Interaction of phosphoinositolglycan(-peptides) with plasma membrane lipid rafts of rat adipocytes.
Insulin receptor-independent activation of the insulin signal transduction cascade in insulin-responsive target cells by phosphoinositolglycans (PIG) and PIG-peptides (PIG-P) is accompanied by redistribution of glycosylphosphatidylinositol (GPI)-anchored plasma membrane proteins (GPI proteins) and dually acylated nonreceptor tyrosine kinases from detergent/carbonate-resistant glycolipid-enriched plasma membrane raft domains of high-cholesterol content (hcDIGs) to rafts of lower cholesterol content (lcDIGs). Here we studied the nature and localization of the primary target of PIG(-P) in isolated rat adipocytes. Radiolabeled PIG-P (Tyr-Cys-Asn-NH-(CH(2))(2)-O-PO(OH)O-6Manalpha1(Manalpha1-2)-2Manalpha1-6Manalpha1-4GluN1-6Ino-1,2-(cyclic)-phosphate) prepared by chemical synthesis or a radiolabeled lipolytically cleaved GPI protein from Saccharomyces cerevisiae, which harbors the PIG-P moiety, bind to isolated hcDIGs but not to lcDIGs. Binding is saturable and abolished by pretreatment of intact adipocytes with trypsin followed by NaCl or with N-ethylmaleimide, indicating specific interaction of PIG-P with a cell surface protein. A 115-kDa polypeptide released from the cell surface by the trypsin/NaCl-treatment is labeled by [(14)C]N-ethylmaleimide. The labeling is diminished upon incubation of adipocytes with PIG-P which can be explained by direct binding of PIG-P to the 115-kDa protein and concomitant loss of its accessibility to N-ethylmaleimide. Binding of PIG-P to hcDIGs is considerably increased after pretreatment of adipocytes with (glycosyl)phosphatidylinositol-specific phospholipases compatible with lipolytic removal of endogenous ligands, such as GPI proteins/lipids. These data demonstrate that in rat adipocytes synthetic PIG(-P) as well as lipolytically cleaved GPI proteins interact specifically with hcDIGs. The interaction depends on the presence of a trypsin/NaCl/NEM-sensitive 115-kDa protein located at hcDIGs which thus represents a candidate for a binding protein for exogenous insulin-mimetic PIG(-P) and possibly endogenous GPI proteins/lipids.
Müller
Günter
G
Aventis Pharma Germany, DG Metabolic Diseases, Industrial Park Höchst, Bldg. H825, 65926, Frankfurt am Main, Germany. Guenter.Mueller@aventis.com
Hanekop
Nils
N
Kramer
Werner
W
Bandlow
Wolfhard
W
Frick
Wendelin
W
D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004822:Epididymis; D005033:Ethylmaleimide / Q000494:pharmacology; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007328:Insulin / Q000502:physiology; D007700:Kinetics; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D015398:Signal Transduction; D014357:Trypsin / Q000494:pharmacology
D017261:Glycosylphosphatidylinositols; D007328:Insulin; D009842:Oligopeptides; D014357:Trypsin; D005033:Ethylmaleimide