The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain

The lymphocyte-specific phosphoprotein LSP1 associates with the cytoplasmic face of the plasma membrane and with the cytoskeleton. Mouse LSP1 protein contains 330 amino acids and contains an NH2-terminal acidic domain of approximately 177 amino acids. The COOH-terminal half of the LSP1 protein is rich in basic residues. In this paper we show that LSP1 protein which is immunoprecipitated with anti-LSP1 antibodies from NP-40-soluble lysates of the mouse B-lymphoma cell line BAL17 is associated with actin. In vitro binding experiments using recombinant LSP1 (rLSP1) protein and rabbit skeletal muscle actin show that LSP1 binds along the sides of F-actin but does not bind to G-actin. rLSP1 does not alter the initial polymerization kinetics of actin. The highly conserved COOH-terminal basic domains of mouse and human LSP1 share a significant homology with the 20-kD COOH-terminal F-actin binding fragment of caldesmon. A truncated rLSP1 protein containing the entire COOH-terminal basic domain from residue 179 to 330, but not the NH2-terminal acidic domain binds to F-actin at least as well as rLSP1. When LSP1/CAT fusion proteins are expressed in a LSP1-negative T-lymphoma cell line, only fusion proteins containing the basic COOH-terminal domain associate with the NP-40-insoluble cytoskeleton. These data show that LSP1 binds F-actin through its COOH-terminal basic domain and strongly suggest that LSP1 interacts with the cytoskeleton by direct binding to F-actin. We propose that LSP1 plays a role in mediating cytoskeleton driven responses in lymphocytes such as receptor capping, cell motility, or cell-cell interactions.     

Human lymphocyte-specific protein 1, the protein overexpressed in neutrophil actin dysfunction with 47-kDa and 89-kDa protein abnormalities (NAD 47/89), has multiple F-actin binding domains

Human lymphocyte-specific protein 1 (LSP1) is an F-actin binding protein, which has an acidic N-terminal half and a basic C-terminal half. In the basic C-terminal half, there are amino acid sequences highly homologous to the actin-binding domains of two known F-actin binding proteins: caldesmon and the villin headpieces (CI, CII, VI, VII). However, the exact numbers and locations of the F-actin binding domains within LSP1 are not clearly defined. In this report, we utilized 125I-labeled F-actin ligand blotting and high-speed F-actin cosedimentation assays to analyze the F-actin binding properties of truncated LSP1 peptides and to define the F-actin binding domains. Results show that LSP1 has at least three and potentially a fourth F-actin binding domain. All F-actin binding domains are located in the basic C-terminal half and correspond to the caldesmon and villin headpiece homologous regions. LSP1 181-245 and LSP1 246-295, containing sequences homologous to caldesmon F-actin binding site I and II, respectively (CI, CII), binds F-actin; similarly, LSP1 306-339 can bind F-actin and contains two inseparable villin headpiece-like F-actin binding domains (VI, VII). Although LSP1 1-305, which does not contain VI and VII regions, retains F-actin binding activity, its binding affinity for F-actin is much weaker than that of full-length LSP1. Site-directed mutagenesis of the basic amino acids in the KRYK (VI) or KYEK (VII) sequences to acidic amino acids create mutants that bind F-actin with lower affinity than full-length wild-type LSP1. High KCl concentrations decrease full-length LSP1 binding to F-actin, suggesting the affinity between LSP1 and F-actin is mainly through electrostatic interaction.     

Lymphocyte-specific Ca2+-binding protein LSP1 is associated with the cytoplasmic face of the plasma membrane.

The gene for LSP1 is a lymphocyte-specific gene previously isolated by us using a subtractive hybridization technique. LSP1 mRNA is found in normal and transformed B lymphocytes and in normal T lymphocytes but not in transformed T lymphocytes. To study the expression of the mouse LSP1 protein, we prepared a polyclonal antiserum specific for the LSP1 protein. Here we report that the gene for LSP1 was expressed in transformed B-lymphoma cell lines and in normal mouse thymocytes as a protein doublet with apparent molecular masses of 52 and 50.5 kilodaltons when analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel. BW5147 cells transfected with an LSP1 cDNA clone expressed only the 52-kilodalton protein. No LSP1 protein was expressed in nine T-lymphoma cell lines tested. Immunofluorescence studies of intact and permeabilized cells and subcellular fractionation experiments showed that the LSP1 protein was associated with the cytoplasmic side of the plasma membrane in transformed B-lymphoma cell lines and in normal thymocytes. Using a simple filter-binding assay, we showed that recombinant LSP1 protein was Ca2+ binding, as predicted on the basis of its deduced amino acid sequence. On the basis of the particular expression pattern, the subcellular localization, and the Ca2+-binding property of the LSP1 protein, we hypothesize that the LSP1 protein is a lymphocyte-specific component of a signal transduction pathway involved in the regulation of lymphocyte growth.     

MAPKAPK2-mediated LSP1 phosphorylation and FMLP-induced neutrophil polarization

In neutrophils, the major substrate of MAPKAPK2 (MK2) is an F-actin binding protein LSP1. Studies using mutants of the two potential Serine phosphorylation sites in LSP1 C-terminal F-actin binding region indicated that the major phosphorylation site for MK2 is Ser243 in murine neutrophils (Ser252 in humans). Human phosphoLSP1 antibodies that recognize phosphoSer252 site were prepared and revealed fMLP-induced neutrophil LSP1 phosphorylation. The phosphorylation was inhibited by p38 MAPK (upstream kinase for MK2) inhibitor SB203580. The antibodies also detect LSP1 phosphorylation in murine neutrophils. Immunostaining revealed that in WT murine neutrophils phosphoLSP1 was localized in F-actin enriched lamellipodia and oriented toward the fMLP gradient while non-phosphoLSP1 failed to colocalize with F-actin. In suspension, WT neutrophils exhibited persistent F-actin polarization following fMLP stimulation, while MK2(-/-) neutrophils exhibited transient F-actin polarization. These studies suggest that MK2-regulated LSP1 phosphorylation is involved in stabilization of F-actin polarization during neutrophil chemotaxis.     

Actin filament binding by a monomeric IQGAP1 fragment with a single calponin homology domain

IQGAP1 is a homodimeric protein that reversibly associates with F-actin, calmodulin, activated Cdc42 and Rac1, CLIP-170, beta-catenin, and E-cadherin. Its F-actin binding site includes a calponin homology domain (CHD) located near the N-terminal of each subunit. Prior studies have implied that medium- to high-affinity F-actin binding (5-50 microM K(d)) requires multiple CHDs located either on an individual polypeptide or on distinct subunits of a multimeric protein. For IQGAP1, a series of six tandem IQGAP coiled-coil repeats (IRs) located past the C-terminal of the CHD of each subunit support protein dimerization and, by extension, the IRs or an undefined subset of them were thought to be essential for F-actin binding mediated by its CHDs. Here we describe efforts to determine the minimal region of IQGAP1 capable of binding F-actin. Several truncation mutants of IQGAP1, which contain progressive deletions of the IRs and CHD, were assayed for F-actin binding in vitro. Fragments that contain both the CHD and at least one IR could bind F-actin and, as expected, removal of all six IRs and the CHD abolished binding. Unexpectedly, a fragment called IQGAP1(2-210), which contains the CHD, but lacks IRs, could bind actin filaments. IQGAP1(2-210) was found to be monomeric, to bind F-actin with a K(d) of approximately 47 microM, to saturate F-actin at a molar ratio of one IQGAP1(2-210) per actin monomer, and to co-localize with cortical actin filaments when expressed by transfection in cultured cells. These collective results identify the first known example of high-affinity actin filament binding mediated by a single CHD.     

Specificity of binding of the plectin actin-binding domain to beta4 integrin

Plectin is a major component of the cytoskeleton and links the intermediate filament system to hemidesmosomes by binding to the integrin beta4 subunit. Previously, a binding site for beta4 was mapped on the actin-binding domain (ABD) of plectin and binding of beta4 and F-actin to plectin was shown to be mutually exclusive. Here we show that only the ABDs of plectin and dystonin bind to beta4, whereas those of other actin-binding proteins do not. Mutations of the ABD of plectin-1C show that Q131, R138, and N149 are critical for tight binding of the ABD to beta4. These residues form a small cavity, occupied by a well-ordered water molecule in the crystal structure. The beta4 binding pocket partly overlaps with the actin-binding sequence 2 (ABS2), previously shown to be essential for actin binding. Therefore, steric interference may render binding of beta4 and F-actin to plectin mutually exclusive. Finally, we provide evidence indicating that the residues preceding the ABD in plectin-1A and -1C, although unable to mediate binding to beta4 themselves, modulate the binding activity of the ABD for beta4. These studies demonstrate the unique property of the plectin-ABD to bind to both F-actin and beta4, and explain why several other ABD-containing proteins that are expressed in basal keratinocytes are not recruited into hemidesmosomes.     

Mouse IgA Fc receptor on CD3+ T cells. Molecular forms of IgA that bind to a 38-kDa Fc alpha R protein and development of an anti-Fc alpha R antisera

Previous studies have shown that splenic T cells from mice that bear IgA myelomas, as well as certain T cell lines, express receptors for the Fc of IgA, and are termed Fc alpha R. In this study, we have isolated and characterized two CD3+ T cell lines derived by fusion of murine Peyer's patch (PP) CD4+ T cells with the BW 5147 lymphoma cell line. These cell lines, designated PPT4-6 and PPT4-16, were shown to bind monomeric or dimeric IgA, whereas the fusion partner did not bind either form of IgA. However, polymeric IgA (m.w. 600,000) bound equally well to all three cell lines. Similar results were also obtained with two known Fc alpha R+ T cell lines, ThHA1 nos. 9 and 10. Immunoprecipitation studies with IgA on PPT4-16 and ThHA1 no. 9 have shown that IgA binds to a 38-kDa protein. A rabbit antiserum was prepared to a 38-kDa fraction of Fc alpha R+ T cell membranes, and heterophilic antibody was removed from the antiserum by adsorption with mouse thymocytes, BW 5147 and R1.1 lymphoma. The antiserum bound to both PPT4-16 and ThHA1 no. 9 as well as to other Fc alpha R+ T cells, but did not bind to thymocytes or to the T lymphomas R1.1 or BW 5147. The antiserum appeared specific for the Fc alpha R, because it failed to block binding of anti-CD3 (145 2C11) or other surface molecule-specific antibodies. Further, competitive inhibition studies with IgA and anti-Fc alpha R (38 kDa) showed that preincubation of Fc alpha R+ T cells with the anti-38-kDa protein completely eliminated IgA binding, whereas IgA partially blocked the binding of the anti-Fc alpha R antibodies to the cell membrane. Immunoisolation with the anti-Fc alpha R antibody of radioiodinated cell membrane proteins from Fc alpha R+ T cells, but not from Fc alpha R- cells, gave a distinct band at 38 kDa. To further test the specificity of this antiserum, we have isolated T cells from spleens of IgA-myeloma bearing mice, and tested the phenotype and IgA binding. A subset consisting of 15 to 20% of CD3+, CD8+ T cells was found that bound monomeric or dimeric IgA. Further, the anti-Fc alpha R antiserum also recognized this CD8+ T cell subset, and preincubation of the cells with antibody resulted in their failure to bind IgA. Our results indicate that the Fc alpha R on T cell lines derived from PP is a 38-kDa protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape. Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling. A number of proteins can bind to the actin cytoskeleton. The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin. The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin. Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.Download video file.^{(136M, mov)}     

Analysis of H-2-linked immune responses involved in resistance to AKR tumor growth

H-2-associated immune response gene(s) govern resistance to growth of a spontaneous AKR lymphoma, BW5147. The antigenic specificities recognized by the anti-BW5147 humoral response have been characterized and include: Thy-1, a T -cell differentiation antigen; gp70, a retroviral envelope protein; and several previously uncharacterized proteins, including a 78 000 molecular mass protein, p78, which is restricted to expression on BW5147 cells and five phosphoproteins with molecular masses of 33 000, 29 000, 23 000, 17 000, and 16 000. Only mice which are able to respond to Thy-1, p78, and the phosphoproteins can survive an inoculation of BW5147. Thus, resistance to BW5147 is complex and involves multiple antigens with possible roles in tumor rejection.Abbreviations used in this paper DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - Ir immune response - MuLV murine leukemia virus - NMS normal mouse serum - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Purification of KBF1, a common factor binding to both H-2 and beta 2-microglobulin enhancers.

An enhancer binding factor, designated KBF1, has been purified from the nuclear extract of mouse BW5147 thymoma cells by five column chromatography steps including a sequence-specific DNA affinity column. Gel retardation and footprint analysis have shown that purified KBF1 has a binding activity specific for both H-2 and beta 2-microglobulin enhancer sequences. After SDS-polyacrylamide gel electrophoresis of the most purified preparation a 48-kd protein showed, after elution and renaturation, a binding activity to both enhancer sequences. These findings suggest that the expression of both H-2 and beta 2-microglobulin genes utilizes a common regulatory mechanism.     

Hair-forming activity of human lymphocyte specific protein 1 requires cooperation between its caldesmon-like domains and the villin headpiece-like domains.

LSP1 is an F-actin binding with multiple F-actin binding domains. Overexpression of LSP1 in NAD 47/89 patient's neutrophils created hair-like projections on the patient's neutrophil cell surfaces and inhibited neutrophil cell motility and transfection of LSP1 in serial cell lines recreate the NAD 47/89 phenotype and produce branching hair-like surface projections. Although LSP1 contains hair-forming ability and LSP1 F-actin binding domains have been defined, the LSP1 domains responsible for its hair-forming activity, the relationship to the F-actin binding domains, and the required domain interactions, if any, for hair formation are not well understood. To define the hair-forming domains of LSP1, the relationship to the known F-actin binding domains, and binding domain interactions, LSP1 truncates, which include or exclude the different F-actin binding domains, were created by PCR. LSP1 mutants were created by site-directed mutagenesis to define the amino acids important for hair formation. Sf9 cells were infected with recombinant baculovirus expressing the cDNA of LSP1 truncates and mutants, and the morphology of infected Sf9 cells was documented by DIC optics. Results show that (1) the hair-forming activity of LSP1 is localized to the basic C-terminal half of the molecule, which contains all of the F-actin binding domains; (2) both the caldesmon-like domains and the villin headpiece-like domains are required for the hair-forming activity of LSP1; (3) basic amino acids in the villin headpiece regions are crucial for the hair-forming activity of LSP1 molecule. The results suggest cooperation between the caldesmon-like domains and the villin headpiece-like domains are required for the hair-forming activity of human LSP1 in cells.     

Studies of the biosynthesis of the mannose 6-phosphate receptor in receptor-positive and -deficient cell lines

Phosphomannosyl residues present on lysosomal enzymes are specifically recognized by the mannose 6-phosphate receptor protein. This interaction results in the selective targeting of lysosomal enzymes to lysosomes. While this pathway is operative in many cell types, we have found four cultured cell lines that are deficient in the ability to bind lysosomal enzymes containing phosphomannosyl residues to their intracellular or surface membranes (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). These cells appear to segregate lysosomal enzymes by an alternate intracellular pathway. To determine the basis for the lack of mannose 6-phosphate receptor activity in these cell lines, we studied the biosynthesis of the receptor in receptor-positive (BW5147) and receptor-deficient (P388D1 and MOPC 315) cells. The cells were labeled with [2-3H]mannose or [35S]methionine and the receptor was immunoprecipitated with an antireceptor antiserum. BW5147 cells synthesize a receptor protein whose size increases after translation/glycosylation. MOPC 315 cells produce an apparently normal receptor and degrade it rapidly. P388D1 cells fail to synthesize any detectable receptor. The receptor from BW5147 and MOPC 315 cells is a glycoprotein with both high mannose and complex asparagine-linked oligosaccharides. The complex-type units become fully sialylated and remain so during long periods of chase.     

F-actin binding region of SPIN90 C-terminus is essential for actin polymerization and lamellipodia formation

We recently reported that SPIN90 is able to bind with several proteins involved in regulating actin cytoskeleton networks, including dynamin, WASP, β PIX, and Nck. Based on these findings, we investigated how SPIN90 regulates the actin cytoskeleton and promotes actin assembly. This study demonstrated that aluminium fluoride-induced localization of SPIN90 to lamellipodia requires amino acids 582-722 at the SPIN90 C-terminus, which is also essential for F-actin binding and Arp2/3 complex mediated polymerization of actin into branched actin filaments. Furthermore, after deletion of the F-actin binding region (582-722 SPIN90) failed to localize at the membrane edge and was unable to promote lamellipodia formation, suggesting that the F-actin binding region in the SPIN90 C-terminus is essential for the formation of branched actin networks and regulation of the actin cytoskeleton at the leading edge of cells.     

Molecular cloning and characterization of a novel mouse actin-binding protein Zfp185

Zinc finger protein (Zfp) 185 is a mouse protein containing a Lin-l1, Isl-1 and Mec-3 (LIM) domains at its C-terminus. It was recognized by comparing the genome sequence between humans and mice in 1997. In this study, we cloned the full-length Zfp185 by means of RACE and RT-PCR. Zfp185 may be closely associated with F-actin in cells as determined by a confocal microscopy. With a series of deletants of Zfp185 and GST-pull-down assay, we determined that N-terminus region (1–144) but not the LIM domain at C-terminus of Zfp185 protein was essential and sufficient to bind to F-actin cytoskeleton. Thus, our data offered evidence for the association of mouse Zfp185 with F-actin, which supports the potential role of Zfp185 in cell fundamental activity.     

Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin

Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein-protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-58(1267N) mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.     

Biochemical characterisation of the actin-binding properties of utrophin

Utrophin is a large ubiquitously expressed cytoskeletal protein that is important for maturation of vertebrate neuromuscular junctions. It is highly homologous to dystrophin, the protein defective in Duchenne and Becker muscular dystrophies. Utrophin binds to the actin cytoskeleton via an N-terminal actin-binding domain, which is related to the actin-binding domains of members of the spectrin superfamily of proteins. We have determined the actin-binding properties of this utrophin domain and investigated its binding site on F-actin. An F-actin cosedimentation assay confirmed that the domain binds more tightly to beta-F-actin than to alpha-F-actin and that the full-length utrophin domain binds more tightly to both actin isoforms than a truncated construct, lacking a characteristic utrophin N-terminal extension. Both domain constructs exist in solution as compact monomers and bind to actin as 1:1 complexes. Analysis of the products of partial proteolysis of the domain in the presence of F-actin showed that the N-terminal extension was protected by binding to actin. The actin isoform dependence of utrophin binding could reflect differences at the N-termini of the actin isoforms, thus localising the utrophin-binding site on actin. The involvement of the actin N-terminus in utrophin binding was also supported by competition binding assays using myosin subfragment S1, which also binds F-actin near its N-terminus. Cross-linking studies suggested that utrophin contacts two actin monomers in the actin filament as does myosin S1. These biochemical approaches complement our structural studies and facilitate characterisation of the actin-binding properties of the utrophin actin-binding domain.     

The COOH terminus of the c-Abl tyrosine kinase contains distinct F- and G-actin binding domains with bundling activity [published erratum appears in J Cell Biol 1994 Mar;124(5):865]

The myristoylated form of c-Abl protein, as well as the P210bcr/abl protein, have been shown by indirect immunofluorescence to associate with F-actin stress fibers in fibroblasts. Analysis of deletion mutants of c-Abl stably expressed in fibroblasts maps the domain responsible for this interaction to the extreme COOH-terminus of Abl. This domain mediates the association of a heterologous protein with F-actin filaments after microinjection into NIH 3T3 cells, and directly binds to F-actin in a cosedimentation assay. Microinjection and cosedimentation assays localize the actin-binding domain to a 58 amino acid region, including a charged motif at the extreme COOH-terminus that is important for efficient binding. F-actin binding by Abl is calcium independent, and Abl competes with gelsolin for binding to F- actin. In addition to the F-actin binding domain, the COOH-terminus of Abl contains a proline-rich region that mediates binding and sequestration of G-actin, and the Abl F- and G-actin binding domains cooperate to bundle F-actin filaments in vitro. The COOH terminus of Abl thus confers several novel localizing functions upon the protein, including actin binding, nuclear localization, and DNA binding. Abl may modify and receive signals from the F-actin cytoskeleton in vivo, and is an ideal candidate to mediate signal transduction from the cell surface and cytoskeleton to the nucleus.     

Alterations in expression and glycosylation pattern of the Thy-1 glycoprotein during maturation and transformation of mouse T lymphocytes

The mouse Thy-1 glycoprotein of normal and transformed lymphoid cells was studied with regard to amount per cell, apparent m.w., and glycosylation characteristics. Thy-1 was measured by a solid-phase radioimmunoassay calibrated with pure mouse brain Thy-1. Thymocytes were shown to contain five times the amount of Thy-1 found in lymph node cells (1 X 10(6) vs 2 X 10(5) molecules per cell), whereas the T cell lymphomas studied (P52-127-166, RBL-5, YWA, Y191, Y274, YAC-1, RL male 1, and BW5147) varied in their Thy-1 content. The apparent m.w. of Thy-1, as determined by SDS-PAGE, was in all cases 25,000 to 30,000. However, the appearance of the Thy-1 bands revealed a size heterogeneity that was less pronounced with material from lymph node cells than from thymocytes. This band broadening seemed to be inversely correlated to the affinity for lentil lectin. Whereas half the Thy-1 molecules from thymocytes were bound to the lectin, lymph nodes Thy-1 showed 75% binding. All T lymphomas but one (BW5147) contained Thy-1 also heterogeneous in lentil lectin binding. The charge, previously shown to be dependent on the sialic acid content, was shifted to more acidic forms for lymph node Thy-1 compared to thymocytes. The T lymphomas possessed Thy-1 with charge properties similar to those of the thymocytes; the only exception was BW5147, which showed more basic forms. These results show that the expression and the glycosylation of Thy-1 is altered when thymocytes mature into immunocompetent cells and after malignant transformation of lymphocytes.     

Dynamics of toxin and lectin receptors on a lymphoma cell line and its toxin-resistant variant using ferritin-conjugated, 125I-labeled ligand

The dynamics of the toxin Ricinus communis agglutinin II (RCAII or ricin) on cells of a murine lymphoma line (BW5147) and a toxin-resistant variant line (BW5147RicR.3) that is 200 times more resistant than the parent to direct RCAII cytotoxicity were examined using ferritin-conjugated, affinity purified, 125I-labeled RCAII (ferritin-125I-RCAII). Ferritin-125I-RCAII was indistinguishable from native RCAII in quantitative binding and cytotoxicity experiments. When RCAII-sensitive BW5147 and -resistant BW5147RicR.3 cells were labeled with ferritin-125I-RCAII at various toxin concentrations (1--10 microgram/ml), no differences in toxin binding were observed. These same cells were examined by electron microscopy. At low ferritin-125I-RCAII concentrations (1-3 microgram/ml RCAII) where only the parental BW5147 cells were significantly more sensitive to RCAII, toxin receptors were internalized by ferritin-125I-RCAII-induced endocytosis. In parallel experiments, ferritin-125I-RCAII that bound to the resistant BW5147RicR.3 cells remained relatively dispersed or clustered, and there was little evidence of transport into cells via endocytosis. At higher ferritin-125I-RCAII concentrations (greater than 7 microgram/ml RCAII) where both parental and resistant variant cells are sensitive to the cytotoxic effects of RCAII, more ferritin-conjugated toxin was bound, and subsequent endocytosis occurred to a similar degree in both cell types. Endocytosis of ferritin-conjugated concanavalin A was indistinguishable on RCAII-sensitive parental and resistant variant cells at all concentrations tested. The results suggest that a specific defect on the selected BW5147RicR.3 cells prevents RCAII entry into these cells a low toxin concentrations, rendering them more resistant to the cytotoxic effects of RCAII.