPK and MEK activation.
• Expression of dominant-negative Ras to block signaling.
• Assessment of Ras GTP-bound levels.
• Investigation of beta gamma subunit and phosphatidylinositol-3 kinase involvement.

Main Results:

• Receptor-mediated activation of PTX-resistant Gi2 led to MAPK and MEK activation.
• Gi2-induced MAPK/MEK activation was blocked by dominant-negative Ras.
• Gi2 activation did not significantly increase GTP-bound Ras.
• Gi2 stimulates the MAPK pathway partly through beta gamma subunit release and phosphatidylinositol-3 kinase activation.

Conclusions:

• Gi2 G protein directly activates the MAPK signaling cascade.
• Ras is involved in Gi2-mediated MAPK activation, but not solely through increased GTP-bound Ras.
• Gi2 signaling to MAPK involves its beta gamma subunit and phosphatidylinositol-3 kinase.