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Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay
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Specific role for p85/p110beta in GTP-binding-protein-mediated activation of Akt

Hiroshi Kubo1, Kaoru Hazeki, Shunsuke Takasuga

  • 1Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan.

The Biochemical Journal
|August 11, 2005
PubMed

Insights

Insulin receptor (IR) and A1 adenosine receptor (A1R) signaling in Chinese hamster ovary cells reveal that p110beta is crucial for A1R-mediated Akt activation, independent of p110alpha. This highlights p110beta

Area of Science:

  • Cellular signaling pathways
  • Molecular biology
  • Receptor pharmacology

Background:

  • Chinese hamster ovary (CHO) cells co-expressing insulin receptor (IR) and A1 adenosine receptor (A1R) were utilized.
  • Both insulin and N6-(2-phenylisopropyl)adenosine (PIA), an A1R agonist, activate Akt via phosphoinositide 3-kinase (PI3K) in these cells.

Purpose of the Study:

  • To elucidate the specific roles of PI3K isoforms, p110alpha and p110beta, in mediating Akt activation downstream of IR and A1R.
  • To investigate the molecular mechanisms underlying A1R-specific signaling.

Main Methods:

  • Gene silencing using short hairpin RNA (shRNA) targeting p110alpha and p110beta.
  • Overexpression of specific PI3K subunits and mutated proteins.
  • Assessment of Akt activity following receptor stimulation.

Main Results:

  • Overexpression of p110beta enhanced PIA-induced Akt activation, while p110alpha overexpression had minimal effect on insulin signaling.
  • Silencing p110beta abolished PIA-induced Akt activation, whereas silencing p110alpha did not impair PIA's effects.
  • PIA-induced signaling in p110alpha-deficient cells was dependent on tyrosine-phosphorylated proteins and Gbetagamma subunits, suggesting a role for p110beta in GPCR signal transduction.

Conclusions:

  • p110beta is essential for A1R-mediated Akt activation, functioning distinctly from p110alpha.
  • Tyrosine-phosphorylated proteins and Gbetagamma subunits are critical for p110beta's function in intact cells.
  • The middle region of p110beta may be involved in receiving signals from G-protein-coupled receptors (GPCRs).

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