USP7 is essential for maintaining Rad18 stability and DNA damage tolerance.

Rad18 functions at the cross-roads of three different DNA damage response (DDR) pathways involved in protecting stressed replication forks: homologous recombination repair, DNA inter-strand cross-link repair and DNA damage tolerance. Although Rad18 serves to facilitate replication of damaged genomes by promoting translesion synthesis (TLS), this comes at a cost of potentially error-prone lesion bypass. In contrast, loss of Rad18-dependent TLS potentiates the collapse of stalled forks and leads to incomplete genome replication. Given the pivotal nature with which Rad18 governs the fine balance between replication fidelity and genome stability, Rad18 levels and activity have a major impact on genomic integrity. Here, we identify the de-ubiquitylating enzyme USP7 as a critical regulator of Rad18 protein levels. Loss of USP7 destabilizes Rad18 and compromises UV-induced PCNA mono-ubiquitylation and Pol η recruitment to stalled replication forks. USP7-depleted cells also fail to elongate nascent daughter strand DNA following UV irradiation and show reduced DNA damage tolerance. We demonstrate that USP7 associates with Rad18 directly via a consensus USP7-binding motif and can disassemble Rad18-dependent poly-ubiquitin chains both in vitro and in vivo. Taken together, these observations identify USP7 as a novel component of the cellular DDR involved in preserving the genome stability.

Retinoic acid receptor-independent mechanism of apoptosis of melanoma cells by the retinoid CD437 (AHPN).

Retinoic acid (RA) induces differentiation of S91 melanoma cells through activation of RA receptor (RAR)gamma without affecting cell viability. The novel RARgamma-agonist CD437 (AHPN), however, also induces concomitant apoptosis through an unknown mechanism which was investigated here. By utilizing DNA microarray analysis, five apoptosis-associated, CD437-induced transcripts (CITs) were identified. Interestingly, all CITs are also regulated by p53 in a DNA damage response, and consistent with this interpretation, CD437 was found to cause DNA adduct-formation. However, p53 is not required for CD437-dependent regulation of CITs. Among this set of genes, induction of p21(WAF1/CIP1) is likely to be responsible for early S-phase growth-arrest of CD437-treated cells, whereas ei24 is a critical mediator of CD437-induced apoptosis in S91 cells. These data suggest an RAR-independent mechanism in which CD437 causes DNA adduct-formation, resulting in induction of a p53-independent DNA damage response, and subsequent growth-arrest and apoptosis. CD437-mediated DNA adduct-formation may also explain its apoptotic effects in other cell types.

ATR inhibition selectively sensitizes G1 checkpoint-deficient cells to lethal premature chromatin condensation.

Premature chromatin condensation (PCC) is a hallmark of mammalian cells that begin mitosis before completing DNA replication. This lethal event is prevented by a highly conserved checkpoint involving an unknown, caffeine-sensitive mediator. Here, we have examined the possible involvement of the caffeine-sensitive ATM and ATR protein kinases in this checkpoint. We show that caffeine's ability to inhibit ATR (but not ATM) causes PCC, that ATR (but not ATM) prevents PCC, and that ATR prevents PCC via Chk-1 regulation. Moreover, mimicking cancer cell phenotypes by disrupting normal G(1) checkpoints sensitizes cells to PCC by ATR inhibition plus low-dose DNA damage. Notably, loss of p53 function potently sensitizes cells to PCC caused by ATR inhibition by a small molecule. We present a molecular model for how ATR prevents PCC and suggest that ATR represents an attractive therapeutic target for selectively killing cancer cells by premature chromatin condensation.

RING3 kinase transactivates promoters of cell cycle regulatory genes through E2F.

RING3 is a novel, nuclear-localized, serine-threonine kinase that has elevated activity in human leukemias. RING3 transforms NIH/3T3 cells and is activated by mitogenic signals, all of which suggest that it may play a role in cell cycle-responsive transcription. We tested this hypothesis with transient transfection of RING3 into fibroblasts and assayed transactivation of the promoters of cyclin D11 cyclin A, cyclin E, and dihydrofolate reductase (dhfr) genes. RING3 transactivates these promoters in a manner dependent on ras signaling. A kinase-deficient point mutant of RING3 does not transactivate. Mutational analysis of the dhfr promoter reveals that transactivation also depends on the presence of a functional E2F binding site. Furthermore, ectopic expression of Rb protein, a negative regulator of E2F activity, suppresses the RING3-dependent transactivation of this promoter. Consistent with a potential role of E2F in RING3-dependent transcription, anti-RING3 immunoaffinity chromatography or recombinant RING3 protein affinity chromatography of nuclear extracts copurified a protein complex that contains E2F-1 and E2F-2. These data suggest that RING3 is a potentially important regulator of E2F-dependent cell cycle genes.

DNA-damaging aryl hydrocarbons induce Mdm2 expression via p53-independent post-transcriptional mechanisms.

During previous studies, we found that mdm2 mRNA levels were elevated in benzo[a]pyrene (BaP, a polycyclic aryl hydrocarbon)-treated cells under conditions of DNA damage-induced cell cycle arrest (Vaziri, C., and Faller, D. V. (1997) J. Biol. Chem. 272, 2762-2769). We have identified potential aryl-hydrocarbon receptor-binding sites in the mdm2 promoter. However, we show that induction of mdm2 mRNA by BaP is entirely dependent upon aryl-hydrocarbon-induced genotoxicity and does not involve direct aryl-hydrocarbon receptor-mediated transcriptional activation of the mdm2 gene. Heterologous mdm2 promoter-reporter constructs containing p53-response elements were not responsive to BaP treatment. Therefore the p53-response elements in the mdm2 promoter are insufficient to confer DNA damage-dependent expression of mdm2. Furthermore, mdm2 transcripts were induced by BaP in p53 null cells from transgenic mice (although both basal and BaP-induced mdm2 expression levels were reduced in these cells relative to p53(+/+) cultures). These data show that p53-mediated mechanisms cannot account for BaP/DNA damage-induced mdm2 expression. Mdm2 promoter-reporter gene assays and nuclear run-off analyses of nascent mdm2 transcripts showed that transcriptional induction was unable to account for the large changes in mdm2 transcript levels following BaP treatment. However, mdm2 mRNA half-life measurements showed stabilization of the mdm2 transcript (from approximately 1 h to >4 h) in response to BaP. To our knowledge, this is the first report of control of mdm2 at the post-transcriptional level and in a p53-independent manner. Transient ectopic expression of mdm2 strongly augmented aryl-hydrocarbon-induced apoptosis, demonstrating that mdm2 levels can have a profound effect on the cellular response to DNA damage. Overall, our results suggest a potentially important link between DNA damage signaling and RNA stability that may be relevant to cell cycle regulation, tumor suppression, and environmental carcinogenesis.

A novel DNA damage checkpoint involving post-transcriptional regulation of cyclin A expression.

The intracellular metabolism of many carcinogenic polycyclic aryl hydrocarbons (PAHs, typified by the ubiquitous pollutant benzo[a]pyrene or B[a]P) generates electrophilic products that react covalently with genomic DNA. Cells that acquire PAH-induced DNA damage undergo growth arrest in a p53-independent manner (Vaziri, C., and Faller, D. V. (1997) J. Biol. Chem. 272, 2762-2769). In this report we have investigated the molecular basis of PAH-induced cell cycle arrest. Mitogenic signaling events involving cyclins D and E, Rb phosphorylation, and transcriptional activation of E2F-responsive genes (including cyclin E and cyclin A) were unaffected in cells containing PAH-damaged DNA. However, PAH-induced growth arrest was associated with post-transcriptional decreases in cyclin A expression. Mitogen-induced expression of cyclin B, an event that is temporally distal to cyclin A expression, was also inhibited in PAH-treated cells. The PAH-induced cell cycle block was transient, and arrested cells resumed DNA synthesis after a prolonged ( approximately 20 h) delay. Resumption of DNA synthesis in PAH-treated cells occurred concomitant with elevated expression of cyclins A and B. PAH-induced cell cycle arrest was overcome by ectopically expressed cyclin A (encoded by a recombinant adenovirus in transiently infected cells). Overall, our results suggest the existence of a DNA damage checkpoint pathway that arrests cell cycle progression via post-transcriptional control of cyclin A expression.

Regulation of platelet-derived growth factor signaling by activated p21Ras.

Elucidating the molecular mechanisms regulating transduction of growth control signals and the discovery of the subversion of these pathways by oncogenes has proven critical in unraveling the biochemical factors leading to cellular transformation. One such line of investigation has been study of the effects of transforming p21Ras on platelet-derived growth factor type-beta receptor (PDGF-betaR) signaling. Platelet-derived growth factor is an important extracellular factor regulating the G0-S phase transition of mesenchymal cells. Expression of activated, oncogenic Kirsten- or Harvey-p21Ras in cells influences PDGF-betaR signaling at multiple levels. At least two separate mechanisms account for defective PDGF-betaR signaling in activated p21Ras-expressing cells: (i) transcriptional down-regulation of PDGF-betaR expression, and (ii) inhibition of ligand-induced PDGF-betaR phosphorylation by a factor which is present in the cellular membrane fraction of fibroblasts expressing activated p21Ras. The state of growth arrest in G0 is associated with increased expression of the PDGF-betaR, and oncogene-transformed cell lines, which fail to undergo growth-arrest following prolonged serum-deprivation, express constitutively low levels of the PDGF-betaR mRNA, and possess greatly reduced numbers of PDGF-BB-binding sites. This repression of PDGF-betaR expression by p21Ras is, at least in large part, transcriptional. The membrane-associated factor induced by oncogenic p21Ras provides a connection between cell morphology and cytoskeletal elements and control of ligand-dependent PDGF-betaR autophosphorylation. Reversion of the transformed phenotype results in the recovery of PDGF-betaR kinase activity. Conversely, disruption of the actin cytoskeleton of untransformed fibroblasts leads to the loss of PDGF-betaR function. These studies define two potential mechanisms for feedback control of PDGF-betaR function by downstream elements in the PDGF signaling pathway. In addition, the connection between cell morphology and the function of the PDGF-betaR established by these studies provides a new mechanistic link between the organization of the cytoskeleton, the Ras-related small G proteins, and the activity of membrane-bound receptor tyrosine kinases.

Butyrate-induced G1 arrest results from p21-independent disruption of retinoblastoma protein-mediated signals.

When treated with millimolar concentrations of butyrate, many cell types undergo growth arrest in the G1 phase of the cell cycle. However, the molecular basis of butyrate-induced G1 arrest has not been elucidated. We have investigated the molecular mechanisms of butyrate-induced G1 arrest in synchronized cultures of untransformed 3T3 fibroblasts. We tested the hypothesis that butyrate-induced growth arrest might be mediated by the p21 cyclin-dependent kinase inhibitor. Sodium butyrate-treated 3T3 cells did, indeed, express elevated levels of p21 mRNA under conditions of G1 arrest. Surprisingly, however, primary cultures of fibroblasts from transgenic p21 "knockout" (p21-/-) mice and fibroblasts from wild-type p21-proficient (p21+/+) mice underwent butyrate-induced G1 arrest with similar dose dependencies. Therefore, p21 expression was not necessary for butyrate-induced G1 arrest. To identify other potential mechanisms of butyrate-induced growth arrest, we analyzed the butyrate sensitivity of key mitogenic signaling events during G1. We found that butyrate inhibited the mitogen-dependent transcriptional induction of cyclin D1 and phosphorylation of retinoblastoma (Rb), both in p21-proficient 3T3 cells and in p21+/+ and p21-/- mouse embryo fibroblasts. Butyrate treatment also prevented mitogen-dependent transcriptional induction of cyclin E and expression of cyclin A, cell cycle events that are temporally distal to expression of cyclin D and are necessary for entry into S phase. Abrogation of a requirement for cyclin D/cyclin-dependent kinase-dependent phosphorylation of Rb (by ectopic expression of the human papilloma virus E7 oncoprotein in 3T3 cells) resulted in decreased sensitivity to the antiproliferative actions of butyrate. Overall, these data show that butyrate-induced G1 arrest is, in large part, independent of p21 induction. Instead, butyrate-induced growth arrest appears to result from perturbation of the Rb signaling axis at the level of or at a stage prior to cyclin D1 expression.

A benzo[a]pyrene-induced cell cycle checkpoint resulting in p53-independent G1 arrest in 3T3 fibroblasts.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor of the basic helix-loop-helix family. Although physiological ligands for the AhR have not been identified, carcinogenic polycyclic aromatic hydrocarbons such as Benzo[a]pyrene (B[a]P) are high affinity AhR ligands that induce nuclear translocation and sequence-specific DNA binding of the AhR. AhR-regulated genes include members of the cytochrome P-450 family that are known to oxidize B[a]P to form genotoxic (DNA-damaging) metabolites. Murine Swiss 3T3 cells express high levels of AhR. Treatment of Swiss 3T3 cells with B[a]P during the G1 phase of the cell cycle resulted in growth arrest, as shown by inhibition of growth factor-stimulated DNA synthesis. By contrast, other murine 3T3 fibroblasts not expressing detectable levels of AhR did not undergo growth arrest in response to B[a]P. The AhR antagonist alpha-naphthoflavone prevented B[a]P-induced growth arrest, further demonstrating that cessation of cell growth was mediated by the activated AhR. A nongenotoxic AhR ligand (2,3,7,8-tetrachlorodibenzo-p-dioxin) did not elicit growth arrest, showing that ligand activation of the AhR alone was insufficient to block cell cycle progression. However, genomic DNA from B[a]P-treated Swiss 3T3 cells contained covalent adducts, whereas that from 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated cells did not, showing that G1 arrest correlated with DNA damage resulting from genotoxic B[a]P metabolites. B[a]P-induced DNA damage and growth arrest was coincident with elevated levels of nuclear p53 protein and induction of the p53-regulated mdm-2 proto-oncogene. However, Swiss 3T3 fibroblasts expressing "dominant negative" mutant p53, as well as primary fibroblasts from p53-/- "knockout" mice, also underwent growth arrest in response to B[a]P. Therefore, B[a]P-induced growth arrest occurs via p53-independent mechanisms.

Expression of the aryl hydrocarbon receptor is regulated by serum and mitogenic growth factors in murine 3T3 fibroblasts.

The aryl-hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates biological responses to planar aromatic hydrocarbons such as benzo[alpha]pyrene. However, no endogenous physiological ligand for the AhR has been identified. Since the AhR regulates bioactivity to common environmental pollutants, and since it is predicted to play an important physiological function, we have investigated the expression of the AhR during the cell cycle of murine 3T3 fibroblasts. We show here that stimulation of growth-arrested 3T3 cells with serum results in increased expression of AhR protein. Serum-induced expression of AhR in synchronized, serum-stimulated cells occurs at the onset of DNA synthesis (S phase) and is maximal at time points corresponding to late S phase. Transient transfections with an AhR-promoter-luciferase construct demonstrate that reporter gene transcription from the AhR promoter is regulated in a serum-dependent manner. Serum-dependent induction of AhR expression is prevented by an inhibitor of tyrosine kinase activity. Ligand-activated growth factor receptors (platelet-derived growth factor receptor basic fibroblast growth factor receptor) as well as an ectopically expressed tyrosine kinase (the v-Src oncoprotein) induce AhR expression in the absence of serum. Therefore, tyrosine kinase signaling is both necessary and sufficient for induction of AhR expression. Studies with the G1 blocker sodium butyrate show that the signal transduction pathways mediating serum-stimulated progression through the cell cycle are distinct from those that induce AhR expression. These data suggest that transcriptional regulation of the AhR is important in determining cellular sensitivity to the actions of AhR ligand(s) and that the AhR may play a role during the cellular proliferative response.

Down-regulation of platelet-derived growth factor receptor expression during terminal differentiation of 3T3-L1 pre-adipocyte fibroblasts.

The transcription and expression of platelet-derived growth factor (PDGF) receptors (PDGFRs) is down-regulated as a consequence of entry into the replicative cell cycle (Vaziri, C., and Faller, D. V. (1995) Mol. Cell. Biol. 15, 1244-1253). In this study, we have investigated the expression of PDGFRs during terminal differentiation, a process in which cells exit from the cell cycle. When treated with appropriate hormonal stimuli, 3T3-L1 fibroblasts initiate a differentiation program resulting in conversion to lipid-accumulating, adipocyte-like cells. Pre-adipocytes express amounts of PDGFalphaR and PDGFbetaR mRNA and protein that are similar to levels expressed in other murine 3T3 fibroblasts. In contrast, the expression of both alpha and beta receptor transcripts is greatly reduced in differentiated 3T3-L1 cells. The loss of PDGFR mRNA following induction of differentiation precedes morphological conversion as well as the induction of many adipocyte-specific genes. The amounts of cell surface PDGFR protein diminish in parallel with the mRNA levels during differentiation, as shown by Western blotting and PDGF-binding assays. The reduced expression of PDGFRs does not reflect a general down-regulation of growth factor receptors, as expression of the type 1 FGFR is unaffected by terminal differentiation. The PDGFbetaR promoter drives strong expression of a luciferase reporter gene in pre-adipocytes, but not in differentiated cells, indicating that the decrease in PDGFR expression following induction of differentiation is a transcriptionally regulated event. Decreased PDGFR expression in differentiated cells is associated with impaired biological responsiveness to PDGF, as shown by reduced activation of mitogen-activated protein-kinase following PDGF stimulation, and decreased chemotactic responsiveness to PDGF. Our data suggest that PDGFR down-regulation is an important mechanism for reducing PDGF-responsiveness in terminally differentiated 3T3-L1 cells.

Repression of platelet-derived growth factor beta-receptor expression by mitogenic growth factors and transforming oncogenes in murine 3T3 fibroblasts.

Platelet-derived growth factor BB (PDGF-BB) is an important extracellular factor for regulating the G0-S phase transition of murine BALB/c-3T3 fibroblasts. We have investigated the expression of the PDGF beta receptor (PDGF beta R) in these cells. We show that the state of growth arrest in G0, resulting from serum deprivation, is associated with increased expression of the PDGF beta R. When the growth-arrested fibroblasts are stimulated to reenter the cell cycle by the mitogenic action of serum or certain specific combinations of growth factors, PDGF beta R mRNA levels and cell surface PDGF-BB-binding sites are markedly downregualted. Oncogene-transformed 3T3 cell lines, which fail to undergo growth arrest following prolonged serum deprivation, express constitutively low levels of the PDGF beta R mRNA and possess greatly reduced numbers of cell surface PDGF receptors, as determined by PDGF-BB binding and Western blotting (immunoblotting). Nuclear runoff assays indicate the mechanism of repression of PDGF beta R expression to be, at least in large part, transcriptional. These data indicate that expression of the PDGF beta R is regulated in a growth state-dependent manner in fibroblasts and suggest that this may provide a means by which cells can modulate their responsiveness to the actions of PDGF.

The turkey erythrocyte beta-adrenergic receptor couples to both adenylate cyclase and phospholipase C via distinct G-protein alpha subunits.

By contrast with mammalian beta-adrenergic receptors, the avian isoform elicits two distinct effector responses, activation of adenylate cyclase and polyphosphoinositide-specific phospholipase C (PLC) leading to the accumulation of both cyclic adenosine monophosphate (cyclic AMP) and inositol phosphates. We have investigated the mechanisms of beta-adrenergic receptor signalling in turkey erythrocytes. Stimulation of adenylate cyclase by the beta-adrenergic-receptor agonist isoprenaline exhibits a 30-fold lower EC50 than that for PLC activation, which may indicate a marked receptor reserve for the former effector. Similar Ki values were obtained for the inhibition of both responses by four beta-adrenergic antagonists, arguing that a single receptor population is responsible for both effects. Antibodies raised against G-protein peptide sequences were used to show that the identity of the G-protein mediating the PLC response was an avian homologue of G11, the level of expression of which was very similar to that of the stimulatory G-protein of adenylate cyclase, Gs. Thus a single population of beta-adrenergic receptors apparently interacts with distinct G-proteins to activate different effectors. The stoichiometries of the receptor-G-protein-effector interactions are therefore similar for both second-messenger responses and the data are discussed in terms of the different efficacies observed for each response.

Direct labelling of hormone-sensitive phosphoinositides by a plasma-membrane-associated PtdIns synthase in turkey erythrocytes.

We have previously characterized phosphatidylinositol (PtdIns) synthase and PtdIns/myo-inositol-exchange enzyme activities in ghost membranes prepared by hypotonic lysis of turkey erythrocytes [McPhee, Lowe, Vaziri and Downes (1991) Biochem. J. 275, 187-192]. Here we show that PtdIns synthase activity is relatively enriched in plasma-membrane preparations of turkey erythrocytes and that inositol phospholipids labelled by both PtdIns synthase and PtdIns myo-inositol exchange enzymes are susceptible to hydrolysis by the receptor- and G-protein-regulated phospholipase C (PLC), which is present also in ghost preparations. Specific-radioactivity measurements of [3H]PtdIns from ghosts labelled to equilibrium under conditions favouring [3H]inositol incorporation by PtdIns synthase activity indicate that PtdIns synthase can directly access approx. 14% of the total erythrocyte ghost PtdIns. Approx. 16% of the [3H]PtdIns labelled by the PtdIns synthase reaction can be phosphorylated to polyphosphoinositides, which are then hydrolysed by the receptor- and G-protein-stimulated PLC. Since the mass of PtdIns declines to a similar extent as [3H]PtdIns during stimulation in the presence of guanine nucleotides and ATP, it is evident that both the labelled and unlabelled phosphoinositides are susceptible to hydrolysis by the relevant PLC. Phosphoinositides present in nuclei-free plasma membranes were also labelled by [3H]inositol under conditions favouring PtdIns synthase and PtdIns/myo-inositol-exchange enzyme activities respectively. These membranes lack PLC activity [Vaziri and Downes (1992) J. Biol. Chem. 267, 22973-22981], but the labelled lipids were sensitive to purinergic-receptor-stimulated hydrolysis in reconstitution assays using partially purified turkey erythrocyte PLC. The results strongly suggest that at least a portion of the PtdIns synthase in turkey erythrocytes is located in the plasma membrane and has direct access to an agonist-sensitive pool of inositol phospholipids.

Association of a receptor and G-protein-regulated phospholipase C with the cytoskeleton.

Approximately 98% of turkey erythrocyte phospholipase C (PLC) is cytosolic and is released by hypotonic lysis of the cells and extensive washing of the resultant erythrocyte ghosts. Well washed turkey erythrocyte ghosts retain a fraction of tightly associated PLC, which is activated by the P2y-purinergic receptor and G-protein present in ghost membranes. The particulate PLC is sufficient to couple to all the available purinergic receptor-regulated G-protein. In contrast to ghosts, turkey erythrocyte plasma membrane preparations contain no detectable PLC. To investigate the subcellular location of the ghost-associated PLC, cytoskeletons were prepared by Triton X-100 extraction of turkey erythrocyte ghosts. The ghost-associated PLC was quantitatively recovered in cytoskeleton preparations. Cytoskeleton-associated PLC was solubilized by sodium cholate extraction, partially purified, and shown to reconstitute with PLC-free plasma membrane preparations in an agonist and guanine nucleotide-dependent fashion, indicating that the cytoskeleton-associated PLC is G-protein-regulated. Dissociation of erythrocyte ghost cytoskeletons with the actin-binding protein DNase 1 resulted in a dose-dependent inhibition of agonist and guanine nucleotide-stimulated PLC responses in ghosts and caused release of PLC from ghost or cytoskeleton preparations. These data demonstrate the specific association of a receptor and G-protein-regulated PLC with a component of the detergent-insoluble cytoskeleton and indicate that the integrity of the actin cytoskeleton is important for localization and effective coupling of PLC to the relevant G-protein.

G-protein-mediated activation of turkey erythrocyte phospholipase C by beta-adrenergic and P2y-purinergic receptors.

Isoprenaline, previously known only to stimulate adenylate cyclase via the stimulatory G-protein, Gs, activates turkey erythrocyte ghost phospholipase C (PLC) in a dose-dependent manner when GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) is present. The effect is specific in that it is abolished by beta-adrenergic-receptor antagonists. Stimulation of adenosine receptors, which also couple to adenylate cyclase via Gs in turkey erythrocytes, does not activate PLC, indicating that the stimulation observed in the presence of isoprenaline is not due to Gs activation. Furthermore, the stimulation seen is independent of cyclic AMP production. Purified turkey erythrocyte PLC is activated in an adenosine 5'-[beta-thio]diphosphate (ADP[S]; a P2y-purinergic-receptor agonist)- or isoprenaline-regulated manner when reconstituted with turkey erythrocyte ghosts, demonstrating that a single species of PLC effector enzyme can be regulated by both the purinergic and the beta-adrenergic receptor populations present in turkey erythrocyte membranes. Pretreatment of intact turkey erythrocytes with the P2y agonist ADP[S] causes decreased PLC responsiveness of subsequent ghost preparations to ADP[S] stimulation, although responses to isoprenaline are unaffected (homologous desensitization). In contrast, pretreatment of intact erythrocytes with isoprenaline results in heterologous desensitization of both the P2y and the beta-adrenergic receptors. These effects occur at the level of receptor-G-protein coupling, since PLC stimulation by GTP[S] (which directly activates G-proteins) in the absence of agonists is unaffected.

Phosphatidylinositol synthase and phosphatidylinositol/inositol exchange reactions in turkey erythrocyte membranes.

Unlike human erythrocytes, those from avian species, such as turkeys and chicks, rapidly incorporate myo-[3H]inositol into membrane phospholipids. The mechanisms regulating [3H]Ins labelling of phosphatidylinositol have been investigated using turkey erythrocyte membranes. In the absence of added nucleotides, [3H]inositol incorporation appears to proceed via phosphatidylinositol/inositol exchange, with a Km for inositol of 0.01 mM. The reaction was dependent upon divalent cations, either Mg2+ or Mn2+, with the latter metal ion being the more effective. [3H]Inositol incorporation was accelerated by CMP, especially when the concentration of Ins was greater than the Km for the exchange reaction. CMP-dependent labelling of PtdIns had a Km for inositol of 0.3 mM and for CMP of 0.015 mM. Divalent cations were also required for this reaction: activity peaked at 0.5 mM-Mn2+ and declined at higher concentrations. At relatively high concentrations, Mg2+ was more effective than Mn2+, with peak activity being achieved above 10 mM. CMP-dependent incorporation of [3H]inositol appears to reflect an exchange reaction catalysed by PtdIns synthase. Definitive evidence for the occurrence of PtdIns synthase in turkey erythrocyte membranes was obtained by demonstrating the formation of [14C]CMP-phosphatidate from [14C]CMP. The radioactivity could be efficiently chased from [14C]CMP-phosphatidate in the presence of unlabelled inositol. The detection of PtdIns synthase activity in morphologically simple turkey erythrocytes should help to clarify the subcellular distribution of this important component of the phosphatidylinositol cycle.