Effect of cholesterol depletion on mitogenesis and survival: the role of caveolar and noncaveolar domains in insulin-like growth factor-mediated cellular function.
The type 1 IGF receptor (IGF-IR) is thought to localize to a subset of lipid rafts, known as caveolae, but the impact on IGF signaling remains controversial. We investigated this potential regulatory mechanism by assessing IGF function in caveolae-positive (3T3L1 and NWTb3) and -negative (HepG2) cells. Coimmunoprecipitation studies demonstrated that IGF-IR and insulin receptor substrate 1 associated with caveolin, a caveolar marker, in 3T3L1 and NWTb3 cells. Subcellular fractionation showed that methyl-cyclodextrin, which disrupts lipid rafts by sequestration of cholesterol, disrupted the colocalization of caveolin and the IGF-IR at the plasma membrane. Methyl-cyclodextrin did not alter IGF-I-induced 3T3L1 or NWTb3 proliferation but significantly impaired the ability of IGF-I to protect these cells from apoptosis. Immunoblotting revealed that methyl-cyclodextrin had no effect on IGF-I-induced activation of the IGF-IR or insulin receptor substrate 1 but increased and decreased the phosphorylation of MAPK and protein kinase B, respectively. In caveolae-negative HepG2 cells, the effect of methyl-cyclodextrin on IGF signaling and cellular function was similar to that observed in caveolae-positive 3T3L1 and NWTb3 cells. Furthermore, transfecting caveolin into HepG2 cells to give morphologically identifiable caveolae made no difference to IGF action, despite a demonstrable interaction between caveolin and the IGF-IR. This suggests that although IGF-IR localizes to caveolin-rich subcellular fractions and coimmunoprecipitates with caveolin, caveolae may not be obligatory for IGF signaling.
Matthews
Laura C
LC
Endocrine Sciences, University of Manchester, UK.
Taggart
Michael J
MJ
Westwood
Melissa
M
D000818:Animals; D021941:Caveolae / Q000502:physiology*; D022461:Caveolins / Q000172:deficiency / Q000378:metabolism; D002460:Cell Line; D002468:Cell Physiological Phenomena; D002470:Cell Survival / Q000502:physiology; D002477:Cells / Q000166:cytology* / Q000378:metabolism; D002784:Cholesterol / Q000172:deficiency*; D006801:Humans; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D008938:Mitosis / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013002:Somatomedins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014018:Tissue Distribution; D047392:beta-Cyclodextrins / Q000494:pharmacology
D022461:Caveolins; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D010750:Phosphoproteins; D013002:Somatomedins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D017526:Receptor, IGF Type 1