Hydrolysis Of Glycosyl-phosphatidylinositol Research Articles

On the possible participation of phosphatidylinositol-3-kinase in activation by insulin and epidermal growth factor phospholipase c, hydrolyzing glycosylphosphatidylinositol

The pleiotropic effect of insulin is based on signal transfer from insulin receptor to a series of intracellular effector systems, one of which is glycosyl phosphatidyl inositol (GPI) specific phospholipase C (PLC), hydrolyzing membranous inositol-containing glycophospholipids to produce diacylglycerol, a phospholipase C activator, and inositolphosphoglycane (IPG), a probable secondary messenger in transfer of insulin metabolic signal. For disclosing the mechanisms of GPI-PLC conjugation with insulin receptors and growth factors, we analyzed GPI hydrolysis in Rati cells and found that insulin and epidermal growth factors (EGF) activate GPI hydrolysis to IPG by 20 and 40%o, respectively (p<0.001), while specific phosphatidylinositol- 3’-kinase (PIj kinase) inhibitor vortmannin cancels this insulin and EGF effect. These data permit us to hypothesize the participation of PIj kinase in signal transfer from insulin and EGF receptor GPI-PLC.

Rab11 mediates selective recycling and endocytic trafficking in Trypanosoma brucei

Trypanosoma brucei possesses a streamlined secretory system that guarantees efficient delivery to the cell surface of the critical glycosyl-phosphatidylinositol (GPI)-anchored virulence factors, variant surface glycoprotein (VSG) and transferrin receptor (TfR). Both are thought to be constitutively endocytosed and returned to the flagellar pocket via TbRab11+ recycling endosomes. We use conditional knockdown with established reporters to investigate the role of TbRab11 in specific endomembrane trafficking pathways in bloodstream trypanosomes. TbRab11 is essential. Ablation has a modest negative effect on general endocytosis, but does not affect turnover, steady state levels or surface localization of TfR. Nor are biosynthetic delivery to the cell surface and recycling of VSG affected. TbRab11 depletion also causes increased shedding of VSG into the media by formation of nanotubes and extracellular vesicles. In contrast to GPI-anchored cargo, TbRab11 depletion reduces recycling of the transmembrane invariant surface protein, ISG65, leading to increased lysosomal turnover. Thus, TbRab11 plays a critical role in recycling of transmembrane, but not GPI-anchored surface proteins. We proposed a two-step model for VSG turnover involving release of VSG-containing vesicles followed by GPI hydrolysis. Collectively, our results indicate a critical role of TbRab11 in the homeostatic maintenance of the secretory/endocytic system of bloodstream T. brucei.

Phosphatidylinositol 3-kinase regulates glycosylphosphatidylinositol hydrolysis through PLC-γ 2 activation in erythropoietin-stimulated cells

Erythropoietin (Epo)-induced glycosylphosphatidylinositol (GPI) hydrolysis was previously described to be correlated with phospholipase C-gamma 2 (PLC-γ2) activation. Here, we analyzed the involvement of phosphatidylinositol (PtdIns) 3-kinase in GPI hydrolysis through PLC-γ2 tyrosine phosphorylation in response to Epo in FDC-P1 cells transfected with a wild type (WT) erythropoietin-receptor (Epo-R). We showed that phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor LY294002 inhibits Epo-induced hydrolysis of endogenous GPI and Epo-induced PLC-γ2 tyrosine phosphorylation in a dose-dependent manner. Wortmannin, another PtdIns 3-kinase inhibitor, also suppressed Epo-induced PLC-γ2 tyrosine phosphorylation. We also present evidence that PLC-γ2 translocation to the membrane fraction on Epo stimulation is completely inhibited by LY294002. Upon Epo stimulation, the tyrosine-phosphorylated PLC-γ2 was found to be associated with the tyrosine-phosphorylated Grb2-associated binder (GAB)2, SHC and SHP2 proteins. LY294002 cell preincubation did not affect GAB2, SHC and SHP2 tyrosine phosphorylation but inhibited the binding of PLC-γ2 to GAB2 and SHP2. Taken together, these results show that PtdIns 3-kinase controls Epo-induced GPI hydrolysis through PLC-γ2.

Purification and Characterization of Insulin-Mimetic Inositol Phosphoglycan-Like Molecules From Grass Pea (Lathyrus sativus) Seeds

Signal transduction through the hydrolysis of glycosyl-phosphatidylinositol (GPI) leading to the release of the water-soluble inositol phosphoglycan (IPG) molecules has been demonstrated to be important for mediating some of the actions of insulin and insulin-like growth factor-I (IGF-I). In the present study, GPI from grass pea (Lathyrus sativus) seeds has been purified and partially characterized on the basis of its chromatographic properties and its compositional analysis. The results indicate that it shows similarities to GPI previously isolated from other sources such as rat liver. IPG was generated from L. sativus seed GPI by hydrolysis with a GPI-specific phospholipase D (GPI-PLD). This IPG inhibited protein kinase A (PKA) in an in vitro assay, caused cell proliferation in explanted cochleovestibular ganglia (CVG), and decreased 8-Br-cAMP-induced phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression in cultured hepatoma cells. Our data indicate that L. sativus seed IPG possess insulin-mimetic activities. This may explain why L. sativus seeds have been used in some traditional medicines to ameliorate diabetic symptoms.

Glycosylphosphatidylinositol (GPI) hydrolysis by transforming growth factor-beta1 (TGF-beta1) as a potential early step in the inhibition of epithelial cell proliferation.

Glycosylphosphatidylinositol (GPI) was previously identified in rabbit articular chondrocytes as being a precursor of inositolphosphate glycan (IPG), released upon (Transforming Growth Factor-beta) (TGF-beta) exposure, and capable of mimicking the proliferative effects of the growth factor. Here, using mink lung epithelial cells (CCL 64), which are known to be growth-inhibited by TGF-beta, we studied the potential role of GPI-derived molecules in the antiproliferative effect of TGF-beta1. We first identified an endogenous pool of GPI material and three different anionic forms of IPG in epithelial cells pre-labeled with [3H]glucosamine. Shortly (8 min) after TGF-beta1 addition, the cells responded by a rapid and transient hydrolysis of GPI, accompanied by the release of the most anionic form of IPG. This TGF-beta-released IPG, after partial purification, was shown to decrease the proliferation of CCL 64 cells. Moreover, anti-IPG antibodies reduced the effects of TGF-beta and blocked the effects of partially purified IPG. These data strongly suggest that GPI hydrolysis may be an early step of the TGF-beta signalling pathway involved in growth inhibition of epithelial cells.

Erythropoietin Induces Glycosylphosphatidylinositol Hydrolysis: POSSIBLE INVOLVEMENT OF PHOSPHOLIPASE C-γ2

We showed that erythropoietin induced rapid glycosylphosphatidylinositol (GPI) hydrolysis and tyrosine phosphorylation of phospholipase C (PLC)-gamma(2) in FDC-P1 cells transfected with the wild-type erythropoietin-receptor. Erythropoietin-induced tyrosine phosphorylation of PLC-gamma(2) was time- and dose-dependent. By using FDC-P1 cells transfected with an erythropoietin receptor devoid of tyrosine residues, we showed that both effects required the tyrosine residues of intracellular domain on the erythropoietin receptor. Erythropoietin-activated PLC-gamma(2) hydrolyzed purified [(3)H]GPI indicating that GPI hydrolysis and PLC-gamma(2) activation under erythropoietin stimulation were correlated. Results obtained on FDC-P1 cells transfected with erythropoietin receptor mutated on tyrosine residues suggest that tyrosines 343, 401, 464, and/or 479 are involved in erythropoietin-induced GPI hydrolysis and tyrosine phosphorylation of PLC-gamma(2), whereas tyrosines 429 and/or 431 seem to be involved in an inhibition of both effects. Thus, our results suggest that erythropoietin regulates GPI hydrolysis via tyrosine phosphorylation of its receptor and PLC-gamma(2) activation.

Phospholipase cleavage of glycosylphosphatidylinositol reconstituted in liposomal membranes

Glycosylphosphatidylinositol (GPI) purified from rat liver lipids was incorporated into lipid bilayers of defined compositions, in the form of large unilamellar vesicles. The GPI concentration in the bilayers was kept constant at 25 mole%, whereas the remaining lipids being phosphatidylcholine, phosphastidylethanolamine, sphingomyelin and/or cholesterol were varied. The resulting liposomes consisted of spherical vesicles, approximately 100 nm in diameter, that could keep their aqueous contents separated from the extravesicular medium. When these liposomes were treated with either Bacillus cereus phosphatidylinositol-phospholipase C, Trypanosoma brucei GPI-phospholipase C, or bovine serum GPI-phospholipase D, GPI was hydrolyzed at different rates, depending on the enzyme and the bilayer lipid composition. These observations open the way to biophysical and biochemical studies of enzymic GPI cleavage under defined conditions. Extensive GPI hydrolysis was observed in certain cases that could allow the use of these systems for the preparation of inositol phosphoglycans, proposed second messengers of a wide variety of hormones, cytokines and growth factors.

ACTH-mediated glucocorticoid and mineralocorticoid production is inhibited by an inositolphosphoglycan and a glycosylphosphatidylinositol-phospholipase C is activated by the hormone in mammalian adrenocortical cells

In the present paper, we report that an inositolphosphoglycan (IPG), derived from a Trypanosoma cruzi glycoinositolphosphoceramide (LPPG), is able to inhibit ACTH-mediated accumulation of a glucocorticoid, cortisol, in calf adrenocortical cells. This IPG is also able to inhibit the stimulation by ACTH of the production of the main glucocorticoid, corticosterone and the main mineralocorticoid, aldosterone, in rat adrenocortical cells. Nitrous acid deamination confirmed that IPG is responsible for this inhibition. In order to study the involvement of glycosylphosphatidylinositol (GPI) in ACTH response in rat adrenal cortex, the activation of a phospholipase that hydrolyzes GPI (GPI-PLC) was evaluated. It was found that the release of alkaline phosphatase, a GPI-anchored enzyme, to the extracellular medium is increased in rat adrenocortical cells by ACTH treatment. In addition, ACTH stimulates the release of ceramide from the glycoinositolphosphoceramide purified from T. cruzi. These data suggest that ACTH activates a GPI-PLC in rat adrenal cortex, which is in agreement with our previous data in calf adrenocortical cells; thus, the hydrolysis of GPI provoked by ACTH takes place in different mammals and the IPG released could inhibit ACTH-mediated synthesis of aldosterone, corticosterone and cortisol.

Subcellular distribution of glycosylphosphatidylinositol-specific phospholipase D in rat liver.

Glycosylphosphatidylinositol (GPI)-hydrolysing enzymes have been described in many mammalian tissues and body fluids; however, their site(s) of action and in vivo functions have remained unclear. In order to identify a possible intracellular site of GPI hydrolysis, we studied the subcellular distribution of GPI-hydrolysing activity in rat liver. We found that purified fractions from rat liver hydrolysed the GPI moieties of two GPI-anchored proteins with the specificity of a phospholipase D. This GPI-specific phospholipase D (GPI-PLD) activity was found to be highly enriched in a lysosomal fraction and showed a similar intracellular distribution to that of typical lysosomal enzymes. Our results indicate that lysosomes may represent a possible intracellular site of GPI-PLD action.

Phosphatidylinositol Hydrolysis by Trypanosoma brucei Glycosylphosphatidylinositol Phospholipase C

Detergent-solubilized glycosylphosphatidylinositol (GPI)-anchored structures can be cleaved by C-type phospholipases isolated from peanuts and bloodstream cells of the African trypanosome, Trypanosoma brucei. The two enzymes differ in their reported ability to hydrolyze phosphatidylinositol (PI); while the peanut enzyme readily hydrolyzes PI in vitro, the T. brucei enzyme was reported to be virtually inactive against PI and consequently named GPI-specific phospholipase C (GPI-PLC). In this paper, we describe experiments in which we reinvestigated the substrate specificity of T. brucei GPI-PLC by incubating the purified enzyme with Triton X-100/PI-mixed micelles and by studying PI hydrolysis. We found that PI hydrolysis occurred in a detergent-dependent fashion over the range of concentrations tested (5 microM to 1 mM PI). At 5 microM PI, hydrolysis was maximal at 0.005% Triton X-100, whereas at 1 mM PI, maximal hydrolysis required 0.05% Triton X-100. Hydrolysis of both PI and GPI was strongly affected by the presence of phospholipids. Endogenous PI was hydrolyzed during osmotic and detergent lysis of trypanosomes under conditions used to obtain quantitative hydrolysis of the GPI-anchored trypanosome variant surface glycoprotein. PI hydrolysis in the lysates was inhibited by sodium p-chloromercuriphenylsulfonate but unaffected by EGTA, consistent with the proposal that hydrolysis is due to GPI-PLC. These results suggest that the function of T. brucei GPI-PLC may be to regulate PI as well as (or instead of) GPI levels.

Role of glycosyl-phosphatidylinositol hydrolysis as a mitogenic signal for epidermal growth factor

We have investigated the role of the hydrolysis of glycosyl-phosphatidylinositol (GPI) as one of the signalling pathways elicited after interaction of epidermal growth factor (EGF) with its specific plasma membrane receptor (EGFR). Endogenous GPI was characterized in both NIH 3T3 mouse fibroblast cells and in EGFR-transfected NIH 3T3 cells (designated EGFR T17). GPI molecules isolated from both cell lines were identical and they incorporated radioactivity from both sugar and fatty acid substrates. Incubation of EGFR T17 cells with EGF, produced a rapid and transient hydrolysis of GPI. Maximum hydrolysis occurred after a 1-min incubation with 50 nM EGF. No such effects of EGF were observed in the parental cell line. Both inositol phosphoglycan (IPG)- and EGF-induced cell proliferation was inhibited in the presence of an IPG-antibody to different extents. The relationship between GPI hydrolysis and the activity of the EGFR was studied using the tyrosine kinase inhibitors tyrphostin (RG50864) and genistein. These agents were able to significantly inhibit EGF-mediated cell proliferation, EGF-dependent hydrolysis of GPI and EGF-regulated autophosphorylation of the EGFR. It is concluded that GPI hydrolysis is one of the earliest intracellular events generated in response to EGF.

Insulin-induced activation of glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin mediator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki rats.

Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosphoglycan (IPG) mediator, prepared from beef liver, bypassed this defect and comparably activated G3PAT in cell-free adipocyte preparations of both diabetic GK and control rats. A myo-inositol-containing IPG mediator did not activate G3PAT. Relative to control adipocytes, labeling of GPI by [3H]glucosamine was diminished by 50% and insulin failed to stimulate GPI hydrolysis in GK adipocytes. In contrast to GPI-dependent G3PAT activation, insulin-stimulated hexose transport was intact in adipocytes and soleus and gastrocnemius muscles of the GK rat, as was insulin-induced activation of mitogen-activated protein kinase and protein kinase C. We conclude that (i) chiro-inositol-containing IPG mediator activates G3PAT during insulin action, (ii) diabetic GK rats have a defect in synthesizing or releasing functional chiro-inositol-containing IPG, and (iii) defective IPG-regulated intracellular glucose metabolism contributes importantly to insulin resistance in diabetic GK rats.

Diacylglycerol/protein kinase C signalling: a mechanism for insulin resistance?

It is proposed that an intracellular cycle exists to limit or terminate the insulin signal. The cycle involves increased synthesis of sn-1,2-diacylglycerol (DAG) in response to insulin. The DAG activates protein kinase C (PKC) which phosphorylates glycogen synthase either directly or through other protein kinases to render it inactive. Protein kinase C may also inhibit the insulin receptor by phosphorylation of receptor serine residues. Insulin resistance could then arise as a consequence of a persistent increase in DAG levels. Such an increase could occur in three different ways. Chronic hyperinsulinaemia could increase DAG levels by de-novo synthesis from phosphatidic acid, by hydrolysis of phosphatidylcholine, or by hydrolysis of glycosyl-phosphatidylinositol; DAG is also formed by hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP2). This reaction, known as the 'PI response,' may be the connection between hypertension and insulin resistance. A third mechanism for an increase in DAG involves neural abnormalities. Thus, muscle denervation in the rat is characterized both by a profound insulin resistance and a large increase in DAG. It is possible that a similar increase occurs in humans and may explain the association between denervation, inactivity, and insulin resistance.

Brain-Derived Neurotrophic Factor and Neurotrophin-3 Support the Survival and Neuritogenesis Response of Developing Cochleovestibular Ganglion Neurons

The effects of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on the differentiation of avian cochleovestibular ganglion and their possible association with the hydrolysis of glycosyl-phosphatidylinositol (GPI) were studied. BDNF and NT-3 (2 ng/ml) promoted neurite outgrowth in explants of both cochlear and vestibular ganglia. This effect on neuritogenesis was stage-dependent, reaching a maximum at E7 for NT-3 and at E9 for BDNF. The magnitude of the response of the vestibular ganglion to BDNF was always smaller than that of the cochlear ganglion of an equivalent stage. BDNF and NT-3 stimulation of neuronal survival and neurite extension was also demonstrated in dissociated neuronal cell cultures. The effect was concentration-dependent with saturation of the response occurring at 4 ng/ml for BDNF and at 2 ng/ml for NT-3, the half-maximal effect occurring at 2 and 1 ng/ml, respectively, for the most sensitive stages of the chick cochlear ganglion. Inositol phosphoglycan (IPG) did not mimic the effects of BDNF or NT-3 on neuronal survival and neurite outgrowth, nor was it able to potentiate their responses. Antibodies raised against IPG did not block the effects of these neurotrophins. The results suggest that BDNF and NT-3 may act in cooperation to establish the innervation pattern of the inner ear. Unlike their early proliferative effects, neurotrophic effects are uncoupled from the GPI/IPG signal transduction system.

Brain-Derived Neurotrophic Factor and Neurotrophin-3 Induce Cell Proliferation in the Cochleovestibular Ganglion through a Glycosyl-Phosphatidylinositol Signaling System

We have investigated the role of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the regulation of cell proliferation in the early developing cochleovestibular ganglion (CVG). Ganglia were isolated from 72-hr chick embryos and cultured for 24 hr. Both BDNF and NT-3 had a powerful mitogenic effect, at doses of 1-5 ng/ml, consistent with an involvement of the high-affinity receptor. Evidence for the participation of the glycosyl-phosphatidylinositol (GPI)/inositol phosphoglycan (IPG) signaling system in the mediation of proliferative effects of BDNF and NT-3 is presented. Both of these neurotrophins elicited a fast and transient hydrolysis of labeled GPI, approximately 60% in 30 sec. The dose-response profile of GPI hydrolysis overlaps the neurotrophin-induced cell proliferation response profile. Anti-IPG antibodies were able to block the growth-promoting effects of BDNF and NT-3. Anti-IPG antibodies immunoprecipitated a CVG-endogenous IPG, induced upon BDNF treatment, which exhibited proliferative stimulating properties. Both BDNF and NT-3 are proposed as potential candidates for regulation of growth during CVG development, with this mitogenic effect being mediated by the GPI/IPG signaling system.

IPG (inositolphosphate glycan) as a cellular signal for TGF‐β1 modulation of chondrocyte cell cycle

The knowledge of transforming growth factor (TGF)-beta receptors has greatly progressed in the recent years. TGF-beta receptors type I and II have been implicated in the modulation of cell proliferation, whereas type III (betaglycan) may act as a component presenting TGF-beta to its signaling receptors. In addition, four other proteins that bind TGF-beta 1 or TGF-beta 2 have been recently identified in some cell lines, three being anchored to the membrane through a glycosylphosphatidylinositol (GPI). Despite this knowledge, the molecular mechanism of signal transduction through the TGF-beta receptors remain an enigma. TGF-beta family does not signal via any of the classical pathways. As GPI anchors of membrane proteins have been implicated in the transduction of some hormonal effects, we investigated the putative role of GPI in signaling the TGF-beta effects on the proliferation of rabbit articular chondrocytes (RAC). We previously showed that TGF-beta 1 increased DNA replication rate of RAC, with a recruitment of cells in G2/M followed by a subsequent mitosis wave. Here, we find that the factor causes specific GPI hydrolysis, with correlated increase of inositolphosphate glycan (IPG). This effect was specifically inhibited by antibodies that bind TGF-beta 1. Using [3H]-inositol labeling and Triton X-114 extraction, we demonstrate that a hydrophobic material from the membrane is cleaved by treatment of cell cultures with phosphatidylinositol specific phospholipase C (PI-PLC) or by exposure to TGF-beta, supporting that a PI-anchored molecule gives rise to IPG by TGF-beta-induced hydrolysis. The biological relevance of this hydrolysis was demonstrated by the enhancing effect of purified IPG on the DNA synthesis rate, which mimicked the TGF-beta action. These results demonstrate that IPG could be an early messenger in the cellular signaling that mediates the effect of TGF-beta on RAC growth.

Partial characterization of a glycosylphosphatidyl-inositol (GPI) in porcine thyroid cultured cells

The aim of this study was to provide information on the structure of glycosylphosphatidylinositol (GPI) and to characterize this novel phospholipid isolated from pig thyroid. We investigated the incorporation of different radioactive precursors: [ 3H]glucosamine, [ 3H]inositol, [ 3H]oleic acid, [ 32P]orthophosphate and demonstrated the presence of these moieties in the structure of GPI. After labelling and hydrochloric acid hydrolysis, the major part of radioactivity comigrated with glucosamine used as marker. Cleavage of GPI by nitrous acid deamination indicated the presence of a glycosidic bond between the lipid moiety and glucosamine. The fatty acid composition of diacylglycerol was also studied by gas chromatography. Oleic acid was found preferentially to other fatty acids. In a previous paper we reported that a chronic treatment with 0.1 mU/ml thyrotropin (TSH) of thyroid cultured cells for 3 days produced a large increase in the turnover rate of GPI and concomitantly the release of a water-soluble product of GPI hydrolysis: an inositolphosphateglycan (IPG). These findings confirm that GPI may play a role in membrane signalling systems and thyroid cell regulation.

Regulation of interleukin 2-dependent growth responses by glycosylphosphatidylinositol molecules.

The molecular mechanism of signal transduction through the interleukin 2 (IL-2) receptor remains an enigma. Glycosylphosphatidylinositol (GPI) lipids were investigated as one component of this process. IL-2 stimulated the rapid (30 sec) loss of greater than 50% of GPI in the IL-2-dependent T-cell line CTLL-2. Half-maximal GPI loss was detected at 40 pM IL-2, coincident with the EC50 (20 pM) for IL-2-induced proliferation of this cell line. This effect was specifically inhibited by antibodies that bind either IL-2 or the IL-2 receptor. The loss of GPI was mirrored by the accumulation of both polar inositolphosphoglycan (IPG) and diacylglycerol lipid fragments within cells. Increases in lipids were initially restricted to myristoyl diacylglycerol but were followed by the accumulation of myristoyl phosphatidic acid. These results are indicative of IL-2-dependent hydrolysis of GPI in T cells. The biological relevance of this hydrolysis was demonstrated by synergism of purified IPG with IL-2 in T-cell proliferation responses. The inclusion of IPG (0.1 microM) in determinations of IL-2-dependent CTLL-2 growth shifted the EC50 from 20 to 7 pM IL-2. IPG had no effect on either the number or affinity of IL-2 receptors; therefore, half-maximal CTLL-2 proliferation was obtained at less than 10% IL-2 receptor occupancy. These results demonstrate that GPI lipids are an important component of the biological response to IL-2.

Hydrolysis of Glycosyl-Phosphatidylinositol in Response to Insulin is Reduced in Cells Bearing Kinase-Deficient Insulin Receptors

A glycosyl-phosphatidylinositol (GPI) has been previously identified that serves as a precursor of the polar head group that mimics and may mediate some of the intracellular actions of insulin. Since many of the biological activities of insulin may depend upon the activity of the insulin receptor kinase, we evaluated the requirement for this activity in insulin-dependent GPI hydrolysis. For the analysis we used stably transfected CHO cell lines, expressing either the wild-type human insulin receptor or a mutant receptor that lacks tyrosine kinase activity (Chou et al., 1987) and a stably transfected CHO cell line, expressing the wild-type human insulin-like growth factor I (IGF-1) receptor (Steele-Perkins et al., 1988). A GPI was identified in both types of transfected cells and in both sets of parental cells by metabolic labeling with [3H]glucosamine or [3H]galactose. The isolated glycolipid was sensitive to hydrolysis by phospholipase C and to deamination by nitrous acid. Insulin induced a time- and dose-dependent hydrolysis of the GPI in the parental line and in the transfected cell types. Cells bearing normal human receptors hydrolyzed up to 70% of their radiolabeled GPI within 2 min of the addition of 0.1 nM insulin, whereas parental cells and cells expressing the mutant receptor hydrolyzed only 20-30% in response to 100 nM insulin. IGF-1 (5-50 nM) had little effect on GPI hydrolysis in these cells as well as in CHO cells expressing the human IGF-1 receptor. It is concluded that insulin-dependent GPI hydrolysis is mediated by the normal but not by a kinase-deficient insulin receptor.