Membrane Association of Lipoprotein Lipase and a cAMP-Binding Ectoprotein in Rat Adipocytes

Abstract

CAMP-binding ectoprotein (Gcel ) 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 myoinositol. Quantitative release from the membrane of LPL and Gcel 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 Gcel 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 Gcel 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-PLCcleaved GPI moiety. This interaction may have implications for the regulated membrane release of GPIproteins and for their possible internalization.GPI-proteins,' a subclass of ectoproteins in both lower and higher eucaryotes, are anchored to the outer leaflet of the plasma membrane by covalently attached GPI glycolipids which are characterized by a high degree of structural conservation from yeast to man. Their linear core structure is built from phosphatidylinositol bound via nonacetylated glucosamine to three mannose residues. The nonreducing end is linked to ethanolamine via a phosphodiester bridge. From this conserved core glycan various carbohydrate and/ or phosphoethanolamine residues may branch off [for a review see Ferguson and Williams (1988), Low (1989), and McConville and Ferguson (1993l. During the biosynthesis of GPI-proteins, the amino group of ethanolamine of the complete preformed GPI structure is coupled in a pseudo transpeptidation reaction to the carboxy terminus of the mature fully translated polypeptide chain, thereby replacing a transmembrane domain present in the protein precursor [for a review see Doering et al. (1990) and Thomas et al. (1990)l.Despite the lack of a cytoplasmic domain someGPI-proteins seem to have important roles in transmembrane signaling

Redistribution of Glycolipid Raft Domain Components Induces Insulin-Mimetic Signaling in Rat Adipocytes

“…The protein composition of DIG and non-DIG areas prepared by the carbonate method from purified plasma membranes or by the detergent method from total-cell lysates of untreated isolated rat adipocytes was analyzed by immunoblotting. The caveolar structural and marker protein, caveolin 1, the GPI proteins Gce1 (glycolipid-anchored cAMPbinding ectoprotein) and 5Ј-nucleotidase (39,43,46), and the dual-acylated NRTK of the Src-class, pp59…”

“…After stimulation and/or electroporation, rat adipocytes (5 ϫ 10 7 cells) were washed once with KRH containing 0.25 M sucrose and 2 mM sodium pyruvate by flotation (200 ϫ g, 2 min) and aspiration of the infranatant and were immediately homogenized in 10 ml of lysis buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.25 mM EGTA, 0.25 M sucrose, 50 mM NaF, 5 mM sodium pyrophosphate, 25 mM glycerol-3-phosphate, and 1 mM sodium orthovanadate, supplemented with protease inhibitors [10 g of leupeptin/ml, 2 M pepstatin, 10 g of aprotinin/ml, 5 M antipain, 5 mM iodoacetate, 100 M phenylmethylsulfonyl fluoride, 4 mM benzamidine]) by using a motor-driven Teflon-in-glass homogenizer (10 strokes with a loosely fitting pestle) at 22°C. The following procedures were performed at 4°C (43). After centrifugation (1,500 ϫ g, 5 min), the postnuclear infranatant was separated from the fat cake, and the pellet fraction (containing adipocyte ghosts and cell debris) was removed by suction with a needle.…”

“…Immune complex kinase assays were performed as described previously (17, 44) Miscellaneous. Phosphatidylinositol 3Ј-kinase (PI-3ЈK) assay, photoaffinity labeling of Gce1 with 8-N 3 -[ 32 P]cyclic AMP (cAMP), immunoprecipitation, and immunoblotting were performed as described previously (17,39,43,44). Protein concentration was determined using the bicinchoninic acid protein determination kit from Pierce (Rockford, Ill.) and BSA as a calibration standard.…”

Glucose-Induced Sequential Processing of a Glycosyl-Phosphatidylinositol-Anchored Ectoprotein in Saccharomyces cerevisiae

“…(b) The amount of radiolabeled hydrophilic Gce1p contained in each gel band was calculated as a percentage of the sum of the radiolabeled hydrophilic and amphiphilic material. s, total 8-N 3 -[ were isolated ([G]PI-PLC-cleaved Gce1p remains associated with the membrane through specific, noncovalent interactions and is dissociable only in the presence of high salt [37,44]) and subjected to TX-114 partitioning. The processed hydrophilic fraction of Gce1p was isolated from the aqueous phase and, in the case of metabolically labeled samples, partially purified by affinity chromatography on cAMP-Sepharose.…”

“…16,1996 GLYCOSYL-PHOSPHATIDYLINOSITOL PROCESSING IN YEAST CELLS 453 based on two principal properties: (i) the ability of GPI-anchored proteins to associate in clusters together with glycosphingolipids and certain transmembrane and long-chain acylated proteins may be involved in the transmission of mitogenic signals across the membrane in vertebrate cells, most importantly in T-cell activation via GPI-anchored receptor proteins (24,47,53) as well as in the formation of caveolae thought to mediate specific endocytotic and transcytotic processes (for reviews, see reference 2 and 3); and (ii) the accessibility of GPI-anchored proteins for cleavage of their GPI anchors by specific phospholipases may provide the basis for controlled cell surface expression and/or degradation of their protein moieties. In some instances, GPI anchorage seems to control the topology of certain amphitropic proteins occurring either at the outer face of the plasma membrane or soluble in the extracellular space via release from the membrane after lipolytic processing of the GPI anchor (32,44,51). Eventually, the change in topology of a GPI-anchored protein with enzymatic function may be accompanied by alterations of its catalytic properties and by accessibility to novel substrates, as has been proven in a few cases (4,5,6,34,38,57).…”