Triton X-100 Insoluble Cytoskeleton Research Articles

Flightless-I regulates proinflammatory caspases by selectively modulating intracellular localization and caspase activity

Caspase-1 and caspase-11 are proinflammatory caspases that regulate cytokine production and leukocyte migration during pathogen infection. In an attempt to identify new intracellular regulators of caspase-11, we found that Flightless-I, a member of the gelsolin superfamily of actin-remodeling proteins, interacts and regulates both caspase-11 and caspase-1. Flightless-I targets caspase-11 to the Triton X-100–insoluble cytoskeleton fraction and the cell leading edge. In addition, Flightless-I inhibits caspase-1 activation and caspase-1–mediated interleukine-1β (IL-1β) maturation. The physiological relevance of these findings is supported by the opposing effects of Flightless-I overexpression and knockdown on caspase-1 activity and IL-1β maturation. Our results suggest that Flightless-I may be a bona fide caspase-1 inhibitor that acts through a mechanism similar to that of cytokine response modifier A, a potent caspase-1 inhibitor from the cowpox virus. Our study provides a new mechanism controlling the localization and activation of proinflammatory caspases.

Force transduction by Triton cytoskeletons.

Force-initiated signal transduction can occur either via membrane-based ionic mechanisms or through changes in cytoskeletal–matrix linkages. We report here the stretch-dependent binding of cytoplasmic proteins to Triton X-100 cytoskeletons of L-929 cells grown on collagen-coated silicone. Triton X-100–insoluble cytoskeletons were stretched by 10% and incubated with biotinylated cytoplasmic proteins. Analysis with two-dimensional gel electrophoresis showed stretch-dependent binding of more than 10 cytoplasmic protein spots. Bound cytoplasmic proteins were purified by a photocleavable biotin tag and stretch-dependent binding of paxillin, focal adhesion kinase, and p130Cas was found, whereas the binding of vinculin was unchanged and actin binding decreased with stretch. Paxillin binding upon stretch was morphologically and biochemically similar in vitro and in vivo, that is, enhanced in the periphery and inhibited by the tyrosine phosphatase inhibitor, phenylarsine oxide. Thus, we suggest that transduction of matrix forces occurs through force-dependent conformation changes in the integrated cytoskeleton.

Effects of cytochalasin H, a potent inhibitor of cytoskeletal reorganisation, on platelet function

Platelets contain a well-developed and dynamic cytoskeleton composed mainly of actin and actin-associated proteins. Upon platelet activation there is rapid polymerisation of actin and a marked reorganisation of the platelet cytoskeleton. Cytochalasins are agents that interfere with the polymerisation of actin, and it has recently been discovered that cytochalasin H (CyH) is particularly effective as an inhibitor of the cytoskeletal reorganisation that occurs in platelets following activation by adenosine diphosphate (ADP). Here we have used CyH to inhibit platelet cytoskeletal reorganisation and to determine its effects on various aspects of platelet function. Experiments were performed in hirudinized platelet-rich plasma (PRP) or whole blood obtained from human volunteers. PRP was treated with 10 w M CyH or vehicle, then activated by ADP. The effect of CyH on cytoskeletal reorganisation was determined by SDS-PAGE of the Triton X-100 insoluble cytoskeletons and quantitated by densitometry. Platelet aggregation and aggregate stability in PRP were measured by monitoring changes in light absorbance; aggregation was measured in whole blood via platelet counting. Shape change, P-selectin expression and changes in intracellular calcium were measured using flow cytometry. CyH prevented the normal incorporation of actin, f -actinin and actin-binding protein into the cytoskeleton that occurred following ADP activation, and incorporation of myosin was markedly reduced. Aggregation was only partially inhibited but, more dramatically, the rate of disaggregation following addition of certain agents that interfere with fibrinogen binding to glycoprotein IIb/IIIa on the surface of platelets was markedly increased. The ADP-induced shape change was also inhibited. CyH had no effect on calcium mobilisation. Curiously, expression of P-selectin was potentiated by CyH, suggesting a modulatory role of the cytoskeleton in platelet secretory activity. The results suggest that cytoskeletal reorganisation plays an important role in platelet shape change and aggregation and contributes in a major way to the stability of the aggregates that form.

Characterization of Newly Identified Four Isoforms for a Putative Cytosolic Protein Tyrosine Phosphatase PTP36

In the course of determining the expression profiles of protein tyrosine phosphatases in lactating mammary gland, we found the expression of an isoform for a putative cytosolic and cytoskeleton-associated protein tyrosine phosphatase PTP36. Further detailed RT-PCR and Northern blot analyses revealed the expression of several isoforms for PTP36 in a tissue-dependent manner. We have cloned the cDNAs encoding four truncated isoforms for PTP36 and designated PTP36-A, -B, -C, and -D, respectively. PTP36-A and -C had new sequences generated due to frameshift, whereas PTP36-B and -D were in-frame variants. Gly- and Glu-rich domains and a putative PTP domain were missing from PTP36-A, but the band 4.1 domain remained. PTP36-B retained the band 4.1 and PTP domains but lacked Pro-, Gly- and Glu-rich domains. Most domain structures were lacking in PTP36-C and -D. Interestingly, PTP36-C contained an incomplete band 4.1 domain, but the newly created sequence exhibited high homology to human nebulette, which was also suggested to associate with cytoskeletons. When transiently expressed in COS7 and HEK293 cells, not only the wild type but also all the isoforms were recovered in Triton X-100-insoluble cytoskeleton-associated fractions and this distribution was not affected by mechanical cell detachment and treatment with a kinase inhibitor staurosporine. Such cellular distribution of PTP36 was also observed in stable COS7 clones. Further studies using deletion mutants suggested that the first 30 amino acids as well as the band 4.1 domain of PTP36 were involved in association with Triton X-100 insoluble cytoskeletons. Tissue-dependent expression and deletion in domain structures might reflect the biological significance of the isoforms for PTP36 in certain physiological conditions.

Nickel Enhances Collagen-induced Platelet Activation Acting by Increasing the Organization of the Cytoskeleton

In keratinocytes, osteoclasts and enterocytes, Ni2+ acts as an agonist working through selective activation of the polyvalent cation-sensing receptor. We report here that while Ni2+ alone had no direct ability to induce platelet aggregation or secretion, Ni2+ pretreatment produced these responses when platelets were stimulated with subthreshold concentrations of collagen. In addition, pretreatment with Ni2+ significantly enhanced collagen-induced phospholipase C activation and calcium mobilization. Platelet adhesion to collagen was increased and the inhibition of collagen-induced adhesion normally seen after cytochalasin D treatment was significantly diminished. When Ni2+ was added to platelets alone, tyrosine phosphorylation of p60src was increased. Moreover, Ni2+ enhanced the amount of protein, especially actin, found in the low-speed Triton X-100 insoluble cytoskeleton. Our results indicate that nickel, possibly acting via a platelet cation sensing receptor analogous to that which has been described in other cell types, may cause a rapid tyrosine kinase-dependent cytoskeleton reorganization leading to enhanced adhesion of platelets to collagen and increasing collagen-dependent responses.

Dependence of Epithelial Intercellular Junction Biogenesis on Thapsigargin-sensitive Intracellular Calcium Stores

Perturbation of potentially regulatable endoplasmic reticulum (ER) calcium stores with the Ca-ATPase inhibitor, thapsigargin (TG), perturbs the formation of desmosomes and tight junctions during polarized epithelial cell biogenesis, despite the development of cell contact. In a Madin-Darby canine kidney cell model for intercellular junction assembly, TG treatment inhibited the development of transepithelial electrical resistance (TER), a measure of tight junction assembly, in a dose-dependent manner. The TG-induced inhibition of tight junction assembly was paralleled by a defect in the sorting of the tight junction protein, ZO-1. An even more dramatic delay in sorting of the desmosomal protein, desmoplakin, was observed in the presence of TG. In addition, while both ZO-1 and desmoplakin-I in control cells were shown to become associated with the Triton X-100 insoluble cytoskeleton during intercellular junction assembly, prior treatment with 100 nM TG diminished this biochemical stabilization into the detergent-insoluble fraction, particularly in the case of ZO-1. Although spectrofluorimetric measurements in fura-2 loaded Madin-Darby canine kidney cells confirmed the occurrence of TG-mediated release of calcium from internal stores, total cytosolic calcium during junction assembly remained similar to untreated cells. Therefore, the presence of cytosolic calcium alone is not sufficient for normal intercellular junction biogenesis if intracellular stores are perturbed by TG. The results indicate the presence of calcium-sensitive intracellular mechanisms involved in the sorting and cytoskeletal stabilization of both tight junction and desmosomes and suggest a role for calcium-dependent signaling pathways at an early (possibly common) step in polarized epithelial biogenesis.

Detergent-Resistant Cytoskeleton of the Surface-Activated Platelet Differs From the Suspension-Activated Platelet Cytoskeleton

AbstractThis study contrasts the protein composition of the detergent-resistant cytoskeleton of platelets fully spread on glass with the cytoskeletal composition of resting platelets and platelets aggregated in suspension with thrombin. Complete Triton X-100–insoluble cytoskeletons were isolated from spread, resting, and suspension-activated platelets in the presence of protease inhibitors, solubilized in sodium dodecyl sulfate/EDTA and analyzed on reduced, one-dimensional polyacrylamide gels. The protein composition of the cytoskeletons differed both qualitatively and quantitatively. Most notable were more extensive incorporation of total protein, talin, and vinculin into the cytoskeleton of spread platelets than the cytoskeleton of suspension-activated platelets. Varying the concentration and time of exposure to thrombin during suspension activation did not mimic the cytoskeletal changes of surface activation. Scanning electron microscopy, measurement of lipid phosphorus content, and varying the duration of Triton extraction did not show incomplete solubilization or nonspecific trapping of constituents in the spread platelet cytoskeleton. Proteolysis of talin was minimal in suspension-activated platelets and in platelets spread for 50 minutes. The differences in the detergent-resistant cytoskeletons of surface- and suspension-activated platelets indicate significant divergence in the physiologies of platelet spreading on surfaces and platelet activation in suspension.

Heat shock protein HSP90 and its association with the cytoskeleton: a morphological study.

To investigate the cellular localization of the 90-kilodalton heat shock protein (HSP90) and its interaction with the cytoskeleton, we performed single- and double-staining immunofluorescence microscopy of cytoskeletal proteins and HSP90 in the absence and presence of cytoskeletal inhibitors. As a model, we used a human endometrial adenocarcinoma cell line (Ishikawa cells), which expresses HSP90. We confirmed the recently reported colocalization of HSP90 with microtubules. However, Ishikawa cells treated with 10(-5) M of the antimicrotubule agents colchicine or triethyl lead showed residual filamentous structures stained with anti-HSP90 antibodies, while no microtubules were visualized with anti-tubulin antibodies. In the presence of 10(-5) M cytochalasin B, the microfilament staining of the cells disappeared, while residual filamentous structures were labeled with anti-HSP90 antibodies. Furthermore, Ishikawa cells treated with 10(-5) M triethyl lead and stained with anti-HSP90 antibodies demonstrated residual filamentous structures, clearly different from those of reorganized vimentin intermediate filaments. Conversely, similar reorganized morphology of filamentous structures stained with both anti-HSP90 and anti-cytokeratins antibodies were observed when Ishikawa cells were treated with 2 x 10(-5) M cytochalasin B and 2 x 10(-5) M colchicine. HSP90 was also present in Ishikawa cell preparations of the Triton X-100 insoluble cytoskeleton. In addition, Triton-insoluble cytoskeleton treated with 10(-5). M triethyl lead and double stained with anti-HSP90 and anti-vimentin antibodies demonstrated clearly different filamentous patterns, when exposed on the same photographic plaque.(ABSTRACT TRUNCATED AT 250 WORDS)

Granulocyte-macrophage colony-stimulating factor induces a staurosporine inhibitable tyrosine phosphorylation of unique neutrophil proteins

Human neutrophils treated with chemotactic peptides or phorbol esters demonstrate tyrosine phosphorylation of a subset of proteins. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced a time- and concentration-dependent increase in the tyrosine phosphorylation of at least seven proteins. Three of these proteins with approximate molecular weights of 150, 95, and 70 Kd were unique to neutrophils treated with GM-CSF, and were not seen to be phosphorylated on tyrosine in neutrophils treated with the agonists FMLP or PMA, or the cytokines G-CSF and tumor necrosis factor. We found the 150-Kd protein to be localized within the cell particulate fraction and the 95- Kd protein within the cell cytosol. The 70-Kd phosphotyrosine protein was found in both fractions. When the neutrophils were treated with Triton X-100 (Sigma Chemical Co, St Louis, MO) to evaluate cytoskeletal associations of proteins, the 150 phosphotyrosine protein partitioned with the Triton X-100 insoluble cytoskeleton (TICS), and the 70-Kd protein partitioned with both the TICS and Triton X-100 soluble proteins. The GM-CSF-induced tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor ST638. This was not seen with the putative C-kinase inhibitor, H-7. However, staurosporine was seen to inhibit tyrosine phosphorylation of neutrophil proteins by GM-CSF and in vitro tyrosine kinase activity of isolated neutrophil cytosol and particulate fractions. These data indicate that the three unique GM-CSF- induced phosphotyrosine-containing proteins may be responsible for the unique actions of GM-CSF and that staurosporine inhibits a tyrosine kinase responsible for the phosphorylation of these proteins.

Granulocyte-Macrophage Colony-Stimulating Factor Induces a Staurosporine Inhibitable Tyrosine Phosphorylation of Unique Neutrophil Proteins

Human neutrophils treated with chemotactic peptides or phorbol esters demonstrate tyrosine phosphorylation of a subset of proteins. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced a time- and concentration-dependent increase in the tyrosine phosphorylation of at least seven proteins. Three of these proteins with approximate molecular weights of 150, 95, and 70 Kd were unique to neutrophils treated with GM-CSF, and were not seen to be phosphorylated on tyrosine in neutrophils treated with the agonists FMLP or PMA, or the cytokines G-CSF and tumor necrosis factor. We found the 150-Kd protein to be localized within the cell particulate fraction and the 95-Kd protein within the cell cytosol. The 70-Kd phosphotyrosine protein was found in both fractions. When the neutrophils were treated with Triton X-100 (Sigma Chemical Co, St Louis, MO) to evaluate cytoskeletal associations of proteins, the 150 phosphotyrosine protein partitioned with the Triton X-100 insoluble cytoskeleton (TICS), and the 70-Kd protein partitioned with both the TICS and Triton X-100 soluble proteins. The GM-CSF-induced tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor ST638. This was not seen with the putative C-kinase inhibitor, H-7. However, staurosporine was seen to inhibit tyrosine phosphorylation of neutrophil proteins by GM-CSF and in vitro tyrosine kinase activity of isolated neutrophil cytosol and particulate fractions. These data indicate that the three unique GM-CSF-induced phosphotyrosine-containing proteins may be responsible for the unique actions of GM-CSF and that staurosporine inhibits a tyrosine kinase responsible for the phosphorylation of these proteins.

Effect of Stimulation on the Stabilization of Platelet-Fibrinogen Interactions

The interaction between fibrinogen and stimulated platelets is a multiphasic process that culminates in the stabilization of ligand binding and reduced accessibility of bound fibrinogen to exogenous antibody. The present study was designed to further explore platelet-fibrinogen interactions by examining the effect of agonist on bound fibrinogen expression and interaction with stimulated platelets as a function of time after ligand binding. Two agents were identified, Zn2+ and phorbol myristate acetate (PMA), which support progressive decreases in bound fibrinogen expression on platelets, but fail to support the stabilization of fibrinogen binding. Sixty min after binding to platelets, approximately 80% of bound fibrinogen remained reversibly associated with Zn(2+)- or PMA-treated platelets and failed to associate with the Triton X-100 insoluble cytoskeleton. In contrast, polyclonal anti-fibrinogen antibody binding decreased by more than 66%. Over the same time course, fibrinogen binding to control platelets, stimulated with thrombin or ADP, was not only accompanied by a 70% decrease in antifibrinogen antibody binding, but also an inability of EDTA or excess exogenous fibrinogen to dissociate more than half of platelet-associated fibrinogen, as well as the progressive association of bound fibrinogen with the platelet cytoskeleton. Costimulation of platelets with ZnCl2 and thrombin or ZnCl2 and ADP enhanced overall fibrinogen binding but not the EDTA-resistant component, and prevented the recovery of irreversibly bound fibrinogen with the Triton X-100 insoluble cytoskeleton. Costimulation of PMA- or Zn(2+)-treated platelets with low doses of A23187, however, restored the stabilization of platelet-fibrinogen interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Previous studies indicated a correlation between the formation of EDTA- resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA- resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA- resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet- fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.

Pentoxifylline inhibits actin polymerization in human neutrophils after stimulation by chemoattractant factor

Pentoxifylline (PTX) has been recently reported to stimulate PMN chemotaxis under dense agarose. The present study was designed to characterize the effect of PTX on actin polymerization before and after stimulation by the chemotactic factor f-MLP- We used two different methods to determine the proportion of actin in the filamentous form: SDS-polyacrylamide gel electrophoresis to study the Triton X-100 insoluble cytoskeleton, and flow cytometry using fluorescent Rhodamine-Phalloidin to study actin conformation. PTX (10(-3) M) did not affect the amount of F-actin (polymerized G-actin) incorporated into the cytoskeleton, but reduced total F-actin in a dose-dependent manner, at all concentrations of f-MLP used. Moreover, this inhibitory effect appeared more clearly in PMN with the higher activation ratios. Thus F-actin is only partially incorporated into the cytoskeleton, and PTX-induced reduction of non-incorporated actin may reduce the stiffness of activated PMN. This could explain the increased chemotaxis of PMN across the small holes of dense agarose.

Epidermal growth factor receptors associated to cytoskeletal elements of epidermoid carcinoma (A431) cells.

The structural interaction of the epidermal growth factor (EGF) receptor and the cytoskeleton of A431 cells has been studied using a monoclonal anti-EGF receptor antibody. This has been done with immunogold labeling using a variety of electron microscopical preparation procedures and EGF binding studies. By providing an image of the membrane-associated cytoskeleton, the dry cleavage method reveals a preferential localization of EGF receptors superimposed upon cytoskeletal filaments. The colocalization of gold particles with cytoskeletal filaments is not affected when pre-labeled cells are extracted with the non-ionic detergent Triton X-100, as visualized by dry cleavage. Using surface replication, this treatment results in visualization of the cytoskeleton. In these latter preparations, it is also observed that EGF receptor-coupled gold particles remain associated with cytoskeletal elements. Moreover, Triton extraction performed before immunogold labeling of EGF receptors demonstrates that isolated cytoskeletons contained binding sites for anti-EGF receptor antibodies. Using stereo micrographs of replica's obtained from these isolated cytoskeletons, it is shown that gold-labeled EGF receptors are exclusively present on the cortical membrane-associated region of the cytoskeleton and not on more intracellular-located filaments. Scatchard analysis of EGF binding to cells fixed with glutaraldehyde and treated with Triton X-100 before and after EGF binding indicates that a high affinity EGF binding site is associated with the Triton X-100 insoluble cytoskeleton.

Identification of some plasma membrane proteins of cultured rat pigment epithelial cells by labeling with 125I

Cultured rat retinal pigment epithelial (PE) cells were labeled with 125I by lactoperoxidase (LPO)-catalyzed radioiodination. Examination of 125I-labeled cells by electron microscopic autoradiography suggested that 125I was incorporated into proteins at both the apical and basal surfaces of the PE cells, and into the extracellular matrix (ECM). Analysis of labeled cells by SDS-polyacrylamide gel electrophoresis and autoradiography showed that 125I was incorporated into at least 15 polypeptides. In order to determine which of these labeled proteins were derived from the plasma membrane, 125I-labeled cells were subjected to differential detergent extraction. Triton X-100 (2% v/v), which removed the cell plasma membrane, solubilized only three of the labeled proteins (1 52 000, 1 38 000, 1 23 000 daltons). SDS (0·25 % w/v) completely removed cells from the tissue culture dish and solubilized all but four of the labeled proteins (225 000, 173 000, 87 000 and 72 100 daltons). When 125I-labeled PE cells were mechanically disrupted, and the resulting cell fractions separated by sucrose density gradient centrifugation, labeled proteins separated into two subcellular fractions. One fraction was especially enriched in the 1 52 000, 1 38 000 and 1 23 000 dalton labeled proteins, in addition to the plasma membrane marker enzymes Na +, K +-ATPase, and alkaline phosphodiesterase I. The second fraction was enriched in the 225 000, 1 96 000 and 1 73 000 dalton labeled proteins, the ECM proteins laminin and fibronectin, and the 43 000 actin band. It is proposed that 125I-labeled proteins in the former cell fraction are truly plasma membrane proteins, while those found in the latter cell fraction are a mixture of 125I-labeled ECM and basal plasma membrane proteins. The 1 52 000, 1 38 000 and 1 23 000 dalton labeled proteins of the plasma membrane fraction are glycoproteins and become firmly anchored to the Triton X-100 insoluble cytoskeleton when labeled cells are treated with concanavalin A.

Alteration of binding properties and cytoskeletal attachment of nerve growth factor receptors in PC12 cells by wheat germ agglutinin.

Incubation of PC12 cells preloaded with 125I-nerve growth factor (NGF) reveals rapidly and slowly dissociating binding components indicative of a heterogeneous population of receptors. If the cells are previously exposed to wheat germ agglutinin (WGA) for 30 min, NGF now binds to an apparently homogeneous receptor population which exhibit slow dissociation kinetics. Total binding is also reduced by 50%. If WGA is added subsequent to 125I-NGF, total binding is not diminished, but rapidly dissociating receptors occupied with NGF are all converted to the slowly dissociating form. This conversion of receptors occurs rapidly, reaching completion within 2 min at 37 degrees or 4 degrees C, and is unaffected by metabolic energy poisons, suggesting that WGA-induced slowly dissociating receptors are not the product of internalization. The effects of the lectin are blocked by the sugar N-acetyl-D-glucosamine, and the lectin-induced slowly dissociating receptors are converted back to rapidly dissociating receptors by addition of this same sugar. WGA also affects the association of the NGF receptor with the Triton X-100 cytoskeleton. Greater than 90% of bound 125I-NGF becomes associated with Triton X-100 insoluble cytoskeletons in the presence of the lectin, compared with less than 20% before lectin addition. Cytoskeleton association of the NGF receptor by WGA shows similar kinetics as the conversion of rapidly to slowly dissociating receptors. This interaction may be involved in the alteration of NGF-receptor binding properties produced by this lectin.

Nerve growth factor receptors on PC12 cells: Evidence for two receptor classes with differing cytoskeletal association

PC12, an NGF responsive cell line, exhibits two classes of NGF receptors which we designate “Fast” and “Slow.” Fast receptors, accounting for 75% of specific NGF binding, are distinguished by their rapid rates for association and dissociation of 125I-NGF. At 37°C, binding of 125I-NGF to Fast receptors is 5-fold more rapid than to Slow receptors and dissociation of 125I-NGF from Fast receptors is 40-fold more rapid than from Slow receptors. No evidence was obtained for a ligand-induced conversion of receptors from Fast to Slow characteristics. Scatchard analysis of binding experiments indicates that PC12 cells possess 60,000 specific receptors for NGF of which 15,000 are of the Slow class. Despite having very different kinetic constants, Slow and Fast receptors have similar equilibrium binding constants (about 2 × 10 −10 M) due to cancelling effects of differing association and dissociation rates. Brief digestion of PC12 cells with trypsin before addition of NGF inactivates essentially all Fast receptors without significantly affecting Slow receptors. Therefore Fast and Slow classes of receptors must exist prior to addition of NGF, and the observed receptor heterogeneity is not due to ligand-induced changes. 125I-NGF bound to Slow receptors is preferentially associated with preparations of Triton X-100 insoluble cytoskeletons, while 125I-NGF bound to Fast receptors is solubilized by this procedure. Cytoskeletally associated NGF is almost exclusively associated with the extranuclear cytoskeletal matrix rather than with the nucleus itself. Preparation of nuclei by various methods suggests that the presence of contaminating cytoskeletal elements should be considered in evaluating the existence of translocation and binding of NGF to the nucleus. Inhibition of endocytotic internalization of NGF either by lowering of temperature to O°C or by preincubation of cells with sodium azide in medium lacking glucose does not reduce the slowly released component of bound NGF, nor alter its cytoskeletal association. The possible functional roles of Slow and cytoskeletal receptors are discussed.