Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes.
The activation of phosphatidylinositol 3-kinase (PI 3-kinase) and production of PtdIns(3,4,5)P(3) 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)P(3) 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)P(3), were fused to green fluorescent protein and used to examine the subcellular localization of PtdIns(3,4,5)P(3) generation in living 3T3-L1 adipocytes. PtdIns(3,4,5)P(3) 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)P(3) 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; Gln(67)-->Leu) to cause glucose transporter GLUT4 translocation suggests that activation of this pathway is not sufficient to cause GLUT4 translocation.
Oatey
P B
PB
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, U.K.
Venkateswarlu
K
K
Williams
A G
AG
Fletcher
L M
LM
Foulstone
E J
EJ
Cullen
P J
PJ
Tavaré
J M
JM
D016475:3T3 Cells; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics; D017667:Adipocytes / Q000187:drug effects*; D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D011232:Chemical Precipitation; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism; D051275:Glucose Transporter Type 4; D049452:Green Fluorescent Proteins; D007328:Insulin / Q000494:pharmacology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D018129:Phosphatidylinositol Phosphates / Q000302:isolation & purification*; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000089982:ADP-Ribosylation Factor 6; D020690:GTPase-Activating Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D008164:Luminescent Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D018129:Phosphatidylinositol Phosphates; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; C495693:Slc2a4 protein, mouse; C102474:cytohesin-2; C060974:phosphatidylinositol 3,4,5-triphosphate; C040985:phosphatidylinositol receptors; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020727:ADP-Ribosylation Factors; C000715727:Arf6 protein, mouse