Active EGF receptors have limited access to PtdIns(4,5)P(2) in endosomes: implications for phospholipase C and PI 3-kinase signaling.
Although prolonged cell signaling is attenuated by internalization and downregulation of active receptors, it is now appreciated that many receptors continue to signal in intracellular compartments. Employing enhanced green fluorescent protein fusion probes, we have investigated the hypothesis that multiple signaling pathways are affected by the differential trafficking of membrane substrates such as PtdIns(4,5)P(2). A phosphotyrosine-specific probe, but not a PtdIns(4,5)P(2)-specific probe, colocalized with internalized EGF as well as transferrin in EGF-stimulated living cells expressing autophosphorylation-competent EGF receptors. Neither probe colocalized with transferrin in the absence of EGF, demonstrating that the reduced level of accessible PtdIns(4,5)P(2) in endosomes is constitutive. Finally, a PtdIns(3,4,5)P(3)-specific probe, which monitors phosphorylation of PtdIns(4,5)P(2) by phosphoinositide 3-kinases, was recruited to the plasma membrane but not to EGF- or transferrin-containing endosomes in response to EGF stimulation. These results suggest that while many internalized receptors continue to engage intracellular enzymes, the phospholipase C and phosphoinositide 3-kinase signaling pathways are abrogated by the constitutive lack of accessible PtdIns(4,5)P(2) in endosomes.
Haugh
Jason M
JM
Department of Chemical Engineering, North Carolina State University, Raleigh, NC 27695-7905, USA. jason_haugh@ncsu.edu
Meyer
Tobias
T
D016475:3T3 Cells; D000818:Animals; D002451:Cell Compartmentation / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics; D014168:Transferrin / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D014168:Transferrin; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D010738:Type C Phospholipases