Epidermal growth factor-mediated signaling of G(i)-protein to activation of phospholipases in rat-cultured hepatocytes.
Hepatocytes were established in tissue culture in order to study the effects of pertussis toxin (PT) on epidermal growth factor (EGF)-mediated cellular responses under in vitro conditions. EGF caused a 3-fold increase of myo-inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) mass and a 50% increase of diacylglycerol mass within the first minute, with the change of diacylglycerol content being 100-fold greater than that of Ins-1,4,5-P3. Diacylglycerol, but not Ins-1,4,5-P3, continued to accumulate over several hours, indicating that EGF increased the hydrolysis of lipids other than phosphatidylinositol 4,5-bisphosphate (PIP2). EGF increased phosphoinositide-specific phospholipase C-gamma (PLC-gamma) tyrosine phosphorylation within 1 min, but no effect was observed with vasopressin, insulin, or glucagon after 5 min. EGF also caused a rapid, tyrosine kinase-dependent association of G(i) alpha with PLC-gamma, which was maximal within 10 min. In contrast to our previous data on fresh hepatocytes, PT had no effect on the EGF-induced tyrosine phosphorylation of PLC-gamma, although Ins-1,4,5-P3 and diacylglycerol production were inhibited. The role of G-proteins in EGF signaling was investigated further by microinjection of G alpha antibodies into single fura-2-loaded hepatocytes. Anti-G(i) alpha (common) antibodies prevented EGF-induced but not vasopressin-induced Ca2+ transients. These results strengthen previous observations that a PT-sensitive G-protein is involved in EGF-mediated phospholipid metabolism in hepatocytes and show that tyrosine phosphorylation of PLC-gamma is an insufficient signal for activation of PIP2 hydrolysis.
Yang
L
L
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104.
Camoratto
A M
AM
Baffy
G
G
Raj
S
S
Manning
D R
DR
Williamson
J R
JR
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D037342:Pertussis Toxin; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000906:Antibodies; D004075:Diglycerides; D007527:Isoenzymes; D009842:Oligopeptides; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D004815:Epidermal Growth Factor; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D002118:Calcium