Human histidine-rich glycoprotein: simultaneous purification with antithrombin III and characterization of its gross structure

The simple and simultaneous purification of histidine-rich glycoprotein (HRG) and antithrombin III (AT III) from human plasma and gross structural characterization of HRG have been performed. The purification method consists of two chromatographic procedures using heparin-agarose and DEAE-Sephadex. The yields of HRG and AT III were 22 mg and 70 mg, respectively, from 1 liter of plasma. The purified HRG is a single-chain polypeptide with a molecular weight (Mr) of 75,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, indicating it was the native form of this protein. Cyanogen bromide cleavage of HRG, followed by analysis of the amino acid composition and determination of the amino-terminal sequence of each purified cyanogen bromide fragment established that the gross structure of HRG consisted of three cyanogen bromide fragments; an amino-terminal CN-50 kDa fragment (Mr 50,000) and a carboxy-terminal small fragment of eight amino acids, and a CN-30 kDa fragment (Mr 30,000) between them. As to the amino acid composition of the CN-30 kDa fragment, it had an unusually high content of histidine (25 mol%), suggesting the presence of a histidine-rich region(s) in the carboxy-terminal half of the molecule. These results together with our previous results (Koide, T., Odani, S., & Ono, T. (1982) FEBS Lett. 141, 222-224) and those of Morgan (Morgan, W.T. (1985) Biochemistry 24, 1496-1501) imply that HRG is composed of at least two domains with distinct functional properties; i.e. an amino-terminal domain with heparin-binding ability and a carboxy-terminal domain with heme- and divalent metal-binding abilities.     

Plasma fatty acid levels may regulate the Zn2+-dependent activities of histidine-rich glycoprotein

Histidine-rich glycoprotein (HRG) is a plasma adaptor protein involved in the formation of protein complexes that regulate a number of biological processes in the blood, most notably coagulation and the immune system. Elevated levels of HRG are clinically linked to thrombotic disorders such as blood vessel occlusion. A large body of evidence suggests that Zn²⁺ ions stimulate HRG-complex formation; however, under normal conditions the vast majority of Zn²⁺ in the blood is bound to human serum albumin (HSA). We have previously demonstrated that high levels of fatty acid act as an allosteric switch which disrupts the major Zn²⁺-binding site on HSA. Transient or sustained elevation of plasma fatty acid levels may therefore increase the proportion of plasma Zn²⁺ associated with HRG. We speculate that this mechanism may potentiate an increased risk of thrombosis in individuals with elevated fatty acid levels such as those associated with cancer, obesity and diabetes.     

Histidine-Rich Glycoprotein Can Prevent Development of Mouse Experimental Glioblastoma

Extensive angiogenesis, formation of new capillaries from pre-existing blood vessels, is an important feature of malignant glioma. Several antiangiogenic drugs targeting vascular endothelial growth factor (VEGF) or its receptors are currently in clinical trials as therapy for high-grade glioma and bevacizumab was recently approved by the FDA for treatment of recurrent glioblastoma. However, the modest efficacy of these drugs and emerging problems with anti-VEGF treatment resistance welcome the development of alternative antiangiogenic therapies. One potential candidate is histidine-rich glycoprotein (HRG), a plasma protein with antiangiogenic properties that can inhibit endothelial cell adhesion and migration. We have used the RCAS/TV-A mouse model for gliomas to investigate the effect of HRG on brain tumor development. Tumors were induced with platelet-derived growth factor-B (PDGF-B), in the presence or absence of HRG. We found that HRG had little effect on tumor incidence but could significantly inhibit the development of malignant glioma and completely prevent the occurrence of grade IV tumors (glioblastoma).     

Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein

Histidine-rich glycoprotein (HRG) is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD). We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD) cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.     

Evaluation of peptide/metal ion interactions by UV laser desorption time-of-flight mass spectrometry.

A relatively recent method developed to determine the molecular weights of intact peptides and proteins, matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), has been evaluated as a new means to investigate the metal ion-binding properties of model synthetic peptides. A contiguous sequence of 25 residues on the surface of the 74 kDa human plasma metal-binding transport protein histidine-rich glycoprotein (HRG) has been identified as a bioactive metal-binding domain. The peptide, (GHHPH)5G, was synthesized and evaluated by LDTOF-MS before and after the addition of Cu(II) in solution with 2,5-dihydroxybenzoic acid as the matrix. In the absence of added Cu(II), the major protonated molecular ion (M + H)+ was observed to have a mass equal to its calculated mass (2904.0 Da). In the presence of Cu(II), however, five additional peaks were observed at mass increments of approximately 63.9 Da. The maximum Cu(II)-binding capacity observed for the 26-residue peptide (5 g-atoms/mol) suggested that up to 1 Cu(II) may be bound per 5-residue internal repeat unit (GHHPH) within this peptide; several other monovalent and divalent metal cations were not bound under identical conditions of analysis. The Cu(II)-binding stoichiometry was verified by spectrophotometric titration and by frontal analyses of the immobilized peptide with a solution of Cu(II) ions. These results demonstrate the ability to verify directly the solution-phase binding capacity of metal-binding peptides by LDTOF-MS.     

Evidence for the Interaction between (−)-Epigallocatechin Gallate and Human Plasma Proteins Fibronectin,Fibrinogen, and Histidine-rich Glycoprotein

Human plasma proteins were subjected to affinity chromatography with (–)-epigallocatechin gallate (EGCg)-agarose, and the bound proteins were examined by sodium dodecylsulfate–polyacrylamide gel electrophoresis. A molecular weight evaluation of the protein bands suggested the presence of three proteins, fibronectin, fibrinogen, and a 75-kDa protein. When human serum was used, the 75-kDa protein dominated the bound fraction. The determination of the partial amino acid sequence of a peptide derived by endopeptidase digestion of this fraction suggested the 75-kDa protein to be histidine-rich glycoprotein (HRG). The presence of these proteins in the bound fraction was confirmed by the immunoblotting method. Affinity chromatography of the individual proteins indicated that fibrinogen and HRG had direct affinity for EGCg. Dot binding assays demonstrated the interaction of EGCg with these proteins. The method also showed that only gallate-containing catechins were bound by these proteins. These data suggest that when EGCg is absorbed in the body through the digestive system, it may interact with these proteins in blood plasma.     

High affinity interaction between histidine-rich glycoprotein and the cell surface type ATP synthase on T-cells

Histidine-rich glycoprotein (HRG) is a plasma protein implicated in the innate immune system. In recent studies, we showed that either HRG, or the Arg23-Lys66 glycopeptide derived from HRG, in concert with concanavalin A (Con A), promotes a morphological change and adhesion of the human leukemic T-cell line MOLT-4 to culture dishes, and that cell surface glycosaminoglycan or Fcγ receptors do not participate in this cellular event. In the present study, we identified the α-subunit of ATP synthase as one of the HRG-binding proteins on the surface of T-cells by HRG-derived glycopeptide affinity chromatography and by a peptide mass finger printing method. HRG specifically interacted with mitochondrial ATP synthase with a dissociation constant of 66 nM. The presence of α- and β-subunits of ATP synthase on the plasma membrane of MOLT-4 cell was demonstrated by immunofluorescent staining and FACS analysis. The HRG/Con A-induced morphological changes of MOLT-4 cells were specifically inhibited by a monoclonal antibody against the β-subunit of ATP synthase. These results strongly suggest that the cell surface ATP synthase functions as a binding protein for HRG on MOLT-4 cells, which is required for the morphological changes observed in MOLT-4 cells following treatment with HRG/Con A.     

Histidine-rich glycoprotein blocks collagen-binding integrins and adhesion of endothelial cells through low-affinity interaction with α2 integrin

The plasma protein histidine-rich glycoprotein (HRG) affects the morphology and function of both endothelial cells (ECs) and monocytes/macrophages in cancer. Here, we examined the mechanism of action of HRG's effect on ECs. HRG suppressed adhesion, spreading and migration of ECs specifically on collagen I (COL I) whereas ECs seeded on other extracellular matrix proteins were insensitive to HRG. HRG did not bind specifically to COL I or to the α-integrin binding site on collagen, GFOGER. Furthermore, HRG's inhibition of EC adhesion was not dependent upon heparan sulfate (HS) moieties as heparitinase-treated ECs remained sensitive to HRG. C2C12 cells expressing α2 integrin, the major collagen-binding α-integrin subunit in ECs, showed increased binding of HRG compared with wild type C2C12 cells lacking the α2 subunit. Recombinant α2 I-domain protein bound HRG and to a higher extent when in active conformation. However, the α2 I-domain bound weakly to HRG compared with COL I and the purified α2β1 ectodomain complex failed to retain HRG. We conclude that HRG binds to α2 integrin through low-affinity interactions in a HS-independent manner, thereby blocking EC-adhesion to COL I.     

A heme- and metal-binding hexapeptide from the sequence of rabbit plasma histidine-rich glycoprotein

Rabbit histidine-rich glycoprotein (HRG) binds low-spin heme and metals tightly at several sites that contain histidine. As part of an on-going effort to define and locate the binding sites for these and the other ligands of HRG, the sequence: NH2-Gly-His-Phe-Pro-Phe-His-Trp-... was found in a 16 kDa heme-binding peptide isolated from HRG. The spacing of the histidyl residues in this peptide, which contains the C-terminal 79 residues of HRG, together with molecular modeling suggested that this sequence might constitute one heme binding site of HRG by accommodating heme in a bis-histidyl linkage. Three peptides based on this sequence (I, HFPFHW; II, WHFPFH; and III, HFGFHW) were synthesized, and their ability to bind heme and metals examined. All three peptides bind heme as demonstrated by the changes produced in the absorbance of heme when mixed with the peptides. Substituting glycine for proline in the central position or moving the location of the tryptophan did not affect heme binding. The apparent Kd's of the mesoheme/peptide I, II and III complexes are 75 +/- 25 microM, indicative of heme binding approximately 100 times less avid than the mesoheme/HRG complex (Kd ca. 1 microM), but nearly 1000 times tighter than that of the mesoheme/histidine complex (Kd ca. 60 mM). The absorbance spectra of the mesoheme/peptide complexes, the loss of binding caused by modification of histidine residues, and the pH dependence of heme binding, all indicate that heme forms a low spin, bis-histidyl type of complex with these peptides, like that formed with HRG itself. Copper, but not cadmium or nickel, was an effective inhibitor of heme binding by the peptides. The sequence of HRG congruent with the sequence of peptide I is proposed to be one heme- and metal-binding site of rabbit HRG.     

Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the gamma'-chain

Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn(2+)-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn(2+)-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn(2+), HRG binds the predominant γ(A)/γ(A)-fibrinogen and the γ-chain elongated isoform, γ(A)/γ'-fibrinogen, with K(d) values of 9 nm. Likewise, (125)I-labeled HRG binds γ(A)/γ(A)- or γ(A)/γ'-fibrin clots with similar K(d) values when Zn(2+) is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ'-peptide, an analog of the COOH terminus of the γ'-chain that mediates the high affinity interaction of thrombin with γ(A)/γ'-fibrin. Thrombin competes with HRG for γ'-peptide binding and displaces (125)I-HRG from γ(A)/γ'-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γ(A)/γ'-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation.     

Metal ion affinity adsorption of a Zn(II)-transport protein present in maternal plasma during lactation: Structural characterization and identification as histidine-rich glycoprotein

A high-affinity Zn(II)-binding protein has been purified to homogeneity (880-fold) from the plasma of lactating women by a single affinity adsorption step on columns of tris(carboxymethyl)ethylenediamine (TED)-agarose loaded with Zn(II) ions. Purity was evaluated by high-performance reverse-phase (phenyl) chromatography and by silver staining after SDS-polyacrylamide gradient gel electrophoresis. The mass of denatured Zn(II)-binding protein was estimated by SDS-polyacrylamide gradient gel electrophoresis to be 75 kDa under both reducing and nonreducing conditions; by matrix-assisted uv laser desorption time-of-flight mass spectrometry the purified protein mass was determined to be 66 kDa. The amino acid composition revealed a high content of His (13 mol%) and Pro (12 mol%). N-terminal amino acid sequence analysis (50 residues) identified the purified protein as histidine-rich glycoprotein (HRG). Immunoblots demonstrated the absence of fragments in the purified product. An enzyme-linked immunosorbent assay was developed; a 75% recovery of intact HRG from the immobilized Zn(II) ion affinity column was documented. The circular dichroism spectra for the purified human HRG in the far uv (260-178 nm) were similar to those published for human and rabbit serum HRG. These results demonstrate that TED-immobilized Zn(II) ions can be used as a new and efficient method for the isolation of structurally intact human plasma HRG.     

Histidine-rich glycoprotein binds to cell-surface heparan sulfate via its N-terminal domain following Zn2+ chelation

Histidine-rich glycoprotein (HRG) is an alpha2-glycoprotein found in mammalian plasma at high concentrations (approximately 150 microg/ml) and is distinguished by its high content of histidine and proline. Structurally, HRG is a modular protein consisting of an N-terminal cystatin-like domain (N1N2), a central histidine-rich region (HRR) flanked by proline-rich sequences, and a C-terminal domain. HRG binds to cell surfaces and numerous ligands such as plasminogen, fibrinogen, thrombospondin, C1q, heparin, and IgG, suggesting that it may act as an adaptor protein either by targeting ligands to cell surfaces or by cross-linking soluble ligands. Despite the suggested functional importance of HRG, the cell-binding characteristics of the molecule are poorly defined. In this study, HRG was shown to bind to most cell lines in a Zn(2+)-dependent manner, but failed to interact with the Chinese hamster ovary cell line pgsA-745, which lacks cell-surface glycosaminoglycans (GAGs). Subsequent treatment of GAG-positive Chinese hamster ovary cells with mammalian heparanase or bacterial heparinase III, but not chondroitinase ABC, abolished HRG binding. Furthermore, blocking studies with various GAG species indicated that only heparin was a potent inhibitor of HRG binding. These data suggest that heparan sulfate is the predominate cell-surface ligand for HRG and that mammalian heparanase is a potential regulator of HRG binding. Using recombinant forms of full-length HRG and the N-terminal N1N2 domain, it was shown that the N1N2 domain bound specifically to immobilized heparin and cell-surface heparan sulfate. In contrast, synthetic peptides corresponding to the Zn(2+)-binding HRR of HRG did not interact with cells. Furthermore, the binding of full-length HRG, but not the N1N2 domain, was greatly potentiated by physiological concentrations of Zn2+. Based on these data, we propose that the N1N2 domain binds to cell-surface heparan sulfate and that the interaction of Zn2+ with the HRR can indirectly enhance cell-surface binding.     

Homologous human blood protein separation using immobilized metal affinity chromatography: protein C separation from prothrombin with application to the separation of factor IX and prothrombin.

Protein C (PC) is a natural anticoagulant and antithrombotic present in human blood at a concentration of 4 microg/mL. Its deficiency can result in excessive clotting and thrombosis. Protein C can be obtained from human blood plasma; however, there are other coagulant proteins in blood, including prothrombin (factor II), which is present in relatively large amounts and is one of the most active components. Protein C and prothrombin are homologous proteins with similar biochemical features; therefore, immunoaffinity chromatography is used for their separation. However, this technology is very expensive, protein C recovery and activity is low, and contamination problems with mouse antibody are likely. Immobilized metal affinity chromatography (IMAC) utilizes the protein metal-binding properties for protein separation. Protein C has twelve surface-accessible histidines, which are the major metal-binding groups for IMAC separation. After investigating metal ion-binding properties of protein C, we used an IDA-Cu column to separate protein C and prothrombin. Following protein adsorption to the column, prothrombin was washed out using a sodium phosphate buffer containing 2 mM imidazole and protein C was recovered with 15 mM imidazole in the buffer. The mild elution condition allows a high protein C activity and a high recovery. Also, this technology introduces no immunoglobulins, and it is relatively inexpensive. IMAC could replace the immunoaffinity technology for the large-scale separation of protein C from blood plasma Cohn Fraction IV-1. In addition, this work demonstrates a significant application of this technology for the separation of factor IX from prothrombin. Prothrombin has proven to be a harmful contaminant in factor IX cocktails that have been administered to humans in the treatment of hemophilia B.     

Plasminogen is tethered with high affinity to the cell surface by the plasma protein, histidine-rich glycoprotein

Plasminogen has been implicated in extracellular matrix degradation by invading cells, but few high affinity cell surface receptors for the molecule have been identified. Previous studies have reported that the plasma protein, histidine-rich glycoprotein (HRG), interacts with plasminogen and cell surfaces, raising the possibility that HRG may immobilize plasminogen/plasmin to cell surfaces. Here we show, based on optical biosensor analyses, that immobilized HRG interacts with soluble plasminogen with high affinity and with an extremely slow dissociation rate. Furthermore, the HRG-plasminogen interaction is lysine-dissociable and involves predominately the amino-terminal domain of HRG, and the fifth kringle domain of plasminogen, but not the carboxyl-terminal lysine of HRG. HRG was also shown to tether plasminogen to cell surfaces, with this interaction being potentiated by elevated Zn(2+) levels and low pH, conditions that prevail at sites of tissue injury, tumor growth, and angiogenesis. Based on these data we propose that HRG acts as a soluble adaptor molecule that binds to cells at sites of tissue injury, tumor growth, and angiogenesis, providing a high affinity receptor for tethering plasminogen to the cell surface and thereby enhancing the migratory potential of cells.     

Histidine-rich glycoprotein protects from systemic Candida infection

Fungi, such as Candida spp., are commonly found on the skin and at mucosal surfaces. Yet, they rarely cause invasive infections in immunocompetent individuals, an observation reflecting the ability of our innate immune system to control potentially invasive microbes found at biological boundaries. Antimicrobial proteins and peptides are becoming increasingly recognized as important effectors of innate immunity. This is illustrated further by the present investigation, demonstrating a novel antifungal role of histidine-rich glycoprotein (HRG), an abundant and multimodular plasma protein. HRG bound to Candida cells, and induced breaks in the cell walls of the organisms. Correspondingly, HRG preferentially lysed ergosterol-containing liposomes but not cholesterol-containing ones, indicating a specificity for fungal versus other types of eukaryotic membranes. Both antifungal and membrane-rupturing activities of HRG were enhanced at low pH, and mapped to the histidine-rich region of the protein. Ex vivo, HRG-containing plasma as well as fibrin clots exerted antifungal effects. In vivo, Hrg(-/-) mice were susceptible to infection by C. albicans, in contrast to wild-type mice, which were highly resistant to infection. The results demonstrate a key and previously unknown antifungal role of HRG in innate immunity.     

富组氨酸糖蛋白(HRG)的结构与功能

富组氨酸糖蛋白(HRG)为一种多结构域血浆糖蛋白,可与多种配体结合而行使多种功能.HRG配体包括锌离子、肝素和硫酸肝素、纤溶酶原、纤溶酶、纤维蛋白原、凝血酶敏感素、原肌球蛋白、IgG、FcγR及补体.在锌离子存在或在低pH的环境中(如组织损伤或肿瘤生长),HRG的富含组氨酸结构域与配体的结合能力加强.HRG的多结构域特点及其与多种配体结合的性质表明,其可以作为细胞外衔接蛋白衔接细胞表面的不同配体.除了细胞表面分子,HRG还可以结合IgG,从而阻止可溶性免疫复合物的产生.HRG与大多数细胞发生结合的功能是在锌离子存在或低pH环境下,通过与细胞表面硫酸肝素蛋白聚糖相互作用实现的.HRG还具有加强凋亡细胞、坏死的吞噬细胞和免疫复合物的清除、抗血管新生、细胞的粘附和迁移、纤维蛋白溶解作用、血凝固、补体激活等生理活动调节等功能.本文针对HRG的分子结构与功能及其在临床上的研究进展进行概述.     

Histidine-rich glycoprotein: A novel adaptor protein in plasma that modulates the immune, vascular and coagulation systems

Histidine-rich glycoprotein (HRG) is an abundant plasma glycoprotein that has a multidomain structure, interacts with many ligands, and has been shown to regulate a number of important biological processes. HRG ligands include Zn(2+) and haem, tropomyosin, heparin and heparan sulphate, plasminogen, plasmin, fibrinogen, thrombospondin, IgG, FcgammaR and complement. In many cases, the histidine-rich region of the molecule enhances ligand binding following interaction with Zn(2+) or exposure to low pH, conditions associated with sites of tissue injury or tumour growth. The multidomain nature of HRG indicates that it can act as an extracellular adaptor protein, bringing together disparate ligands, particularly on cell surfaces. HRG binds to most cells primarily via heparan sulphate proteoglycans, binding which is also potentiated by elevated free Zn(2+) levels and low pH. Recent reports have shown that HRG can modulate angiogenesis and additional studies have shown that it may regulate other physiological processes such as cell adhesion and migration, fibrinolysis and coagulation, complement activation, immune complex clearance and phagocytosis of apoptotic cells. This review outlines the molecular, structural, biological and clinical properties of HRG as well as describing the role of HRG in various physiological processes.     

Histidine-Rich Glycoprotein Uptake and Turnover Is Mediated by Mononuclear Phagocytes

Histidine-rich glycoprotein (HRG) is implicated in tumor growth and metastasis by regulation of angiogenesis and inflammation. HRG is produced by hepatocytes and carried to tissues via the circulation. We hypothesized that HRG''s tissue distribution and turnover may be mediated by inflammatory cells. Biodistribution parameters were analyzed by injection of radiolabeled, bioactive HRG in the circulation of healthy and tumor-bearing mice. ¹²⁵I-HRG was cleared rapidly from the blood and taken up in tissues of healthy and tumor-bearing mice, followed by degradation, to an increased extent in the tumor-bearing mice. Steady state levels of HRG in the circulation were unaffected by the tumor disease both in murine tumor models and in colorectal cancer (CRC) patients. Importantly, stromal pools of HRG, detected in human CRC microarrays, were associated with inflammatory cells. In agreement, microautoradiography identified ¹²⁵I-HRG in blood vessels and on CD45-positive leukocytes in mouse tissues. Moreover, radiolabeled HRG bound in a specific, heparan sulfate-independent manner, to differentiated human monocytic U937 cells in vitro. Suppression of monocyte differentiation by systemic treatment of mice with anti-colony stimulating factor-1 neutralizing antibodies led to reduced blood clearance of radiolabeled HRG and to accumulation of endogenous HRG in the blood. Combined, our data show that mononuclear phagocytes have specific binding sites for HRG and that these cells are essential for uptake of HRG from blood and distribution of HRG in tissues. Thereby, we confirm and extend our previous report that inflammatory cells mediate the effect of HRG on tumor growth and metastatic spread.     

Growth factor regulation of the molecular chaperone calnexin.

Heregulin-beta1 (HRG) is a regulatory polypeptide having several distinct biological effects in mammary epithelial cells. To address the hypothesis that HRG selectively regulates gene expression, we performed differential display screening using cells grown in the presence or absence of HRG. One cDNA clone upregulated by HRG was identical to human calnexin, a protein with molecular chaperone function. This is the first demonstration of the regulation of calnexin mRNA and protein expression by a physiologically relevant polypeptide factor in human breast cancer cells. HRG stimulation also caused a rapid redistribution of calnexin from vesicle-like structures in the cell cytoplasm to the perinuclear area and to the cell membrane. Furthermore, HRG induced colocalization and physical interaction of calnexin with the HER2 growth factor receptor. Finally, calnexin protein levels were increased in progressive stages of human breast cancer. These findings suggest that stimulation of calnexin expression by HRG may constitute a mechanism of protein redistribution and facilitate downstream signaling events in growth-factor-activated cells.