The activation of phosphatidylinositol 3-kinase (PI 3-kinase) and production of PtdIns(3,4,5)P3 is crucial in the actions of numerous extracellular stimuli, including insulin-stimulated glucose uptake. Platelet-derived growth factor (PDGF) also stimulates PI 3-kinase, but only weakly promotes glucose uptake when compared with insulin. Insulin and PDGF have thus been proposed to have differential effects on the subcellular targeting of PI 3-kinase. However, owing to a lack of suitable methodologies, the subcellular localization of the PtdIns(3,4,5)P3 generated has not been examined. The pleckstrin-homology (PH) domains of the nucleotide exchange factors, ADP-ribosylation factor nucleotide-binding-site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1), which have a high affinity and specificity for PtdIns(3,4,5)P3, were fused to green fluorescent protein and used to examine the subcellular localization of PtdIns(3,4,5)P3 generation in living 3T3-L1 adipocytes. PtdIns(3,4,5)P3 was produced almost exclusively in the plasma membrane in response to both agonists, although the response to insulin was greater in magnitude and occurred in considerably more cells. The results suggest that the greater ability of insulin to stimulate glucose uptake may be the result of its ability to generate significantly more plasma-membrane PtdIns(3,4,5)P3 than PDGF. ARNO and GRP1 are nucleotide exchange factors for the small GTP-binding protein ADP-ribosylation factor 6 (ARF6). The inability of a constitutively active GTPase-deficient mutant of ARF6 (ARF6-Q67L; Gln67 → Leu) to cause glucose transporter GLUT4 translocation suggests that activation of this pathway is not sufficient to cause GLUT4 translocation.
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December 1999
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Research Article|
November 24 1999
Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes
Paru B. OATEY;
Paru B. OATEY
1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, U.K.
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Kanamarlapudi VENKATESWARLU;
Kanamarlapudi VENKATESWARLU
1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, U.K.
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Alan G. WILLIAMS;
Alan G. WILLIAMS
1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, U.K.
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Laura M. FLETCHER;
Laura M. FLETCHER
1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, U.K.
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Emily J. FOULSTONE;
Emily J. FOULSTONE
1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, U.K.
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Peter J. CULLEN;
Peter J. CULLEN
1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, U.K.
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Jeremy M. TAVARÉ
Jeremy M. TAVARÉ
1
1Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, U.K.
1To whom correspondence should be addressed (e-mail j.tavare@bristol.ac.uk).
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Biochem J (1999) 344 (2): 511–518.
Article history
Received:
July 05 1999
Revision Received:
September 10 1999
Accepted:
September 23 1999
Citation
Paru B. OATEY, Kanamarlapudi VENKATESWARLU, Alan G. WILLIAMS, Laura M. FLETCHER, Emily J. FOULSTONE, Peter J. CULLEN, Jeremy M. TAVARÉ; Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes. Biochem J 1 December 1999; 344 (2): 511–518. doi: https://doi.org/10.1042/bj3440511
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