The lysine-rich C-terminal tail of heparin affin regulatory peptide is required for mitogenic and tumor formation activities

Heparin affin regulatory peptide (HARP) is a 18-kDa heparin-binding polypeptide that is highly expressed in developing tissues and in several primary human tumors. It seems to play a key role in cellular growth and differentiation. In vitro, HARP displays mitogenic, angiogenic, and neurite outgrowth activities. It is a secreted protein that is organized in two beta-sheet domains, each domain containing a cluster of basic residues. To assess determinants involved in the biological activities of HARP, C-terminally truncated proteins were produced in Chinese hamster ovary-K1 cells and tested for their mitogenic, tumor formation in nude mice and neurite outgrowth activities. Our data clearly indicate that the residues 111-136 of the lysine-rich C-terminal domain are involved in the mitogenic and tumor formation activities of HARP. Correlatively, no signal transduction was detected using the corresponding mutant, suggesting the absence of HARP binding to its high affinity receptor. However, this C-terminal domain of HARP is not involved in the neurite outgrowth activity. We also demonstrate that HARP signal peptide cleavage could led to two maturated forms that are both but differentially mitogenic.     

A basic peptide derived from the HARP C-terminus inhibits anchorage-independent growth of DU145 prostate cancer cells

Heparin affin regulatory peptide (HARP) is an 18 kDa heparin-binding protein that plays a key role in tumor growth. We showed previously that the synthetic peptide P(111-136) composed of the last 26 HARP amino acids inhibited HARP-induced mitogenesis. Here, to identify the exact molecular domain involved in HARP inhibition, we investigated the effect of the shorter basic peptide P(122-131) on DU145 cells, which express HARP and its receptor protein tyrosine phosphatase beta/zeta (RPTPbeta/zeta). P(122-131) was not cytotoxic; it dose-dependently inhibited anchorage-independent growth of DU145 cells. Binding studies using biotinylated P(122-131) indicated that this peptide interfered with HARP binding to DU145 cells. Investigation of the mechanisms involved suggested interference, under anchorage-independent conditions, of P(122-131) with a HARP autocrine loop in an RPTPbeta/zeta-dependent fashion. Thus, P(122-131) may hold potential for the treatment of disorders involving RPTPbeta/zeta.     

Effect of herparin on bovine epithelial lens cell proliferation induced by heparin affin regulatory peptide

HARP (heparin affin regultory peptide) is an 18 kDa heparin binding protein, also known as HB-GAM or pleiotrophin (PTN) which has been primarily isolated from brain and uterus, and displays neurite outgrowth, angiogenic and mitogenic activities. Previously, we have expressed the human cDNA encoding human HARP in NIH 3T3 cells. Purified recombinant HARP displayed mitogenic activity for endothelial cells. Its NH2-terminal sequence indicates that the HARP molecule possesses a three amino acid extension from the signal peptide more than the NH2-terminal described. For HB-GAM or PTN, these three amino acids may be essential for the stability and the mitogenic activity of this growth factor. In an attempt to further study the mode of action of this growth factor, we have investigated the mitogenic effect of HARP on various cell types. In contrast to FGF-2, HARP failed to induce stimulation of DNA synthesis on a CCL39 cell line. However, we found that in quiescent bovine epithelial lens (BEL) cells, the stimulation of DNA synthesis induced by HARP is dose-dependent (EC₅₀: 2.5 ng/ml) and maximal stimulation is as potent as that induced by FGF-2 (EC₅₀: 25 pg/ml). Interestingly, when BEL cells were allowed to quiesce in the presence of serum, the stimulation induced by HARP is considerably less potent. In this highly responsive cell system, heparin could potentiate the mitogenic activity of HARP at very low doses (0.1-1 m?g/ml) and inhibit this activity at concentrations of 10 m?g/ml. In contrast to its protective effect on FGF-1 and -2, heparin was unable to preserve HARP from tryptic and chymotryptic degradations. © 1995 Wiley-Liss, Inc.     

Heparin affin regulatory peptide in milk: its involvement in mammary gland homeostasis

HARP (heparin affin regulatory peptide) is a heparin binding growth factor implicated in cellular growth and differentiation. Previously, HARP had been localized in the human mammary, in both alveolar epithelial and myoepithelial cells although HARP mRNAs were only expressed by myoepithelial cells [J. Histochem. Cytochem. 45 (1997) 1]. In the present study, we demonstrate that HARP is secreted in human mature milk with concentrations ranging from 17.68+/-6.4ng/ml in mature milk to 59.9+/-11.22ng/ml in colostrum. In vitro, HARP was found to be mitogenic on human mammary epithelial and myoepithelial cell lines and correlated with the expression of its high affinity receptor tyrosine kinase ALK (anaplastic lymphoma kinase). In vivo, ALK is expressed in both mammary epithelial and myoepithelial cells, suggesting that HARP could act in vivo as a paracrine and autocrine growth factor in the regulation of the mammary gland development and its homeostatic maintenance during pregnancy and lactation.     

HARP induces angiogenesis in vivo and in vitro: implication of N or C terminal peptides

HARP (heparin affin regulatory peptide) is a growth factor displaying high affinity for heparin. In the present work, we studied the ability of human recombinant HARP as well as its two terminal peptides (HARP residues 1-21 and residues 121-139) to promote angiogenesis. HARP stimulates endothelial cell tube formation on matrigel, collagen and fibrin gels, stimulates endothelial cell migration and induces angiogenesis in the in vivo chicken embryo chorioallantoic membrane assay. The two HARP peptides seem to be involved in most of the angiogenic effects of HARP. They both stimulate in vivo angiogenesis and in vitro endothelial cell migration and tube formation on matrigel. We conclude that HARP has an angiogenic activity when applied exogenously in several in vitro and in vivo models of angiogenesis and its NH(2) and COOH termini seem to play an important role.     

Neuroblastoma cells express c-sis and produce a transforming growth factor antigenically related to the platelet-derived growth factor.

Mouse neuroblastoma Neuro-2A cells produce transforming growth factors during exponential growth in a defined hormone-free medium, which, on Bio-Gel columns in 1 M HAc, elute at a molecular size of 15 to 20 kilodaltons (kDa). These neuroblastoma-derived transforming growth factors have strong mitogenic activity, but they do not compete with epidermal growth factor for receptor binding (E. J. J. van Zoelen, D. R. Twardzik, T. M. J. van Oostwaard, P. T. van der Saag, S. W. de Laat, and G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 81:4085-4089, 1984). In this study approximately 80% of the mitogenic activity was immunoprecipitated by antibodies raised against platelet-derived growth factor (PDGF). Immunoblotting indicated a true molecular size of 32 kDa for this PDGF-like growth factor. Analysis of poly(A)+ RNA from Neuro-2A cells demonstrated the expression of the c-sis oncogene in this cell line, whereas in vitro translation of the RNA yielded a 20-kDa protein recognized by anti-PDGF antibodies. Separation by reverse-phase high-pressure liquid chromatography demonstrated the presence of two distinct mitogenic activities in neuroblastoma-derived transforming growth factor preparations, one of which is antigenically related to PDGF. Both activities had the ability to induce anchorage-independent growth in normal rat kidney cells, both in the presence and in the absence of epidermal growth factor. It is concluded that Neuro-2A cells express c-sis with concomitant production and secretion of a PDGF-like growth factor, which plays a role in the induction of phenotypic transformation on normal rat kidney cells.     

Inhibition of the mitogenic, angiogenic and tumorigenic activities of pleiotrophin by a synthetic peptide corresponding to its C-thrombospondin repeat-I domain

Pleiotrophin (PTN), is a heparin-dependent growth factor involved in angiogenesis and tumor growth. PTN contains a thrombospondin repeat-I (TSR-I) motif in its two beta-sheet domains that are involved in its binding to heparin and its neurite outgrowth activity. Based on the importance of the binding of PTN to heparin in its dimerization and biological activities, we have designed two synthetic peptides, P(13-39) and P(65-97) corresponding to a part of the N-terminal and C-terminal TSR-I motif of PTN, respectively. P(65-97) inhibited the mitogenic, tumorigenic and angiogenic activities of PTN, as well as the mitogenic and an angiogenic activity of fibroblast growth factor-2 (FGF-2). However, P(65-97) had no effect on the mitogenic activity of epidermal growth factor, which does not bind heparin. P(65-97) but not P(13-39) inhibited the binding of PTN and to a lesser extent of FGF-2 to heparin using an immunoassay and an optical biosensor assay and bound directly to heparin with a K(d) of 120 nM. These findings suggest that P(65-97), containing amino acids 65-97 of the TSR-I motif of the C-terminal domain of PTN, inhibits the activities of PTN and FGF-2 by virtue of its ability to bind heparin very effectively and so compete with the growth factors for their polysaccharide co-receptor.     

Superagonistic activation of ErbB-1 by EGF-related growth factors with enhanced association and dissociation rate constants

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) are mitogenic hormones that exert their activity primarily by binding to the EGF receptor, also known as ErbB-1. We have recently characterized a set of EGF/TGFalpha chimeric molecules with similar high affinity for ErbB-1 as EGF and TGFalpha and shown that three of these chimeras induce mitogenic cell stimulation at already a 10-fold lower concentration than their wild-type counterparts (Lenferink, A. E., Kramer, R. H., van Vugt, M. J., K?nigswieser, M., DiFiore, P. P., van Zoelen, E. J., and van de Poll, M. L. (1997) Biochem. J. 327, 859-865). In the present study we show that these so-called superagonistic chimeras do not differ from EGF and TGFalpha in their ability to induce ErbB-1 tyrosine phosphorylation but are considerably more potent in activation of mitogen-activated protein kinase phosphorylation. Direct cell binding studies and analysis of ligand-receptor interaction by surface plasmon resonance measurements revealed that both the association rate constant (k(on)) and the dissociation rate constant (k(off)) of these superagonists is 3-5-fold higher in comparison with the wild-type ligands and nonsuperagonistic chimeras. These data indicate that the dynamic on and off rate constants for receptor binding may be more specific parameters for determining the mitogenic activity of peptide hormones than their constants for equilibrium receptor binding.     

Glycosaminoglycans promote HARP/PTN dimerization

Heparin affin regulatory peptide (HARP), also called pleiotrophin (PTN), is a secreted polypeptide which binds to heparin and plays a key role in cellular growth and differentiation. In order to assess the determinants potentially important to its biological activity, we tested the ability of HARP to oligomerize, a process involved in mitogenic activity of the heparin-binding fibroblast growth factor. Using dissuccinimidyl suberate cross-linking experiments and affinity chromatography, we report that human HARP forms noncovalent dimers. Dimerization is dependent on the presence of heparin or other sulfated glycosaminoglycans, as chlorate treatment of cells inhibits this process. In vitro, different glycosaminoglycans, such as dermatan sulfate and chondroitin sulfate-C, also induce a dimer assembly of HARP. The relevance of this process was supported by experiments demonstrating that HARP is secreted as a dimer in conditioned medium of NIH-3T3 cells that overexpressed this growth factor and is also associated to the cell surface or to the extracellular matrix.     

Toxicity of diphtheria toxin-related proteins produced by suppression of nonsense mutations

The Mr = 62,000 diphtheria toxin-related proteins produced from the suppression of nonsense mutations within the tox gene of corynephage beta were purified by affinity chromatography. Except for the toxin 111-sup2-62, the Mr = 62,000 polypeptides were found to have the same specific toxicity as does wild type toxin. 111-sup2-62 was found to have a prolonged lag period prior to the onset of inhibition of protein synthesis and ADP-ribosylation of elongation factor 2. 111-sup2-62 differs from wild type toxin by an amino acid substitution at a site approximatley 47,000 daltons from the NH2 terminus. The data presented provide genetic support for the Boquet-Pappenheimer model (Boquet, P., and Pappenheimer, A. M. Jr (1978) J. Biol. Chem. 251, 5770-5778) of fragment A translocation into the eukaryotic cell cytosol.     

Identification of heparin affin regulatory peptide domains with potential role on angiogenesis

Heparin affin regulatory peptide (HARP) is a growth factor displaying high affinity for heparin. It is present in the extracellular matrix of many tissues, interacting with heparan sulfate and dermatan/chondroitin sulfate glycosaminoglycans. We have previously shown that HARP is implicated in the control of angiogenesis and its effects are mimicked, at least in part, by synthetic peptides that correspond to its N and C termini. In the present work, we show that HARP is cleaved by plasmin, leading to the production of five peptides that correspond to distinct domains of the molecule. Heparin, heparan sulfate and dermatan sulfate, at various HARP to glycosaminoglycan ratios, partially protect HARP from plasmin degradation. The molecules with higher affinity to HARP are the more protective, heparin being the most efficient. The peptides that are produced from cleavage of HARP by plasmin, affect in vivo and in vitro angiogenesis and modulate the angiogenic activity of vascular endothelial growth factor on human umbilical vein endothelial cells. Similar results were obtained in vitro with recombinant HARP peptides, identical to the peptides generated after treatment of HARP with plasmin. These results suggest that different regions of HARP may induce or inhibit angiogenesis.     

Characterization of insulin-like growth factor I (IGF-I) receptor mutants for their effects on IGF-I- and interleukin 4-mediated DNA synthesis of 32D cells

Recently we demonstrated that overexpression of the wild type insulin-like growth factor I receptor (IGF-IRWT) in 32D myeloid progenitor cells led to cell proliferation in response to interleukin 4 (IL-4) as well as insulin-like growth factor I (IGF-I) in the absence of insulin receptor substrate expression (Soon, L., Flechner, L., Gutkind, J. S., Wang, L. H., Baserga, R., Pierce, J. H., and Li, W. (1999) Mol. Cell. Biol. 19, 3816-3828). To understand the structural importance of insulin-like growth factor I receptor (IGF-IR) in mediating IL-4- and IGF-I-induced DNA synthesis, we transfected various mutants of IGF-IR to 32D cells. Our results show that most mutants, including Y1250F, Y1251F, Y1250F/Y1251F, S1280A/S1281A/S1282A/S1283A, Y1316F, and 1245d, still retained mitogenic response toward IGF-I or IL-4. However, the Y950F, Y1131F, and Y1135F mutants were not able to respond to either ligand. The H1293F/K1294R and 1293d mutants reduced response toward IGF-I but not to IL-4. Phosphorylation of Shc was greatly reduced in those three mutants that lost mitogenic response. The MAPK activity was much lower in Y1131F and Y1135F mutants, indicating the importance of the Shc/MAPK pathway in IGF-I-induced mitogenesis. Importantly, the synergistic effect of these two factors on DNA synthesis was not affected in cells expressing most of the mutants, even in those three that had lower mitogenic response toward a single ligand. These results suggest that an unidentified pathway(s) may be induced upon co-addition of IGF-I and IL-4 that sustains the intact mitogenesis.     

Distinct antitumor properties of a type IV collagen domain derived from basement membrane

Vascular basement membrane is an important structural component of blood vessels. During angiogenesis this membrane undergoes many alterations and these changes are speculated to influence the formation of new capillaries. Type IV collagen is a major component of vascular basement membrane, and recently we identified a fragment of type IV collagen alpha2 chain with specific anti-angiogenic properties (Kamphaus, G. D., Colorado, P. C., Panka, D. J., Hopfer, H., Ramchandran, R., Torre, A., Maeshima, Y., Mier, J. W., Sukhatme, V. P., and Kalluri, R. (2000) J. Biol. Chem. 275, 1209-1215). In the present study we characterize two different antitumor activities associated with the noncollagenous 1 (NC1) domain of the alpha3 chain of type IV collagen. This domain was previously discovered to possess a C-terminal peptide sequence (amino acids 185-203) that inhibits melanoma cell proliferation (Han, J., Ohno, N., Pasco, S., Monboisse, J. C., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). In the present study, we identify the anti-angiogenic capacity of this domain using several in vitro and in vivo assays. The alpha3(IV)NC1 inhibited in vivo neovascularization in matrigel plug assays and suppressed tumor growth of human renal cell carcinoma (786-O) and prostate carcinoma (PC-3) in mouse xenograft models associated with in vivo endothelial cell-specific apoptosis. The anti-angiogenic activity was localized to amino acids 54-132 using deletion mutagenesis. This anti-angiogenic region is separate from the 185-203 amino acid region responsible for the antitumor cell activity. Additionally, our experiments indicate that the antitumor cell activity is not realized until the peptide region is exposed by truncation of the alpha3(IV)NC1 domain, a requirement not essential for the anti-angiogenic activity of this domain. Collectively, these results effectively highlight the distinct and unique antitumor properties of the alpha3(IV)NC1 domain and the potential use of this molecule for inhibition of tumor growth.     

Endothelial cell proliferation induced by HARP: implication of N or C terminal peptides

HARP (Heparin Affin Regulatory Peptide) is a 18-kDa secreted protein displaying high affinity for heparin. It has neurite outgrowth-promoting activity, while there are conflicting results regarding its mitogenic activity. In the present work, we studied the effect of human recombinant HARP expressed in bacterial cells as well as two peptides (HARP residues 1-21 and residues 121-139) on the proliferation of three endothelial cell types derived from human umbilical vein (HUVEC), rat adrenal medulla (RAME), and bovine brain capillaries (BBC) either added as a soluble form in the cell culture medium or coated onto the culture plate. HARP added in a soluble form in the culture medium had no effect on the proliferation of BBC, HUVEC, and RAME cells. However, when immobilized onto the cell culture plate, HARP had a concentration-dependent mitogenic effect on both BBC cells and HUVEC. The peptides presented as soluble factor induced a significant concentration-dependent mitogenic effect on BBC cells but only a small effect on HUVEC and RAME cells. When they were immobilized onto the cell culture plate, the mitogenic effect was much greater. The most responsive cells were BBC that expressed and secreted in the culture medium the higher amounts of HARP.     

Characterization of heparin affin regulatory peptide signaling in human endothelial cells

Heparin affin regulatory peptide (HARP) is an 18-kDa secreted growth factor that has a high affinity for heparin and a potent role on tumor growth and angiogenesis. We have previously reported that HARP is mitogenic for different types of endothelial cells and also affects cell migration and differentiation (12). In this study we examined the signaling pathways involved in the migration and tube formation on matrigel of human umbilical vein endothelial cells (HUVEC) induced by HARP. We report for the first time that receptor-type protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), which is a receptor for HARP in neuronal cell types, is also expressed in HUVEC. We also document that HARP signaling through RPTPbeta/zeta leads to activation of Src kinase, focal adhesion kinase, phosphatidylinositol 3-kinase, and Erk1/2. Sodium orthovanadate, chondroitin sulfate-C, PP1, wortmannin, LY294002, and U0126 inhibit HARP-mediated signaling and HUVEC migration and tube formation. In addition, RPTPbeta/zeta suppression using small interfering RNA technology interrupts intracellular signals and HUVEC migration and tube formation induced by HARP. These results establish the role of RPTPbeta/zeta as a receptor of HARP in HUVEC and elucidate the HARP signaling pathway in endothelial cells.     

A peptide corresponding to the C-terminal region of pleiotrophin inhibits angiogenesis in vivo and in vitro

Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both α(ν) β(3) integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN(112-136) ) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN(112-136) inhibited binding of PTN to α(ν) β(3) integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of β(3) . Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing α(ν) β(3) -selective antibody. PTN(112-136) did not affect binding of PTN to RPTPβ/ζ in endothelial cells and induced β(3) Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPβ/ζ by siRNA or by c-src inhibition, suggesting that PTN(112-136) may interact with RPTPβ/ζ. NMR spectroscopy studies showed that PTN(112-136) was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123-132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN(112-136) induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to α(ν) β(3) integrin.     

The negative charge of Glu-111 is required to activate the cleavage center of EcoRI endonuclease

King et al. (King, K., Benkovic, S. J., and Modrich, P. (1989) 264, 11807-11815) have shown that Glu-111 is required for DNA cleavage by EcoRI endonuclease and have suggested that this residue is required for activation of the cleavage center upon specific recognition. We have substituted Gln or Asp for Glu-111 by oligonucleotide-directed mutagenesis. First and second strand cleavage rate constants are reduced by a factor of more than 10(4) by the Gln-111 substitution. However, these rate constants are enhanced 9-fold when pH is increased from 7.6 to 8.5, which enhances strand cleavage at EcoRI sites by wild type endonuclease to a similar degree. The specific affinity of Gln-111 endonuclease for EcoRI sites is 1000 times greater than that of wild type enzyme reflecting a decrease in the rate constant governing specific complex dissociation. In contrast to Gln-111 endonuclease, the equilibrium specific affinity of Asp-111 endonuclease for the EcoRI sequence is similar to that of wild type enzyme, and first and second strand cleavage rate constants are reduced only 100-fold relative to wild type enzyme. These results suggest that a negative charge on residue 111 is required for strand cleavage and are consistent with participation of Glu-111 in activation of the DNA cleavage center, with energy associated with specific sequence recognition driving this process.     

Identification of candidate angiogenic inhibitors processed by matrix metalloproteinase 2 (MMP-2) in cell-based proteomic screens: disruption of vascular endothelial growth factor (VEGF)/heparin affin regulatory peptide (pleiotrophin) and VEGF/Connective tissue growth factor angiogenic inhibitory complexes by MMP-2 proteolysis

Matrix metalloproteinases (MMPs) exert both pro- and antiangiogenic functions by the release of cytokines or proteolytically generated angiogenic inhibitors from extracellular matrix and basement membrane remodeling. In the Mmp2^−/− mouse neovascularization is greatly reduced, but the mechanistic aspects of this remain unclear. Using isotope-coded affinity tag labeling of proteins analyzed by multidimensional liquid chromatography and tandem mass spectrometry we explored proteome differences between Mmp2^−/− cells and those rescued by MMP-2 transfection. Proteome signatures that are hallmarks of proteolysis revealed cleavage of many known MMP-2 substrates in the cellular context. Proteomic evidence of MMP-2 processing of novel substrates was found. Insulin-like growth factor binding protein 6, follistatin-like 1, and cystatin C protein cleavage by MMP-2 was biochemically confirmed, and the cleavage sites in heparin affin regulatory peptide (HARP; pleiotrophin) and connective tissue growth factor (CTGF) were sequenced by matrix-assisted laser desorption ionization-time of flight mass spectrometry. MMP-2 processing of HARP and CTGF released vascular endothelial growth factor (VEGF) from angiogenic inhibitory complexes. The cleaved HARP N-terminal domain increased HARP-induced cell proliferation, whereas the HARP C-terminal domain was antagonistic and decreased cell proliferation and migration. Hence the unmasking of cytokines, such as VEGF, by metalloproteinase processing of their binding proteins is a new mechanism in the control of cytokine activation and angiogenesis.     

Ligand binding and functional properties of betaglycan, a co-receptor of the transforming growth factor-beta superfamily. Specialized binding regions for transforming growth factor-beta and inhibin A

Betaglycan, also known as the transforming growth factor-beta (TGF-beta) type III receptor, is a membrane-anchored proteoglycan that binds TGF-beta via its core protein. Deletion mutagenesis analysis has revealed two regions of betaglycan ectodomain capable of binding TGF-beta: one at the amino-terminal half, the endoglin-related region (López-Casillas, F., Payne, H., Andres, J. L., and Massagué, J. (1994) J. Cell Biol. 124, 557-568), and the other at the carboxyl-terminal half, the uromodulin-related region (Pepin, M.-C., Beauchemin, M., Plamondon, J., and O'Connor-McCourt, M. D. (1994) Proc. Natl. Acad. Sci. U. S. A 91, 6997-7001). In the present work we have functionally characterized these ligand binding regions. Similar to the wild type receptor, both regions bind TGF-beta2 with higher affinity than TGF-beta1. However, only the endoglin-related region increases the TGF-beta2 labeling of the TGF-beta type II receptor, the so-called "TGF-beta -presentation" function of the wild type receptor. Despite this preference, both regions as well as the wild type receptor mediate the TGF-beta2-dependent Smad2 phosphorylation, indicating that they can function indistinguishably as TGF-beta-enhancing co-receptors. On the other hand, we found that the recently described ability of the wild type betaglycan to bind inhibin A is a property of the core protein that resides in the uromodulin-related region. Binding competition experiments indicate that this region binds inhibin and TGF-beta with the following relative affinities: TGF-beta2 > inhibin A > TGF-beta1. All together, the present results suggest that betaglycan ectodomain is endowed with two bona fide independent ligand binding domains that can perform specialized functions as co-receptors of distinct members of the TGF-beta superfamily.     

Biochemical and mitogenic properties of the heparin-binding growth factor HARP

Heparin affin regulatory peptide (HARP), also called Pleiotrophin (PTN), is a polypeptide that displays a high affinity for heparin and that shares approximately 50% sequence homology with Midkine (MK). According to this structural homology, these two molecules constitute a new family of heparin-binding proteins. The biological properties of HARP and MK remain largely a subject of debate. Both proteins have been described as neurite outgrowth promoting agents whereas until recently the mitogenic activity has been controversial. The aim of this review is to summarize the information on HARP with special focus on the recent data relating to its mitogenic properties.