How CD95 stimulates invasion

CD95 is best known for its capacity to induce apoptosis, but also activates multiple non-apoptotic signalling pathways. In particular, CD95 promotes migration and tissue invasion of apoptosis-resistant cell types, and this plays a central role in inflammation, neurobiology, and tumor biology. CD95 induces invasion by stimulating the expression of extracellular matrix (ECM)-degrading proteases, and by stimulating the formation of actin-driven cell protrusions through Rac and the cofilin pathway. In this review we discuss how CD95-initiated signalling pathways may cooperate to facilitate cell migration and tissue invasion. -     

Epidermal growth factor-regulated activation of Rac GTPase enhances CD44 cleavage by metalloproteinase disintegrin ADAM10

Invasive tumour cells, such as gliomas, frequently express EGF (epidermal growth factor) receptor at a high level and they exhibit enhanced cell migration in response to EGF. We reported previously that tumour cell migration is associated with ectodomain cleavage of CD44, the major adhesion molecule that is implicated in tumour invasion and metastasis, and that the cleavage is enhanced by ligation of CD44. In the present study, we show that EGF promotes CD44 cleavage and CD44-dependent cell migration. Introduction of a dominant-negative mutant of the small GTPase Rac1 or depletion of Rac1 by RNAi (RNA interference) abrogated CD44 cleavage induced by EGF. Treatment with PD98059, an inhibitor for MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase), also suppressed the CD44 cleavage. Furthermore, RNAi studies showed that EGF induced ADAM10 (a disintegrin and metalloproteinase 10)-dependent CD44 cleavage and cell migration. These results indicate that EGF induces ADAM10-mediated CD44 cleavage through Rac1 and mitogen-activated protein kinase activation, and thereby promotes tumour cell migration and invasion.     

The Shigella flexneri type three secretion system effector IpgD inhibits T cell migration by manipulating host phosphoinositide metabolism

Shigella, the Gram-negative enteroinvasive bacterium that causes shigellosis, relies on its type III secretion system (TTSS) and injected effectors to modulate host cell functions. However, consequences of the interaction between Shigella and lymphocytes have not been investigated. We show that Shigella invades activated human CD4(+) T lymphocytes. Invasion requires a functional TTSS and results in inhibition of chemokine-induced T cell migration, an effect mediated by the TTSS effector IpgD, a phosphoinositide 4-phosphatase. Remarkably, IpgD injection into bystander T cells can occur in the absence of cell invasion. Upon IpgD-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)), the pool of PIP(2) at the plasma membrane is reduced, leading to dephosphorylation of the ERM proteins and their inability to relocalize at one T cell pole upon chemokine stimulus, likely affecting the formation of the polarized edge required for cell migration. These results reveal a bacterial TTSS effector-mediated strategy to impair T cell function.     

Phospholipases stimulate secretion in RBL mast cells

Roles for glycerophospholipids in exocytosis have been proposed, but remain controversial. Phospholipases are stimulated following the activation of the high-affinity receptor for immunoglobulin E (IgE) in mast cells. To study the biochemical sequelae that lead to degranulation, broken cell systems were employed. We demonstrate that the addition of three distinct types of exogenous phospholipases (i.e., bcPLC, scPLD, and tfPLA(2)), all of which hydrolyze phosphatidylcholine (PC), trigger degranulation in permeabilized RBL-2H3 cells, a mucosal mast cell line. Production of bioactive lipids by these phospholipases promotes release of granule contents through the plasma membrane and acts downstream of PKC, PIP(2), and Rho subfamily GTPases in regulated secretion. These exogenous phospholipase-induced degranulation pathways circumvent specific factors activated following stimulation of the IgE receptor as well as in ATP- and GTP-dependent intracellular pathways. Taken together, these results suggest that regulated secretion may be achieved in vitro in the absence of cytosolic factors via phospholipase activation and that products of PC hydrolysis can promote exocytosis in mast cells.     

Mechanism of PLC-mediated Kir3 current inhibition

A large number of ion channels maintain their activity through direct interactions with phosphatidylinositol bisphosphate (PIP2). For such channels, hydrolysis of PIP2 causes current inhibition. It has become controversial whether the inhibitory effects on channel activity represent direct effects of PIP2 hydrolysis or of downstream PKC action. We studied Phospholipase C (PLC)-dependent inhibition of G protein-activated inwardly rectifying K+ (Kir3) channels. By monitoring simultaneously channel activity and PIP2 hydrolysis, we determined that both direct PIP2 depletion and PKC actions contribute to Kir3 current inhibition. We show that the PKC-induced effects strongly depend on PIP2 levels in the membrane. At the same time, we show that PKC destabilizes Kir3/PIP2 interactions and enhances the effects of PIP2 depletion on channel activity. These results demonstrate that PIP2 depletion and PKC-mediated effects reinforce each other and suggest that both of these interdependent mechanisms contribute to Kir3 current inhibition. This mechanistic insight may explain how even minor changes in PIP2 levels can have profound effects on Kir3 activity. We also show that stabilization of Kir3/PIP2 interactions by Gbetagamma attenuates both PKC and Gq-mediated current inhibition, suggesting that diverse pathways regulate Kir3 activity through modulation of channel interactions with PIP2.     

Phospholipase C isoforms are localized at the cleavage furrow during cytokinesis

It has recently been demonstrated that phosphatidylinositol 4,5-bisphosphate (PIP2) is localized at the cleavage furrow in dividing cells and its hydrolysis is required for complete cytokinesis, suggesting a pivotal role of PIP2 in cytokinesis. Here, we report that at least three mammalian isoforms of phosphoinositide-specific phospholipase C (PLC), PLCdelta1, PLCdelta3 and PLCbeta1, are localized to the cleavage furrow during cytokinesis. Targeting of the delta1 isoform to the furrow depends on the specific interaction between the PH domain and PIP2 in the plasma membrane. The necessity of active PLC in animal cell cytokinesis was confirmed using the specific inhibitors for PIP2 hydrolysis. These results support the model that activation of selected PLC isoforms at the cleavage furrow controls progression of cytokinesis through regulation of PIP2 levels: induction of the cleavage furrow by a contractile ring consisting of actomyosin is regulated by PIP2-dependent actin-binding proteins and formation of specific lipid domains required for membrane separation is affected by alterations in the lipid composition of the furrow.     

Induction of apoptosis by IFNgamma in human neuroblastoma cell lines through the CD95/CD95L autocrine circuit.

The CD95 (APO-1/Fas) system can mediate apoptosis in immune cells as well as in tumour cells, where it may contribute to tumour immune-escape. On the other hand, its induction by anticancer drugs may lead to tumour reduction. Interferongamma (IFNgamma) increases the sensitivity of tumour cell lines to anti-CD95 antibody-mediated apoptosis. We describe induction of apoptosis by IFNgamma through the expression of CD95 and its ligand (CD95L) in human neuroblastoma cell lines. Neuroblastoma cells showed low constitutive expression of CD95 and CD95L. Subsequent to IFNgamma-modulated increase in CD95 and CD95L mRNA as well as protein levels, apoptosis was observed. Our results demonstrated that cytokine-mediated apoptosis was mediated through the activation of the CD95/CD95L autocrine circuit since: (i) cell death occurred following CD95/CD95L expression and correlated with CD95 and CD95L expression levels, (ii) failed to occur in a clone which weakly upregulated CD95 and lacked CD95L induction after IFNgamma stimulation, (iii) was at least partially inhibited by using blocking F(ab')2 anti-CD95 antibody fragments and the recombinant Fas-Fc protein, that prevented the interaction between CD95 and CD95L. The intracellular molecular mechanisms elicited by IFNgamma are clearly highly complex, with several signalling pathways being activated, including the CD95 system. These findings suggest that IFNgamma may have a significant potential in the therapy of neuroblastoma in vivo.     

Sustained diacylglycerol formation from inositol phospholipids in angiotensin II-stimulated vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells to stimulate phospholipase C-mediated hydrolysis of membrane phosphoinositides and subsequent formation of diacylglycerol and inositol phosphates. In intact cells, angiotensin II induces a dose-dependent increase in diglyceride which is detectable after 5 s and sustained for at least 20 min. Angiotensin II (100 nM)-stimulated diglyceride formation is biphasic, peaking at 15 s (227 +/- 19% control) and at 5 min (303 +/- 23% control). Simultaneous analysis of labeled inositol phospholipids shows that at 15 s phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP) decline to 52 +/- 6% control and 63 +/- 5% control, respectively, while phosphatidylinositol (PI) remains unchanged. In contrast, at 5 min, PIP2 and PIP have returned toward control levels (92 +/- 2 and 82 +/- 4% control, respectively), while PI has decreased substantially (81 +/- 2% control). The calcium ionophore ionomycin (15 microM) stimulates diglyceride accumulation but does not cause PI hydrolysis. 4 beta-Phorbol 12-myristate 13-acetate, an activator of protein kinase C, inhibits early PIP and PIP2 breakdown and diglyceride formation, without inhibiting late-phase diglyceride accumulation. Thus, angiotensin II induces rapid transient breakdown of PIP and PIP2 and delayed hydrolysis of PI. The rapid attenuation of polyphosphoinositide breakdown is likely caused by a protein kinase C-mediated inhibition of PIP and PIP2 hydrolysis. While in vascular smooth muscle stimulated with angiotensin II inositol 1,4,5-trisphosphate formation is transient, diglyceride production is biphasic, suggesting that initial and sustained diglyceride formation from the phosphoinositides results from different biochemical and/or cellular processes.     

Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization

Phosphatidylinositol bisphosphate (PIP2) directly regulates functions as diverse as the organization of the cytoskeleton, vesicular transport and ion channel activity. It is not known, however, whether dynamic changes in PIP2 levels have a regulatory role of physiological importance in such functions. Here, we show in both native cardiac cells and heterologous expression systems that receptor-regulated PIP2 hydrolysis results in desensitization of a GTP-binding protein-stimulated potassium current. Two receptor-regulated pathways in the plasma membrane cross-talk at the level of these channels to modulate potassium currents. One pathway signals through the betagamma subunits of G proteins, which bind directly to the channel. Gbetagamma subunits stabilize interactions with PIP2 and lead to persistent channel activation. The second pathway activates phospholipase C (PLC) which hydrolyses PIP2 and limits Gbetagamma-stimulated activity. Our results provide evidence that PIP2 itself is a receptor-regulated second messenger, downregulation of which accounts for a new form of desensitization.     

Protein kinase C activation in B cells by indolactam inhibits anti-Ig-mediated phosphatidylinositol bisphosphate hydrolysis but not B cell proliferation

In order to examine the role of phosphatidylinositol bisphosphate (PIP2) hydrolysis in B cell activation, we studied the effect of various classes of protein kinase C (PKC) activators on anti-Ig-mediated B cell stimulation. Anti-Ig-stimulated PIP2 hydrolysis, elevations in [Ca2+]i, and induction of DNA synthesis were inhibited by PMA (a phorbol ester) as previously reported. In contrast, indolactam (an alkaloid PKC activator) inhibited PIP2 hydrolysis and elevations in [Ca2+]i, but stimulated rather than inhibited cellular proliferation. In order to examine whether the binding avidity of the PKC activators to PKC played a role in determining their activity to stimulate or inhibit B cell activation, we studied two other PKC activators, bryostatin and mezerein. Again, both inhibited anti-Ig mediated PIP2 hydrolysis and elevations in [Ca2+]i, whereas only the former inhibited induction of DNA synthesis. These data suggest that a) high levels of PIP2 hydrolysis and elevations in [Ca2+]i are not essential for anti-Ig-mediated induction of B cell DNA synthesis and b) activation of PKC may induce both stimulatory and inhibitory pathways of B cell activation, and whether stimulation or inhibition of cell activation is observed may depend on the combined intensity of these two signals.     

Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant-activated human polymorphonuclear leukocytes

Binding of chemoattractants to specific cell surface receptors on polymorphonuclear leukocytes (PMNs) initiates a series of biochemical responses leading to cellular activation. A critical early biochemical event in chemoattractant (CTX) receptor-mediated signal transduction is the phosphodiesteric cleavage of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), with concomitant production of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer, and the protein kinase C activator, 1,2-diacylglycerol (DAG). The following lines of experimental evidence collectively suggest that CTX receptors are coupled to phospholipase C via a guanine nucleotide binding (G) protein. Receptor-mediated hydrolysis of PIP2 in PMN plasma membrane preparations requires both fMet-Leu-Phe and GTP, and incubation of intact PMNs with pertussis toxin (which ADP ribosylates and inactivates some G proteins) eliminates the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in isolated plasma membranes. Studies with both PMN particulate fractions and with partially purified fMet-Leu-Phe receptor preparations indicate that guanine nucleotides regulate CTX receptor affinity. Finally, fMet-Leu-Phe stimulates high-affinity binding of GTP gamma S to PMN membranes as well as GTPase activity. A G alpha subunit has been identified in phagocyte membranes which is different from other G alpha subunits on the basis of molecular weight and differential sensitivity to ribosylation by bacterial toxins. Thus, a novel G protein may be involved in coupling CTX receptors to phospholipase C. Studies in intact and sonicated PMNs demonstrate that metabolism of 1,4,5-IP3 proceeds via two distinct pathways: 1) sequential dephosphorylation to 1,4-IP2, 4-IP1 and inositol, or 2) ATP-dependent conversion to inositol 1,3,4,5-tetrakisphosphate (IP4) followed by sequential dephosphorylation to 1,3,4-IP3, 3,4-IP2, 3-IP1 and inositol. Receptor-mediated hydrolysis of PIP2 occurs at ambient intracellular Ca2+ levels; but metabolism of 1,4,5-IP3 via the IP4 pathway requires elevated cytosolic Ca2+ levels associated with cellular activation. Thus, the two pathways for 1,4,5-IP3 metabolism may serve different metabolic functions. Additionally, inositol phosphate production appears to be controlled by protein kinase C, as phorbol myristate acetate (PMA) abrogates PIP2 hydrolysis by interfering with the ability of the activated G protein to stimulate phospholipase C. This implies a physiologic mechanism for terminating biologic responses via protein kinase C mediated feedback inhibition of PIP2 hydrolysis.     

Trafficking and function of the tetraspanin CD63

Tetraspanins comprise a large superfamily of cell surface-associated membrane proteins characterized by four transmembrane domains. They participate in a variety of cellular processes, like cell activation, adhesion, differentiation and tumour invasion. At the cell surface, tetraspanins form networks with a wide diversity of proteins called tetraspanin-enriched microdomains (TEMs). CD63 was the first characterized tetraspanin. In addition to its presence in TEMs, CD63 is also abundantly present in late endosomes and lysosomes. CD63 at the cell surface is endocytosed via a clathrin-dependent pathway, although recent studies suggest the involvement of other pathways as well and we here present evidence for a role of caveolae in CD63 endocytosis. In late endosomes, CD63 is enriched on the intraluminal vesicles, which by specialized cells are secreted as exosomes through fusion of endosomes with the plasma membrane. The complex localization pattern of CD63 suggests that its intracellular trafficking and distribution must be tightly regulated. In this review we discuss the latest insights in CD63 trafficking and its emerging function as a transport regulator of its interaction partners. Finally, the involvement of CD63 in cancer will be discussed.     

Selection of T cell receptor expression mutants through the functionally linked Ly-6A

Ly-6A is a glycosyl-phosphatidylinositol (GPI)-anchored molecule that participates in murine T cell activation. Activation of T cell hybridomas with anti-Ly-6A monoclonal antibody (mAb) leads to production of interleukin-2 (IL-2), but also to a paradoxical growth inhibition, which was used to select for signaling mutants. Fifteen subclones derived from two independent mutageneses and anti-Ly-6A selection were characterized. Thirteen subclones responded poorly or not at all to soluble anti-Ly-6A mAb. Although the selective pressure was exerted through Ly-6A, only one mutant did not express the Ly-6A antigen. Interestingly, 10 of the 15 subclones expressed either nondetectable or a very low level of T cell receptor/CD3 complex (TCR/CD3). Preferential expansion of TCR/CD3 expression mutants following anti-Ly-6A selection further established functional linkage between Ly-6A and TCR/CD3 complex. The mechanism of the functional coupling was investigated by analyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), one of the early events in T cell activation. We showed that PIP2 was not hydrolyzed in response to anti-Ly-6A in TCR/CD3-negative mutants. Aluminum fluoride, which activates G protein directly, did induce PIP2 hydrolysis in these cells. These data suggest that activation signals originated from Ly-6A must be transmitted first to TCR/CD3 complex, which then couples to the G protein/phospholipase C system. A similar requirement also applies to the Thy-1 protein and lectin receptors. Thus, the TCR/CD3 complex plays a central role in the integration and transmission of activation signals that originated from several T cell surface molecules.     

Non-genomic oestrogen receptor signal in B lymphocytes: An approach towards therapeutic interventions for infection,autoimmunity and cancer

The non-genomic membrane bound oestrogen receptor (mER) regulates intracellular signals through receptor-ligand interactions. The mER, along with G-protein coupled oestrogen receptor GPR 30 (GPER), induces diverse cell signalling pathways in murine lymphocytes. The mER isoform ER-alpha46 has recently been demonstrated in human B and T lymphocytes as an analogue receptor for chemokine CCL18, the signalling events of which are not clearly understood. Ligand-induced mER and GPER signalling events are shared with BCR, CD19 mediated intracellular signalling through phospholipase C, PIP2/IP3/PI3 mediated activation of Akt, MAP kinase, and mTOR. Oestrogen has the ability to induce CD40-mediated activation of B cells. The complete signalling pathways of mER, GPR30 and their interaction with other signals are targeted areas for novel drug development in B cells during infection, autoimmunity and cancer. Therefore, an in depth investigation is critical for determining shared signal outputs during B cell activation. Here, we focus on the mode of action of membrane bound ER in B cells as therapeutic checkpoints.     

Homophilic interactions of Tetraspanin CD151 up-regulate motility and matrix metalloproteinase-9 expression of human melanoma cells through adhesion-dependent c-Jun activation signaling pathways

The tetraspanin membrane protein CD151 has been suggested to regulate cancer invasion and metastasis by initiating signaling events. The CD151-mediated signaling pathways involved in this regulation remain to be revealed. In this study, we found that stable transfection of CD151 into MelJuSo human melanoma cells lacking CD151 expression significantly increased cell motility, matrix metalloproteinase-9 (MMP-9) expression, and invasiveness. The enhancement of cell motility and MMP-9 expression by CD151 overexpression was abrogated by inhibitors and small interfering RNAs targeted to focal adhesion kinase (FAK), Src, p38 MAPK, and JNK, suggesting an essential role of these signaling components in CD151 signaling pathways. Also, CD151-induced MMP-9 expression was shown to be mediated by c-Jun binding to AP-1 sites in the MMP-9 gene promoter, indicating AP-1 activation by CD151 signaling pathways. Meanwhile, CD151 was found to be associated with alpha(3)beta(1) and alpha(6)beta(1) integrins in MelJuSo cells, and activation of associated integrins was a prerequisite for CD151-stimulated MMP-9 expression and activation of FAK, Src, p38 MAPK, JNK, and c-Jun. Furthermore, CD151 on one cell was shown to bind to neighboring cells expressing CD151, suggesting that CD151 is a homophilic interacting protein. The homophilic interactions of CD151 increased motility and MMP-9 expression of CD151-transfected MelJuSo cells, along with FAK-, Src-, p38 MAPK-, and JNK-mediated activation of c-Jun in an adhesion-dependent manner. Furthermore, C8161 melanoma cells with endogenous CD151 were also shown to respond to homophilic CD151 interactions for the induction of adhesion-dependent activation of FAK, Src, and c-Jun. These results suggest that homophilic interactions of CD151 stimulate integrin-dependent signaling to c-Jun through FAK-Src-MAPKs pathways in human melanoma cells, leading to enhanced cell motility and MMP-9 expression.     

Engagement of CD44 promotes Rac activation and CD44 cleavage during tumor cell migration

CD44 is a major cell surface adhesion molecule for hyaluronan, a component of the extracellular matrix, and is implicated in tumor metastasis and invasion. We reported previously that hyaluronan oligosaccharides induce CD44 cleavage from tumor cells. Here we show that engagement of CD44 promotes CD44 cleavage and tumor cell migration, both of which were suppressed by a metalloproteinase inhibitor KB-R7785 and tissue inhibitor of metalloproteinases-1 (TIMP-1) but not by TIMP-2. We also present evidence that blockade of metalloproteinase-disintegrin ADAM10 (a disintegrin and metalloproteinase 10) by RNA interference suppresses CD44 cleavage induced by its ligation. Engagement of CD44 concurrently induced activation of the small GTPase Rac1 and led to drastic changes in cell morphology and actin cytoskeleton with redistribution of CD44 to newly generated membrane ruffling areas. A fluorescence resonance energy transfer approach to visualize GTP-bound Rac1 in living cells revealed the localization of the active Rac1 in the leading edge of the membrane ruffling areas upon ligation of CD44. Taken together, our results indicate that the cleavage of CD44 catalyzed by ADAM10 is augmented by the intracellular signaling elicited by engagement of CD44, through Rac-mediated cytoskeletal rearrangement, and suggest that CD44 cleavage contributes to the migration and invasion of tumor cells.     

Differential functions of phospholipid binding and palmitoylation of tumour suppressor EWI2/PGRL

The tumour suppressor EWI2 associates with tetraspanins and regulates tumour cell movement and proliferation. The short cytoplasmic domain of EWI2 is positively charged; five out of the ten residues of this domain are basic. In the present study we demonstrated that the EWI2 cytoplasmic tail interacts specifically with negatively charged PIPs (phosphatidylinositol phosphates), but not with other membrane lipids. The PIPs that interact with EWI2 cytoplasmic tail include PtdIns5P, PtdIns4P, PtdIns3P, PtdIns(3,5)P(2) and PtdIns(3,4)P2. The binding affinity of PIPs to the EWI2 tail, however, is not solely based on charge because PtdIns5P, PtdIns4P and PtdIns3P have a higher affinity to EWI2 than PtdIns(3,5)P(2) and PtdIns(3,4)P(2) do. Mutation of either of two basic residue clusters in the EWI2 cytoplasmic tail abolishes PIP binding, and PIP binding is also determined by the position of basic residues in the EWI2 cytoplasmic tail. In addition, EWI2 is constitutively palmitoylated at the cytoplasmic cysteine residues located at the N-terminal of those basic residues. The PIP interaction is not required for, but appears to regulate, the palmitoylation, whereas palmitoylation is neither required for nor regulates the PIP interaction. Functionally, the PIP interaction regulates the stability of EWI2 proteins, whereas palmitoylation is needed for tetraspanin-EWI2 association and EWI2-dependent inhibition of cell migration and lamellipodia formation. For cell-cell adhesion and cell proliferation, the PIP interaction functions in opposition to the palmitoylation. In conclusion, the EWI2 cytoplasmic tail actively engages with the cell membrane via PIP binding and palmitoylation, which play differential roles in EWI2 functions.     

Studies of endogenous polyphosphoinositide hydrolysis in human platelet membranes. Evidence that polyphosphoinositides remain inaccessible to phosphodiesterase in the native membrane

Human platelet plasma membranes incubated in the presence of [gamma-32P]ATP and 15 mM MgCl2 incorporated radioactivity mostly into phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP), which represented together over 90% of the total lipid radioactivity. After washing, reincubation of prelabelled membranes revealed some hydrolysis of the two compounds by phosphomonoesterase(s), as detected by the release of radioactive inorganic phosphate (Pi) from the two phospholipids. This degradation attained 40%/30 min for PIP in the presence of 2 mM calcium and cytosol. The effect of calcium was observed at concentrations equal to or greater than 10(-4) M. In no case did calcium alone facilitate the formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). In contrast, simultaneous addition of 2 mM calcium and 2 mg/ml sodium deoxycholate promoted the formation of IP3 and IP2, indicating phosphodiesteratic cleavage of PIP2 and PIP. Phospholipase C activity was detected at calcium concentrations as low as 10(-7) M, in which case PIP2 hydrolysis was slightly more pronounced compared to PIP. Addition of cytosol increased to some extent the phospholipase C activity, suggesting that the low amount of enzyme remaining in the membrane is sufficient to promote submaximal degradation of PIP2 and PIP. We conclude that platelet polyphosphoinositides are present in the plasma membrane in a state where they remain inaccessible to phospholipase C, which is still fully active even at basal calcium concentrations, i.e., 10(-7) M. These results support the view that phosphodiesteratic cleavage of PIP2 promotes and thus precedes calcium mobilization brought about by IP3. The in vitro model presented here may prove very useful in future studies dealing with the mechanism rendering polyphosphoinositides accessible to phospholipase C attack upon agonist-receptor binding.