Activation of phospholipase D by bradykinin and sphingosine 1-phosphate in A549 human lung adenocarcinoma cells via different GTP-binding proteins and protein kinase C delta signaling pathways

Phospholipase D (PLD) is involved in the signaling by many extracellular ligands, and its regulation appears to be quite complex. We investigated the signaling pathways initiated by bradykinin (BK) or sphingosine 1-phosphate (S1P) in A549 cells to define molecular mechanisms responsible for their additive effects on PLD activity. BK and S1P each elicited a sustained increase in phosphatidic acid content through a rapid and transient activation of PLD. The two pathways demonstrated rapid homologous downregulation, but heterologous desensitization was not observed. Action of both agonists required protein kinase C (PKC) activation and Ca(2+) influx but was mediated by different heterotrimeric G proteins. In membranes, inhibition of PKCdelta by rottlerin enhanced BK activation of PLD but inhibited that by S1P. Rottlerin inhibited activation of PLD in nuclei by both BK and S1P. By in situ immunofluorescence or cell fractionation followed by immunoblotting, PLD1 was concentrated primarily in nuclei, whereas the membrane fraction contained PLD2 and PLD1. Moreover, PKCdelta specifically phosphorylated recombinant PLD2, but not PLD1. BK and S1P similarly enhanced RhoA translocation to nuclei, whereas BK was less efficacious than S1P on RhoA relocalization to membranes. Effects of both agonists on the nuclear fraction, which contains only PLD1, are compatible with a RhoA- and PKCdelta-dependent process. In membranes, which contain both PLD1 and PLD2, the stimulatory effect of S1P on PLD activity can best be explained by RhoA- and PKCdelta-dependent activation of PLD1; in contrast, the effects of BK on RhoA translocation and enhancement of BK-stimulated PLD activity by PKC inhibition are both consistent with PLD2 serving as its primary target.     

Induction of cyclooxygenase-2 synthesis by ligation of the macrophage alpha(2)-macroglobulin signalling receptor

We have studied the induction of cyclooxygenase-2 (COX-2) in macrophages consequent to ligating the alpha(2)-macroglobulin (alpha(2)M) signalling receptor (alpha(2)MSR) with receptor-recognized forms of alpha(2)M (alpha(2)M*). Macrophage stimulation with alpha(2)M* increased total cellular and nuclear COX-2 two- to threefold. The maximal increase in COX-2 occurred at a ligand concentration of 50-100 pM and after 2 h. Modulation of intracellular Ca(2+) levels or incubation of [35S] methionine-labelled macrophages with actinomycin D, prior to treatment with alpha(2)M*, markedly reduced the induction of total cellular and nuclear COX-2. Protein kinase C (PKC) or phospholipase A(2) (PLA(2)) inhibition in alpha(2)M*-stimulated macrophages or inhibition of the p21(ras)-dependent mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI 3-kinase) signalling pathways also significantly reduced alpha(2)M*-induced total cellular and nuclear COX-2 expression. Thus, COX-2 induction is dependent on cPLA(2) activity, Ca(2+) mobilization, and PKC activity and requires participation of both the p21(ras)-dependent MAPK and PI 3-kinase signalling pathways. COX-2 activation may mediate alpha(2)M*-induced mitogenesis, which we have previously observed in this and other cell types.     

Activation of bovine neutrophil oxidase in a cell free system. GTP-dependent formation of a complex between a cytosolic factor and a membrane protein

The effect of guanine nucleotides on activation of the O2-. generating oxidase in a cell free system consisting of bovine neutrophils membranes, cytosol and arachidonic acid has been studied. In a complete system, GTP-gamma-S was stimulatory and GDP-beta-S inhibitory. When cytosol was omitted, both nucleotides acted as inhibitors. Activation parameters have been explored in a preincubation step prior to the oxidase assay. Stimulation was found to be maximal at 7 to 100 microM GTP-gamma-S. Whereas the time course of activation was monophasic when activation was performed at room temperature, it became biphasic at 2 degrees C, with a first plateau of activation attained after 1 min, followed by a slow rise lasting for more than 30 min. The following lines of evidence demonstrated that oxidase activation resulted from the formation of a complex between cytosolic factor(s) and a target protein in the plasma membrane. 1/ When activated membranes, in a suspension containing cytosol, arachidonic acid and GTP-gamma-S, were separated from soluble components by centrifugation and washed, their oxidase remained fully active. 2/ The activity of the washed membranes was lost upon addition of GDP-beta-S, urea and deoxycholate, but was preserved by addition of glutaraldehyde, a cross-linking reagent. The results of experiments in which cytosol and membrane fractions were incubated separately with GTP-gamma-S, suggested that GTP-gamma-S first interacts with a factor present in the cytosol, before reacting with a target protein in the plasma membrane.     

Potentiation of calcium levels by extracellular arachidonic acid in nuclei isolated from macrophages stimulated with receptor-recognized forms of alpha(2)-macroglobulin

Ligation of macrophage alpha(2)-macroglobulin signalling receptors (alpha(2)MSR) with activated alpha(2)-macroglobulin (alpha(2)M*) increases intracellular Ca(2+), and cytosolic phospholipase A(2) (cPLA(2)) and phospholipase D activities. In view of the relationship between cellular Ca(2+) and mitogenesis, we examined the effect of the product of cPLA(2) activity, arachidonic acid (AA), on nuclear Ca(2+) levels in macrophages stimulated with alpha(2)M*, platelet derived growth factor, and bradykinin. AA addition increased Ca(2+) levels in Fura-2/AM loaded nuclei from both buffer-treated and agonist-stimulated cells, but the increase in stimulated macrophages was 2-4-fold higher. Preincubation of Fura-2/AM loaded nuclei with EGTA or BAPTA/AM abolished AA-induced increase in nuclear Ca(2+) levels. Preincubation of nuclei with indomethacin did not affect AA-induced increase in nuclear Ca(2+) in agonist-stimulated nuclei. It is concluded that in macrophages stimulated with various agonists, AA, derived from cPLA(2)-dependent hydrolysis of phospholipids, plays a significant role in regulating nuclear Ca(2+) levels and thus nuclear functions.     

Isolation and characterization of a 66-kDa protein from rat liver plasma membrane with RhoA-stimulated phospholipase D activity.

A 66-kDa molecular weight protein with phospholipase D activity was solubilized and partially purified from rat liver plasma membrane. The activity and regulation of this phospholipase D have been characterized. Immunoblot analyses indicated that the enzyme was distinct from hPLD1 and PLD2, but was recognized by an antibody to the 12 terminal amino acids of PLD1. PLD activity was stimulated by 1-100 microM Ca(2+) and Mg(2+) and displayed a pH optimum of 7.5. Activity was inhibited by both saturated and unsaturated fatty acids. This PLD was activated in an ATP-independent manner by the PKC isozymes alpha and betaII but not activated by other PKC isozymes. It was also stimulated by the small G-proteins RhoA and ARF. RhoA stimulated the greatest activation, followed by ARF and PKC(alpha). This enzyme was further activated in a synergistic manner when combinations of PKC(alpha) and RhoA or ARF were used. This enzyme displayed a greater response activation by RhoA than to activation by ARF. While a potential breakdown product of PLD1, activation by RhoA indicates that the PLD characterized here is distinct from the other PLDs cloned or isolated to date.     

Downregulation of phospholipase D by protein kinase A in a cell-free system of human neutrophils

Agents which elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD) in human neutrophils. The PLD activity of human neutrophils requires protein factors in both membrane and cytosolic fractions. We have studied the regulation of PLD by the catalytic subunit of protein kinase A (cPKA) in a cell-free system. cPKA significantly inhibited GTPgammaS-stimulated PLD activity but had no effect on phorbol ester-activated PLD activity. Pretreatment of plasma membranes with cPKA and ATP inhibited subsequent PLD activation upon reconstitution with untreated cytosol. RhoA, which is known to be a plasma membrane activator of PLD, was dissociated from PKA-treated plasma membrane by addition of cytosol. Plasma membrane-associated RhoA in human neutrophils was phosphorylated by cPKA. The PKA-phosphorylated form of RhoA was more easily extracted from membranes by RhoGDI than the unphosphorylated form. These results suggest that inhibition of neutrophil PLD by PKA may be due to phosphorylation of RhoA on the plasma membrane.     

A novel receptor function for the heat shock protein Grp78: silencing of Grp78 gene expression attenuates alpha2M*-induced signalling

The activated proteinase inhibitor alpha2-macroglobulin (alpha2M*) binds to two receptors, the low density lipoprotein receptor-related protein (LRP-1) and the alpha2M* signalling receptor (alpha2MSR). Silencing LRP-1 gene expression in macrophages by RNA interference does not block alpha2M* activation of signalling cascades. We now demonstrate that transfection of macrophages with a double-stranded RNA homologous in sequence to the Grp78 gene markedly decreased induction of inositol 1,4,5-trisphosphate (IP3) and subsequent IP3-dependent elevation of [Ca2+]i induced by alpha2M*. Concomitantly, alpha2M*-induced increase in [3H]thymidine uptake was abolished in these transfected cells. Insulin treatment significantly upregulates alpha2MSR and it also caused a marked increase in Grp78 expression which could be blocked by RNA interference. alpha2M* treatment of cells activates the Ras- and PI 3-kinase-dependent signalling pathways. Suppressing Grp78 expression leads to the loss of these activation events in transfected macrophages. We thus conclude that Grp78 is the alpha2M* signalling receptor.     

Activation of the O2(.-)-generating oxidase in plasma membrane from bovine polymorphonuclear neutrophils by arachidonic acid, a cytosolic factor of protein nature, and nonhydrolyzable analogues of GTP

A reconstitution system for activation of the O2(.-)-generating oxidase from bovine polymorphonuclear neutrophils (PMN) is described. This system consisted of three components, namely, a particulate fraction enriched in plasma membrane, a supernatant fluid (cytosolic fraction) recovered by high-speed centrifugation from sonicated resting bovine PMN, and arachidonic acid. The pH optimum (7.8) and the Km value for NADPH (45 microM) of the activated oxidase were virtually the same as those found in the purified enzyme. All three components had to be present during the preincubation for elicitation of oxidase activity. A further enhancement of oxidase activity was observed with the addition of nonhydrolyzable GTP analogues, such as guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) and guanosine 5'-(beta, gamma-imidotriphosphate) (GMP-PNP), to the preincubation medium. In contrast, GDP-beta-S drastically decreased oxidase activation. In a two-stage experiment, a 9-min preincubation of PMN membranes with arachidonic acid and GTP-gamma-S followed by a 1-min contact with the cytosolic fraction led to a more marked activation than did preincubation of the cytosol with arachidonic acid and GTP-gamma-S for 9 min followed by a 1-min contact with membranes, suggesting the presence of a G-protein in the membrane fraction. In the absence of added cations, the reconstitution system exhibited a substantial oxidase activity which was totally prevented by ethylenediaminetetraacetic acid (EDTA). Mg2+ added at a concentration of 0.5-1 mM enhanced oxidase activation by about 30%, indicating that endogenous Mg2+ or other activating cations were sufficient to ensure 70% of maximal activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective upregulated expression of the alpha2-macroglobulin signaling receptor in highly metastatic 1-LN prostate carcinoma cells

Cellular binding of receptor-recognized forms of alpha2-macroglobulin (alpha2M*) is mediated by the low-density lipoprotein receptor related protein (LRP) and the alpha2M signaling receptor (alpha2MSR). In nonmalignant cells, ligation of alpha2MSR promotes DNA synthesis and cellular proliferation. Here, we report that insulin treatment of highly metastatic 1-LN human prostate carcinoma selectively increases alpha2MSR expression and binding of alpha2M* to 1-LN cells. alpha2M* induces transient increases in intracellular calcium and inositol 1,4,5-trisphosphate in insulin-treated 1-LN cells, consistent with activation of alpha2MSR. Inhibition of signaling cascades activated by insulin blocks upregulation of alpha2MSR. By contrast, alpha2M* does not bind to nor induce intracellular signaling in PC-3 cells, even though 1-LN cells were subcloned from PC-3 cells. We suggest that alpha2M* behaves like a growth factor in these highly malignant cells. The 1-LN metastatic phenotype may result, in part, from aberrant expression of alpha2MSR, indicating the possible involvement of alpha2M* in tumor progression.     

G protein regulation of phospholipase C activity in a membrane-solubilized system occurs through a Mg2(+)- and time-dependent mechanism

GTP-binding proteins have been implicated to function as key transducing elements in the mechanism underlying receptor activation of a membrane-associated phospholipase C activity. In the present study, the regulation of phospholipase C activity by GTP-binding proteins has been characterized in a detergent-solubilized system derived from bovine brain membranes. Guanosine-5'-(3-O-thio)triphosphate (GTP-gamma-S) and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) stimulated a dose-dependent increase in phospholipase C activity with half-maximal activation at 0.6 microM and 10 microM, respectively. The maximal degree of stimulation due to Gpp(NH)p or GTP-gamma-S was comparable. 100 microM GTP had only a slight stimulatory effect on phospholipase C activity. Adenine nucleotides, 100 microM adenylyl-imidodiphosphate and ATP, did not stimulate phospholipase C activity, indicating that specific guanine nucleotide-dependent regulation of phospholipase C activity was preserved in the solubilized state. Gpp(NH)p or GTP-gamma-S stimulation of phospholipase C activity was time-dependent and required Mg2+.Mg2+ regulated the time course for activation of phospholipase C by guanine nucleotides and the ability of guanine nucleotides to promote an increase in the Ca2+ sensitivity of phospholipase C. 200 microM GDP-beta-S or 5 mM EDTA rapidly reversed the activation due to GTP-gamma-S or Gpp(NH)p. These findings demonstrate that G protein regulation of phospholipase C activity in a bovine brain membrane- solubilized system occurs through a Mg2+ and time-dependent mechanism. Activation is readily reversible upon addition of excess GDP-beta-S or removal of Mg2+.     

Activation of O2.- generating oxidase of bovine neutrophils in a cell-free system. Interaction of a cytosolic factor with the plasma membrane and control by G nucleotides

Activation of the O2.- -generating oxidase of bovine neutrophils was studied in a cell-free system, consisting of a particulate fraction enriched in plasma membrane, cytosol, arachidonic acid, and the non-hydrolyzable nucleotide GTP-gamma-S. Activation of the membrane-bound oxidase was accompanied by the disappearance of the activating factor from the cytosol. Above a cytosol to membrane ratio of 25, the excess of added cytosolic factor remained in active state in the soluble fraction. The process could be partially reversed by serum albumin. Disappearance of the cytosolic factor was promoted by unsaturated long-chain fatty acids, but not by saturated ones, and occurred not only in the presence of GTP-gamma-S but also in the presence of GDP-beta-S or in the absence of Mg ions, although in the latter cases activation of O2.- production was seriously impaired. This suggests that the disappearance of the activating factor from the cytosol and the triggering effect of GTP-gamma-S are related, but distinct, events in the oxidase activation process. The disappearance of the activating factor from cytosol can be explained by translocation of the cytosolic factor to the membrane fraction. Yet under some conditions, including the presence of GDP-beta-S or EDTA, inactivation was prevailing and could be an alternative explanation for the results. Specific binding of radiolabeled GTP-gamma-S could be demonstrated both in the membrane and in the cytosolic fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coordinate regulation of the alpha(2)-macroglobulin signaling receptor and the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor by insulin.

We have studied insulin-dependent regulation of macrophage alpha(2)-macroglobulin signaling receptors (alpha(2)MSR) and low density lipoprotein receptor-related protein/alpha(2)M receptors (LRP/alpha(2)MR) employing cell binding of (125)I-alpha(2)M*, inhibition of binding by receptor-associated protein (RAP) or Ni(2+), LRP/alpha(2)MR mRNA levels, and generation of second messengers. Insulin treatment increased the number of alpha(2)M* high (alpha(2)MSR) and low (LRP/alpha(2)MR) affinity binding sites from 1, 600 and 67,000 to 2,900 and 115,200 sites per cell, respectively. Neither RAP nor Ni(2+) blocked the binding of (125)I-alpha(2)M* to alpha(2)MSR on insulin- or buffer-treated cells, but they both blocked binding to LRP/alpha(2)MR. Insulin significantly increased LRP/alpha(2)MR mRNA levels in a dose- and time-dependent manner. Insulin-augmented (125)I-alpha(2)M* binding to macrophages was severely reduced by wortmannin, LY294002, PD98059, SB203580, or rapamycin. The increase in alpha(2)MSR receptor synthesis was reflected by augmented generation of IP(3) and increased [Ca(2+)](i) levels upon receptor ligation. Incubation of macrophages with wortmannin, LY294002, PD98059, SB203580, rapamycin, or antibodies against insulin receptors before insulin treatment and alpha(2)M* stimulation significantly reduced the insulin-augmented increase in IP(3) and [Ca(2+)](i) levels. Pretreatment of cells with actinomycin D or cycloheximide blocked the synthesis of new alpha(2)MSR. In conclusion, we show here that insulin coordinately regulates macrophage alpha(2)MSR and LRP/alpha(2)MR, utilizing both the PI 3-kinase and Ras signaling pathways to induce new synthesis of these receptors.     

Heterotrimeric Galphaq11 co-immunoprecipitates with surface-anchored GRP78 from plasma membranes of alpha2M*-stimulated macrophages

We have previously shown that a fraction of newly expressed GRP78 is translocated to the cell surface in association with the co-chaperone MTJ-1. Proteinase and methylamine-activated alpha(2)M (alpha(2)M*) bind to cell surface-associated GRP78 activating phosphoinositide-specific phospholipase C coupled to a pertussis toxin-insensitive heterotrimeric G protein, generating IP(3)/calcium signaling. We have now studied the association of pertussis toxin-insensitive Galphaq11, with GRP78/MTJ-1 complexes in the plasma membranes of alpha(2)M*-stimulated macrophages. When GRP78 was immunoprecipitated from plasma membranes of macrophages stimulated with alpha(2)M*, Galphaq11, and MTJ-1 were co-precipitated. Likewise Galphaq11 and GRP78 co-immunoprecipitated with MTJ-1 while GRP78 and MTJ-1 co-immunoprecipitated with Galphaq11. Silencing GRP78 expression with GRP78 dsRNA or MTJ-1 with MTJ-1 dsRNA greatly reduced the levels of Galphaq11 co-precipitated with GRP78 or MTJ-1. In conclusion, we show here that plasma membrane-associated GRP78 is coupled to pertussis toxin-insensitive Galphaq11 and forms a ternary signaling complex with MTJ-1.     

Cytosolic phospholipase A(2) activity associated with nuclei is not inhibited by arachidonyl trifluoromethyl ketone in macrophages stimulated with receptor-recognized forms of alpha(2)-macroglobulin

We have studied the translocation of cytosolic phospholipase A(2) (cPLA(2)) to nuclei in macrophages stimulated with receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*). Translocation of phosphorylated cPLA(2) to nuclei was determined by immunoprecipitation of cPLA(2) in (32)P(i)-labeled cells. The identity of cPLA(2) was established by comparing its mobility on gels with an authentic cPLA(2) standard. cPLA(2) activity was quantified by measuring the release of [(14)C]arachidonic acid from the substrate 1-palmitoyl-2-[1-(14)C]arachidonyl-sn-glycerophosphatidylcholine. alpha(2)M* caused a two- to threefold increase in cPLA(2) phosphorylation and its translocation to nuclei. The p38 MAPK inhibitor SB203580, PKC inhibitor chelerythrin, or depletion of intracellular Ca(2+) profoundly decreased cPLA(2) activity in nuclei isolated from agonist-stimulated cells. The requirement for Ca(2+), PKC, and p38 MAPK activation appears to be of major importance for nuclear cPLA(2) activity. In contrast to cellular cPLA(2) activity, nuclear cPLA(2) activity was not inhibited by arachidonyl trifluoromethyl ketone (AACOCF(3)) in agonist-stimulated cells. It is concluded that the association of cPLA(2) with nuclear membranes in agonist-stimulated cells modifies the activity and the sensitivity of the enzyme to inhibition by AACOCF(3) in this phospholipid environment.     

Ligation of cell surface-associated glucose-regulated protein 78 by receptor-recognized forms of alpha 2-macroglobulin: activation of p21-activated protein kinase-2-dependent signaling in murine peritoneal macrophages

Previous studies of the plasma proteinase inhibitor alpha2-macroglobulin (alpha2M) demonstrated that alpha2M-proteinase complexes (alpha2M*) modulate immune responses and promotes macrophage locomotion and chemotaxis. Alpha2M* binds to cell surface-associated glucose-regulated protein 78 (GRP78), which activates downstream signaling events. The role of p21-activated protein kinase-1 and -2 (PAK-1 and -2) in promoting cellular motility is well documented. In the current study, we examined the ability of alpha2M* to activate PAK-1 and PAK-2. Upon macrophage stimulation with alpha2M*, PAK-2 is autophosphorylated, resulting in increased kinase activity; however, PAK-1 is negligibly affected. Alpha2M*-stimulated macrophages showed a marked elevation in the levels of Rac x GTP. Receptor tyrosine phosphorylation upon binding of alpha2M* to GRP78, recruits PAK-2 to the plasma membrane via the adaptor protein NCK. Consistent with this hypothesis, silencing of GRP78 gene expression greatly attenuated the levels of membrane-associated PAK-2 and NCK. PAK-2 activity was markedly decreased by inhibition of tyrosine kinases and PI3K before alpha2M* stimulation. We further demonstrate that phosphorylation of Lin-11, Isl-1, Mec-3 (LIM) kinase and cofilin is promoted by treating macrophages with alpha2M*. Thus, alpha2M* regulates activation of the PAK-2-dependent motility mechanism in these cells.     

Partially Purified RhoA-Stimulated Phospholipase D Activity Specifically Binds to Phosphatidylinositol 4,5-Bisphosphate

Abstract: Phosphatidylinositol 4,5-bisphosphate (PIP₂) is absolutely required for the ADP-ribosylation factor-stimulated phospholipase D (PLD) activity. In the present study, partially purified rat brain PLD was found to be activated by another PLD activator, RhoA, when PIP₂, but not other acidic phospholipids, was included in vesicles comprising phosphatidylethanolamine (PE) and the PLD substrate phosphatidylcholine (PC) (PE/PC vesicles), demonstrating the absolute requirement of PIP₂ for the RhoA-stimulated PLD activation, too. It is interesting that the RhoA-dependent PLD activity in the partially purified preparation was drastically decreased after the preparation was incubated with and separated from PE/PC vesicles containing PIP₂. The PLD activity was extracted by higher concentrations of NaCl from the vesicles containing PIP₂ that were incubated with and then separated from the partially purified PLD preparation. These results demonstrate that RhoA-dependent PLD binds to PE/PC vesicles with PIP₂. The degree of binding of the RhoA-dependent PLD activity to the vesicles was totally dependent on the amount of PIP₂ in the vesicles and correlated well with the extent of the enzyme activation. Furthermore, it was found that a recombinant peptide of the pleckstrin homology domain of β-adrenergic receptor kinase fused to glutathione S-transferase, which specifically binds to PIP₂, inhibited the PIP₂-stimulated, RhoA-dependent PLD activity in a concentration-dependent manner. From these results, it is concluded that in vitro rat brain PLD translocates to the vesicles containing PIP₂, owing to its specific interaction with PIP₂, to access its substrate PC, thereby catalyzing the hydrolysis of PC. PLD appears to localize exclusively on plasma membranes of cells and tissues. An aminoglycoside, neomycin, that has high affinity for PIP₂ effectively extracted the RhoA-dependent PLD activity from rat brain membranes. This indicates that PIP₂ serves as an anchor to localize PLD on plasma membranes in vivo.     

Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea.

Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly puridied nuclei exhibited specific binding with 125I-labeled human choriogonadotropin, [3H]prostaglandin E1 and [3H]prostaglandin F2 alpha. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E1) and 8.9% (prostaglandin F2 alpha) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F2 alpha binding site and 5'-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21--26% (prostaglandins) of original specific binding despite virtual disappearance of 5'-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound 125I-labeled human choriogonadotropin and [3H]prostaglandin F2 alpha to the same extent or significantly more ([3H]prostaglandin E1, P less than 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.     

Inducible expression of the alpha2-macroglobulin signaling receptor in response to antigenic stimulation: a study of second messenger generation.

Thioglycollate (TG)-elicited murine, peritoneal macrophages express two receptors for activated forms of the proteinase inhibitor alpha2-macroglobulin (alpha2M*)--namely, the low density lipoprotein receptor-related protein (LRP) and the alpha2M signaling receptor (alpha2MSR). We now report that resident peritoneal macrophages express only 400+/-50 alpha2MSR receptors/cell compared to 5000+/-500 receptor/TG-elicited macrophage. By contrast, LRP expression is only 2-2.5-fold greater on elicited cells. The low level of alpha2MSR expression by resident cells is insufficient to trigger signal transduction in contrast to TG-elicited cells which when exposed to alpha2M* demonstrate a rapid rise in inositol 1,4,5-trisphosphate and a concomitant increase in cytosolic free Ca2+. We then studied a variety of preparations injected subcutaneously for their ability to upregulate alpha2MSR. Macroaggregated bovine serum albumin (macroBSA) injection upregulated alpha2MSR and triggered signaling responses by splenic macrophages. Nonaggregated BSA injection alone or in the presence of alum, by contrast, did not alter alpha2MSR expression. Recombivax (hepatitis B antigen adsorbed to alum) injection also upregulated alpha2MSR on splenic macrophages while the alum carrier had no effect. We conclude that macrophage alpha2M* receptors are inducible and their expression may be regulated, in part, by potential antigens.