Ligand binding characteristics and aggregation behavior of purified cow's milk folate binding protein depends on the presence of amphiphatic substances including cholesterol,phospholipids, and synthetic detergents

Folate binding protein was purified from cow's milk by a combination of cation exchange chromatography and methotrexate-AH-sepharose affinity chromatography. Dilution of the preparation to concentrations of protein less than 10 nM resulted in drastic changes of radioligand (folate) binding characteristics, i.e., a decrease in binding affinity with a change from upward to downward convex Scatchard plots and increased ligand dissociation combined with appearance of weak-affinity aggregated forms of the binding protein on gel filtration. These findings, consistent with a model predicting dimerization between unliganded and liganded monomers, were reversed in the presence of material eluted from the affinity column after adsorption of the protein(cofactor) or cholesterol, phospholipids, and synthetic detergents. The latter amphiphatic substances form micelles and lipid bilayers which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers in the surrounding aqueous medium and thereby prevent association between these monomeric forms prevailing at low concentrations of the protein. Our data have some bearings on studies which show that cholesterol and phospholipids are necessary for the clustering of folate receptors in the cell membrane; a process required for optimum receptor function and internalization of folate.     

Characterization of a High Affinity Folate Binding Protein in Porcine Serum: Ionic Charge, Concentration—Dependent Polymerization and Ligand Binding Mechanism

The folate binding protein in porcine serum, present at concentrations of 50-100 nM, is cationic at near neutral pH as evidenced by ion exchange chromatography. The gel filtration profile of the protein isolated from porcine serum by methotrexate affinity chromatography exhibited one peak at 48 kDa and an additional peak of 91 kDa at higher protein concentrations. This could suggest the involvement of concentration-dependent polymerization phenomena. Binding of [3H] folate was of a high-affinity type with upward convex Scatchard plots and Hill coefficients > 1.0 indicative of apparent positive cooperativity. However, binding to protein isolated from porcine serum after affinity chromatography was biphasic (high/low-affinity) in the absence of Triton X-100, 1 g/l. These findings which are similar to those reported for purified milk folate binding proteins are consistent with a model predicting association between unliganded and liganded monomers to weak-ligand affinity heterodimers. Amphiphatic substances, e.g. Triton X-100, form micelles which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers are hydrophilic in the liganded state) thereby preventing hetecrodimerization. The folate analogue N10 methyl folate was a potent and competitive inhibitor of [3H] folate binding to the folate binding protein, and moreover changed the binding type to apparent negative cooperativity.     

A combination of cation exchange and ligand-affinity chromatography for purification of two molecular species of the folate binding protein in human milk,one equipped with a hydrophobic glycosyl phosphatidylinositol tail: characterization of hydrophobicity and electrical charge

Cation exchange chromatography combined with ligand (methotrexate) affinity chromatography on a column desorbed with a pH-gradient was used for separation and large scale purification of two folate binding proteins in human milk. One of the proteins, which had a molecular size of 27 kDa on gel filtration and eluted from the affinity column at pH 5-6 was a cleavage product of a 100 kDa protein eluted at pH 3-4 as evidenced by identical N-terminal amino acid sequences and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidyl-inositol tail that inserts into Triton X-100 micelles. Chromatofocusing showed that both proteins possessed multiple isoelectric points within the pH range 7-9. The 100 kDa protein exhibited a high affinity to hydrophobic interaction chromatographic gels, whereas this was only the case with unliganded forms of the 27 kDa protein indicative of a decrease in the hydrophobicity of the protein after ligand binding.     

Ligand binding characteristics of two molecular forms,one equipped with a hydrophobic glycosyl phosphatidylinositol tail,of the folate binding protein purified from human milk

Two molecular forms of the folate binding protein were isolated and purified from human milk by a combination of cation exchange- and affinity chromatography. One protein (27 kDa) was a cleavage product of the other 100 kDa protein as evidenced by N-terminal amino acid sequence homology and a reduction in the molecular size of the latter protein to 27 kDa after cleavage of its hydrophobic glycosylphosphatidylinositol tail by phosphatidylinositol-specific phospholipase C. High-affinity binding of [³H]folate was characterized by upward convex Scatchard plots and increasing ligand binding affinity with decreasing concentrations of both proteins. Downward convex Scatchard plots and binding affinities showing no dependence on the protein concentration were, however, observed in highly diluted solutions of both proteins. Radioligand binding was inhibited by folate analogs, and dissociation of radioligand was slow at pH 7.4 but rapid and complete at pH 5.0 and 3.5. Ligand binding quenched the tryptophan fluorescence of the 27 kDa protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. The 27 kDa protein representing soluble folate binding protein exhibited a greater affinity for ligand binding than the 100 kDa protein which possesses a hydrophobic tail identical to the one that anchors the folate receptor to the cell membrane.     

Ionic Charge, Hydrophobicity and Tryptophan Fluorescence of the Folate Binding Protein Isolated from Cow's Milk

A high-affinity folate binding protein was isolated and purified from cow's milk by a combination of cation exchange chromatography and methotrexate affinity chromatography. Chromatofocusing studies revealed that the protein possessed isoelectric points in the pH-interval 8–7. Polymers of the protein prevailing at pH values close to the isoelectric points seemed to be more hydrophobic than monomers present at pH 5.0 as evidenced by hydrophobic interaction chromatography and turbidity (absorbance at 340 nm) in aqueous buffer solutions (pH 5–8). Ligand binding seemed to induce a conformation change that decreased the hydrophobicity of the protein. In addition, Ligand binding quenched the tryptophan fluorescence of folate binding protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. There was a noticeable discordance between the ability of individual folate analogues to compete with folate for binding and the quenching effect.     

Folate Receptors in Malignant and Benign Tissues of Human Female Genital Tract

We have characterized the folate receptor in malignant and benign tissues of human female genital tract (Fallopian tube and benign and malignant tissues of uterus). Radioligand binding displayed characteristics similar to those of other folate binding proteins. Those include a high-affinity type of binding (K=10¹⁰M^–1), apparent positive cooperativity, a slow dissociation at pH 7.4 becoming rapid at pH 3.5, and inhibition of binding by folate analogues. The gel filtration profile of Triton X-100 solubilized tissue contained two large peaks of ³H-folate labelled protein (>=130 and 100kDa) as well as a 25 kDa peak. Only a single band of 70 kDa was seen on SDS-PAGE immunoblotting. The large molecular size forms on gel filtration appear to represent folate receptors having a hydrophobic membrane anchor inserted into Triton X-100 micelles. The folate receptor of female genital tract showed cross-reactivity in ELISA and positive immunostaining with rabbit antibodies against human milk folate binding protein. Variations in the ratio of immunoresponse to total high affinity folic acid binding suggests the presence of multiple isoforms of the receptor in different types of malignant and benign tissues.     

Ligand Binding Characteristics of a Glycosylphosphatidyl Inositol Membrane-Anchored HeLa Cell Folate Receptor Epitope-Related to Human Milk Folate Binding Protein

The folate receptor (FR) in HeLa cells was characterized as to ligandbinding mechanism, antigenic properties and membrane anchor in order toobtain information to be used for the design of biological agentstargeting FR in malignant tumors. The receptor displayed the followingbinding characteristics in equilibrium dialysis experiments(37°C, pH 7.4) with [³H] folate: a high-affinity type of bindingthat exhibited positive cooperativity with a Hill coefficient >1.0and an upward convex Scatchard plot, a slow radioligand dissociation atpH 7.4 becoming rapid at pH 3.5 and inhibition in the presence of otherfolates. The molecular size of the receptor was 100 kDa on gel filtrationwith Triton X-100, or similar to that of high molecular weight human milkfolate binding protein (FBP). The latter protein represents a 25 kDamolecule which equipped with a hydrophobic glycosylphosphatidylinositol (GPI) membrane anchor susceptible to cleavage byphosphatidylinositol specific phospholipase C (PI-PLC) formsmicelles of 100 kDa size with Triton X-100. The HeLa cell FRimmunoreacted with antibodies against purified human milk FBP inELISA, and in a fluorescence activated cell sorting system, whereHeLa cells exposed to increasing concentrations of antibody showed adose-dependent response. Exposure to PI-PLC decreased the fraction ofimmunolabeled cells indicating a linkage of FR to cell membranes by aGPI anchor. HeLa cells incubated with radiofolate showed a continuousuptake with time, however, with a complete suppression of uptake in thepresence of an excess of cold folate. Prewash of cells at acidic pH toremove endogenous folate increased the uptake. Binding and uptake of [³H]folate was increased in cells grown in a folate-deprived medium. The HeLaFR seems to be epitope related to human milk FBP.     

Binding proteins for linear renin-inhibiting peptides in basolateral plasma membranes of rat liver.

A linear hydrophobic peptide, (Code no. EMD 55068), a synthetic renin-antagonist, competitively inhibits the uptake of taurocholate and of another linear peptide (EMD 51921) but not of oleic acid, serine or thiamin hydrochloride into isolated rat liver cells. EMD 55068 was attached to a gel matrix at a position that is not involved in the protein ligand interaction. The gel matrix used did not interact nonspecifically with solubilized proteins from rat liver. The quantity of bound ligand was determined to be 3.6 mg/ml of gel matrix. In the fraction of EDTA extracted hydrophilic membrane-associated proteins, no binding proteins were detected. Affinity chromatography of integral plasma membrane proteins resulted in four protein bands with molecular masses of 46, 49, 53 and 56 kDa in SDS-PAGE. In contrast, solubilized plasma membrane proteins from AS-30D ascites hepatoma cells, which are unable to transport bile acids and linear peptides, did not bind specifically to the affinity matrix.     

Purification and characterization of folate binding proteins from rat placenta

Rat placenta contains virtually no unsaturated (i.e., apo-form) folate binding protein. However, by lowering the pH of a solubilized membrane preparation of this tissue to 3.5, the endogenous bound folate was dissociated from the protein and adsorbed to charcoal. The apo-form of the folate binding protein thus obtained was purified by affinity chromatography using pteroylglutamic acid covalently coupled to Sepharose 4B. A single protein band with an apparent M_r of 36 000 was observed by SDS-polyacrylamide gel electrophoresis of the eluate from the affinity matrix. Western blot of this preparation using a rabbit antiserum raised with the affinity eluate also identified a single 36 kDa protein band. However, peptide sequencing of the N-terminal region of the proteins in the affinity eluate established that it contained two homologous proteins. Computer alignment of the first 22 N-terminal amino acids of each rat placental protein with human, bovine milk and mouse folate binding proteins showed 50–64% identical homology and 27% homology when the eight proteins were aligned together. The affinity of both rat proteins is highest for pteroylglutamic acid (K_a = 1.6 − 109 l/mol) lower for N⁵-methyltetrahydrofolate and substantially lower for N⁵-formyltetrahydrofolate. In the dose-response range studied there was no apparent affinity for methotrexate. The folate binding proteins could be released from a preparation of placental membranes using phospholipase C indicating that these proteins belong to the class of proteins anchored to the plasma membrane by a glycosyl phosphatidylinositol adduct.     

Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei

Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (K_d) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. K_d measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca²⁺, Mg²⁺, and ethylenediammonium²⁺ ions). Monovalent cations (e.g., Na⁺, K⁺, and Tris⁺) were also effective, but only at concentrations in the millimolar range. The K_d for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl₂. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.     

Characterization of a High-Affinity Folate Receptor in Normal and Malignant Human Testicular Tissue

We have characterized the folate receptor in normal and malignant tissue from male gonads. Radioligand binding displayed characteristics typical of other folate receptors. Those included a high-affinity type of binding (K = 10^{10 M–1}), apparent positive cooperativity changing into non-cooperativity at low receptor concentrations, a tendency to increased binding affinity with decreasing receptor concentrations, a slow dissociation at pH 7.4 becoming rapid at pH 3.5 and inhibition by folates, in particular oxidized forms. The gel filtration profile of Triton X-100 solubilized tissue contained a 25 and 100 kDa peak of radioligand-receptor. The latter peak could represent receptor equipped with a hydrophobic membrane anchor that inserts into Triton X-100 micelles. The concentration of radiolabelled receptor ranged from 0.41 nmol/g protein to 1.68 nmol/g protein in specimens of normal testicular tissue from patients with prostatic carcinomas and from 1.54 nmol/g protein to 3.82 nmol/g protein in testicular tissue from young individuals. Compared to normal testicular tissue the concentration of receptor in seminoma tissue was low (0.38–1.27 nmol/g protein) but showed a higher degree of immunoreactivity in the presence of antibodies against human milk folate binding protein as evidenced by ELISA and immunohistochemistry data. Hence a folate receptor isoform homologous to human milk folate binding protein is apparently expressed in seminomas where the total expression of receptor, however, seems to be lower than in normal testicles.     

Competitive Interactions of Ligands and Macromolecular Crowders with Maltose Binding Protein

Cellular signaling involves a cascade of recognition events occurring in a complex environment with high concentrations of proteins, polysaccharides, and other macromolecules. The influence of macromolecular crowders on protein binding affinity through hard-core repulsion is well studied, and possible contributions of protein-crowder soft attraction have been implicated recently. Here we present direct evidence for weak association of maltose binding protein (MBP) with a polysaccharide crowder Ficoll, and that this association effectively competes with the binding of the natural ligand, maltose. Titration data over wide ranges of maltose and Ficoll concentrations fit well with a three-state competitive binding model. Broadening of MBP ¹H­¹⁵N TROSY spectra by the addition of Ficoll indicates weak protein-crowder association, and subsequent recovery of sharp NMR peaks upon addition of maltose indicates that the interactions of the crowder and the ligand with MBP are competitive. We hypothesize that, in the Escherichia coli periplasm, the competitive interactions of polysaccharides and maltose with MBP could allow MBP to shuttle between the peptidoglycan attached to the outer membrane and the ATP-binding cassette transporter in the inner membrane.     

The ligand-mediated affinity of brain-type fatty acid-binding protein for membranes determines the directionality of lipophilic cargo transport

The intracellular transport of lipophilic cargoes is a highly dynamic process. In eukaryotic cells, the uptake and release of long-chain fatty acids (LCFAs) are executed by fatty-acid binding proteins. However, how these carriers control the directionality of cargo trafficking remains unclear. Here, we revealed that the unliganded archetypal Drosophila brain-type fatty acid-binding protein (dFABP) possesses a stronger binding affinity than its liganded counterpart for empty nanodiscs (ND). Titrating unliganded dFABP and nanodiscs with LCFAs rescued the broadening of FABP cross-peak intensities in HSQC spectra from a weakened protein–membrane interaction. Two out of the 3 strongest LCFA contacting residues in dFABP identified by NMR HSQC chemical shift perturbation (CSP) are also part of the 30 ND-contacting residues (out of the total 130 residues in dFABP), revealed by attenuated TROSY signal in the presence of lipid ND to apo-like dFABP. Our crystallographic temperature factor data suggest enhanced αII helix dynamics upon LCFA binding, compensating for the entropic loss in the βC-D/βE-F loops. The aliphatic tail of bound LCFA impedes the charge-charge interaction between dFABP and the head groups of the membrane, and dFABP is prone to dissociate from the membrane upon ligand binding. We therefore conclude that lipophilic ligands participate directly in the control of the functionally required membrane association and dissociation of FABPs.     

Binding of the dystrophin second repeat to membrane di-oleyl phospholipids is dependent upon lipid packing

Dystrophin is the genetically deficient protein in Duchenne Muscular Dystrophy. Its C- and N-terminal ends interact with cytoskeletal and membrane proteins, establishing a link between the cytoskeleton and the extracellular matrix. In a previous study, we showed that there is an interaction between the second repeat of the rod domain and membrane phospholipids, which places tryptophan residues in close contact with the membrane. Here, we examine the binding of the dystrophin repeat-2 to small unilamellar vesicles with varying composition. We find that the protein binds predominantly to di-oleyl-phosphatidylserine. The binding as a function of increasing mol% of DOPS appears to be cooperative due to reduction of dimensionality, greatly enhanced in the absence of salts, and partly modulated by pH. Substituting small by large unilamellar vesicles induces a 30-fold lower affinity of the protein for the membrane phospholipids. However, modifying the packing of the acyl chains by introducing lipids such as phosphatidylethanolamine and cholesterol to the vesicle leads to an approximately 7-fold increase in affinity. Taken together, these results show that the binding involves electrostatic forces in addition to hydrophobic ones.     

Anionic phospholipids are involved in membrane association of FtsY and stimulate its GTPase activity

FtsY, the Escherichia coli homologue of the eukaryotic signal recognition particle (SRP) receptor alpha-subunit, is located in both the cytoplasm and inner membrane. It has been proposed that FtsY has a direct targeting function, but the mechanism of its association with the membrane is unclear. FtsY is composed of two hydrophilic domains: a highly charged N-terminal domain (the A-domain) and a C-terminal GTP-binding domain (the NG-domain). FtsY does not contain any hydrophobic sequence that might explain its affinity for the inner membrane, and a membrane-anchoring protein has not been detected. In this study, we provide evidence that FtsY interacts directly with E.coli phospholipids, with a preference for anionic phospholipids. The interaction involves at least two lipid-binding sites, one of which is present in the NG-domain. Lipid association induced a conformational change in FtsY and greatly enhanced its GTPase activity. We propose that lipid binding of FtsY is important for the regulation of SRP-mediated protein targeting.     

Characterization of the folate-binding proteins associated with the plasma membrane of rat liver

Unsaturated folate-binding proteins (i.e., apo forms) have been identified with the plasma membranes of rat liver by the binding of [3H]pteroylglutamic acid. Normal rat liver contains very little of the folate-binding apoproteins, but the folate-binding capacity increases substantially when the rats are made folate-deficient. This increase appears to be due to unsaturation of the folate-binding holoproteins rather than to synthesis of additional protein, because the binding capacity of the plasma membranes from normal rat liver following dissociation of the bound folate is equivalent to the binding capacity of the preparation from folate-deficient liver. Two molecular forms of folate-binding protein were identified by gel filtration of the solubilized plasma membrane fraction, a high-molecular-weight form (Mr less than 100,000), representing 25% of the binding capacity, and a smaller protein (Mr approximately equal to 55,000), representing 75% of the binding capacity. Whereas the larger species can be solubilized only with a detergent, the smaller form appears to be hydrophilic and dissociates spontaneously from the membrane preparation. The binding of [3H]pteroylglutamic acid by the membrane preparation was specific, saturable, and pH- and temperature-dependent. Scatchard analysis of the binding could be fitted to a curvo-linear plot, indicating at least two orders of binding sites which probably correspond to the two molecular forms identified by gel filtration. Competitive inhibition by folate analogues demonstrated that the apoproteins have higher affinity for oxidized folate than for N5-methyltetrahydrofolate and virtually no affinity for N5-formyltetrahydrofolate or methotrexate.     

Identification of a Mg2+-dependent protease in human placenta which cleaves hydrophobic folate-binding proteins to hydrophilic forms

Hydrophobic folate-binding proteins (FBPs), which are only 5-10 kDa larger than 40-kDa hydrophilic FBPs, bind significant quantities of Triton X-100 micelles and elute as apparent 160-kDa species on Sephacryl S-200 gel filtration in Triton X-100. Detergent-solubilized placental membranes release a major (greater than 95%) 40-kDa hydrophilic FBP species as well as a minor apparent 160-kDa folate binding species when similarly analyzed. We tested the hypothesis that this recovery of predominantly hydrophilic FBPs was mediated by a putative hydrophobic FBP-specific placental protease. When placenta was solubilized in the presence of increasing concentrations of EDTA, there was a progressive increase in apparent 160-kDa folate binding moieties concomitant with a decrease in 40-kDa FBPs. At 20 mM EDTA, a single apparent 160-kDa folate binding species was recovered and the 40-kDa FBPs could not be detected by radioligand binding or specific radioimmunoassay. The apparent 160-kDa species specifically bound radiolabeled folates and were specifically immunoprecipitated by rabbit anti-40-kDa FBP antiserum. On 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose and probing with anti-40-kDa FBP antiserum, the apparent 160-kDa FBPs electrophoresed as 45-kDa species. Detergent binding studies indicated that apparent 160-kDa FBPs were hydrophobic, thus accounting for the molecular weight discrepancy in gel filtration in Triton X-100 versus sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The EDTA-mediated inhibition of conversion of hydrophobic FBPs to hydrophilic FBPs by protease was reversed in a dose-dependent manner by Mg2+. If this protease is physiologically relevant, it could play an important regulatory role in folate transport by influencing the net number of hydrophobic FBPs on the cell surface.     

A Folate Binding Protein in Ascitic Fluid, Serum and Ovarian Tissue of Patients with Ovarian Adenocarcinoma Immunoreacts with Antibodies Against Human Milk Folate Binding Protein

The presence of a folate binding protein which immunoreacts with antibodies against human milk folate binding protein was demonstrated in ascitic fluids from seven patients with ovarian adenocarcinoma. Ascitic fluids collected from two patients with other malignancies contained non-immunoreactive FBP. Tumor tissue specimens from five patients with ovarian carcinoma contained immunoreactive FBP. By contrast to normal ovaries ovarian carcinoma tissue showed positive immunostaining on immunohistochemistry. Ascitic fluids from two patients with ovarian carcinoma exhibited single distinct bands on SDS-PAGE immunoblotting. The gel filtration profile of ovarian carcinoma tissue homogenate from two patients contained 25 and 100 kDa peaks of radioligand-bound and immunoreactive folate binding protein, while ascitic fluid from one of the patients exhibited a large 100 kDa immunoreactive peak with no radioligand binding activity. The immunoreactive non-functional 100 kDa FBP could represent unprocessed precursor FBP. Future studies are necessary to evaluate whether determination of immunoreactive FBP in ovarian adenocarcinomatosis is of any diagnostic value.     

Binding of human C-reactive protein to mouse macrophages is mediated by distinct receptors

Human C-reactive protein (CRP) is known to activate mouse macrophages (M phi) to a tumoricidal state and to serve as an opsonin for M phi. Therefore, cell surface receptors for CRP on mouse M phi were characterized and their relationship to the IgG FcR determined. The specific binding of 125I-CRP to resident or elicited mouse M phi was saturable, reversible, and involved both a high and a low affinity receptor population. Binding of CRP to the mouse M phi cell lines PU5 1.8 and J774 was nearly identical to that observed with peritoneal M phi. The high affinity receptor population had a calculated K of 10 nM and a receptor density of approximately 10(5) sites per cell. Mouse Ig of the IgG2a, IgG2b, or IgG1 isotypes inhibited binding of 125I-CRP to PU5 1.8 cells at concentrations five-fold greater than that of the homologous ligand. In the converse experiment, unlabeled CRP failed to inhibit specific binding of 125I-labeled IgG2a, IgG2b or IgG1. Isolation of CRP binding proteins from surface iodinated PU5 1.8 cells by ligand-affinity chromatography or chemical cross-linking yielded a major protein band of 57 to 60 kDa which appeared to be distinct from the IgG1/IgG2b FcR (FcR-II) membrane proteins. Removal of radiolabeled IgG2b/IgG1 binding membrane proteins by affinity chromatography did not remove CRP-binding proteins. The rat mAb 2.4G2 which inhibits binding of radiolabeled mouse IgG2b, did not inhibit the binding of CRP. A rat polyclonal antiserum to CRP-binding membrane proteins of PU5 1.8 cells inhibited 125I-CRP binding, but not 125IgG2b binding. The rat polyclonal antibody reacted with two 57 to 60 kDa membrane proteins from PU5 1.8 cells that appear to be of a similar size on Western blots. The 125I-CRP was internalized via endosomes and intact CRP subunits could be detected intracellularly. The findings suggest that binding of CRP occurs through a receptor that is distinct from the IgG FcRs, but that CRP-R activity may be influenced by an association with an IgG FcR.