Membrane association of lipoprotein lipase and a cAMP-binding ectoprotein in rat adipocytes.
cAMP-binding ectoprotein (Gce1) and lipoprotein lipase (LPL) are anchored to plasma membranes of rat adipocytes by glycosylphosphatidylinositol (GPI) moieties as demonstrated by cleavage by bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), reactivity with anti-crossreacting determinant antibodies (anti-CRD), and metabolic labeling with radiolabeled palmitic acid and myo-inositol. Quantitative release from the membrane of LPL and Gce1 requires both lipolytic removal of their GPI anchors and the presence of either 2 M NaCl or 1 mM inositol 1,2-cyclic monophosphate or inositol 1-monophosphate. PI-PLC-cleaved and released LPL or Gce1 reassociates with isolated plasma membranes of rat adipocytes and, less efficiently, with membranes of 3T3 fibroblasts. The specificity of the reassociation is demonstrated (i) by its inhibition after pretreatment of the membranes with trypsin, (ii) by its competition with inositol 1,2-cyclic monophosphate and inositol 1-monophosphate in a concentration-dependent manner, and (iii) by the limited number of binding sites. Enzymic or chemical removal as well as masking with anti-CRD antibodies of the terminal inositol (cyclic) monophosphate moiety of hydrophilic Gce1 and LPL significantly impairs the reassociation. These data suggest that in rat adipocytes GPI-proteins are not readily released from the cell surface upon lipolytic cleavage, but remain associated through a receptor which specifically recognizes the terminal inositol (cyclic) monophosphate epitope of the (G)PI-PLC-cleaved GPI moiety. This interaction may have implications for the regulated membrane release of GPI-proteins and for their possible internalization.
Müller
G
G
Hoechst Aktiengesellschaft Frankfurt am Main, SBU Metabolic Diseases H 825, Germany.
Wetekam
E M
EM
Jung
C
C
Bandlow
W
W
D016475:3T3 Cells / Q000378:metabolism; D017667:Adipocytes / Q000737:chemistry / Q000201:enzymology* / Q000648:ultrastructure; D000818:Animals; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D008071:Lipoprotein Lipase / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D009608:Nitrous Acid / Q000032:analysis; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D017208:Rats, Wistar
D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D008071:Lipoprotein Lipase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D009608:Nitrous Acid