Cell surface heparan sulfates mediate internalization of the PWWP/HATH domain of HDGF via macropinocytosis to fine-tune cell signalling processes involved in fibroblast cell migration

HDGF (hepatoma-derived growth factor) stimulates cell proliferation by functioning on both sides of the plasma membrane as a ligand for membrane receptor binding to trigger cell signalling and as a stimulator for DNA synthesis in the nucleus. Although HDGF was initially identified as a secretory heparin-binding protein, the biological significance of its heparin-binding ability remains to be determined. In the present study we demonstrate that cells devoid of surface HS (heparan sulfate) were unable to internalize HDGF, HATH (N-terminal domain of HDGF consisting of amino acid residues 1-100, including the PWWP motif) and HATH(K96A) (single-site mutant form of HATH devoid of receptor binding activity), suggesting that the binding of HATH to surface HS is important for HDGF internalization. We further demonstrate that both HATH and HATH(K96A) could be internalized through macropinocytosis after binding to the cell surface HS. Interestingly, HS-mediated HATH(K96A) internalization is found to exhibit an inhibitory effect on cell migration and proliferation in contrast with that observed for HATH action on NIH 3T3 cells, suggesting that HDGF exploits the innate properties of both cell surface HS and membrane receptor via the HATH domain to affect related cell signalling processes. The present study indicates that MAPK (mitogen-activated protein kinase) signalling pathways could be affected by the HS-mediated HATH internalization to regulate cell migration in NIH 3T3 fibroblasts, as judged from the differential effect of HATH and HATH(K96A) treatment on the expression level of matrix metalloproteases.     

PWWP module of human hepatoma-derived growth factor forms a domain-swapped dimer with much higher affinity for heparin

Hepatoma-derived growth factor (hHDGF)-related proteins (HRPs) comprise a new growth factor family sharing a highly conserved and ordered N-terminal PWWP module (residues 1-100, previously referred to as a HATH domain) and a variable disordered C-terminal domain. We have shown that the PWWP module is responsible for heparin binding and have solved its structure in solution. Here, we show that under physiological conditions, both the PWWP module and hHDGF can form dimers. Surface plasmon resonance (SPR) studies revealed that the PWWP dimer binds to heparin with affinity that is two orders of magnitude higher (K(d)=13 nM) than that of the monomeric PWWP module (K(d)=1.2 microM). The monomer-dimer equilibrium properties and NMR structural data together suggest that the PWWP dimer is formed through a domain-swapping mechanism. The domain-swapped PWWP dimer structures were calculated on the basis of the NMR data. The results show that the two PWWP protomers exchange their N-terminal hairpin to form a domain-swapped dimer. The two monomers in a dimer are linked by the long flexible L2 loops, a feature supported by NMR relaxation data for the monomer and dimer. The enhanced heparin-binding affinity of the dimer can be rationalized in the framework of the dimer structure.     

NMR characterization of the electrostatic interaction of the basic residues in HDGF and FGF2 during heparin binding

Electrostatic interaction is a major driving force in the binding of proteins to highly acidic glycosaminoglycan, such as heparin. Although NMR backbone chemical shifts have generally been used to identify the heparin-binding site on a protein, however, there is no correlation between the binding free energies and the perturbed backbone chemical shifts for individual residues. The binding event occurs at the end of a side chain of basic residue, and does not require causing significant alterations in the backbone environment at a distance of multiple bonds. We used the H₂CN NMR pulse sequence to detect heparin binding through the side-chain resonances Hε–Cε–Nζ of Lys and Hδ–Cδ–Nε of Arg in the two proteins of hepatoma-derived growth factor (HDGF) and basic fibroblast growth factor (FGF2). H₂CN titration experiments revealed chemical shift perturbations in the side chains, which were correlated with the free energy changes in various mutants. The residues K19 in HDGF and K125 in FGF2 demonstrated the most significant perturbations, consistent with our previous observation that the two residues are crucial for binding. The result suggests that H₂CN NMR provides a precise evaluation for the electrostatic interactions. The discrepancy observed between backbone and side chain chemical shifts is correlated to the solvent accessibility of residues that the K19 and K125 backbones are highly buried with the restricted backbone conformation and are not strongly affected by the events at the end of the side chains.     

CXCR3 and heparin binding sites of the chemokine IP-10 (CXCL10)

The chemokine IP-10 (interferon-inducible protein of 10 kDa, CXCL10) binds the G protein-coupled receptor CXCR3, which is found mainly on activated T cells and NK cells, and plays an important role in Th1-type inflammatory diseases. IP-10 also binds to glycosaminoglycans (GAGs), an interaction thought to be important for its sequestration on endothelial and other cells. In this study, we performed an extensive mutational analysis to identify the CXCR3 and heparin binding sites of murine IP-10. The mutants were characterized for heparin binding, CXCR3 binding, and the ability to induce chemotaxis, Ca(2+) flux, and CXCR3 internalization. Double mutations neutralizing adjacent basic residues at the C terminus did not lead to a significant reduction in heparin binding, indicating that the main heparin binding site of IP-10 is not along the C-terminal alpha helix. Alanine exchange of Arg-22 had the largest effect on heparin binding, with residues Arg-20, Ile-24, Lys-26, Lys-46, and Lys-47 further contributing to heparin binding. A charge change mutation of Arg-22 resulted in further reduction in heparin binding. The N-terminal residue Arg-8, preceding the first cysteine, was critical for CXCR3 signaling. Mutations of charged and uncharged residues in the loop regions of residues 20-24 and 46-47, which caused reduced heparin binding, also resulted in reduced CXCR3 binding and signaling. CXCR3 expressing GAG-deficient Chinese hamster ovary cells revealed that GAG binding was not required for IP-10 binding and signaling through CXCR3, which suggests that the CXCR3 and heparin binding sites of IP-10 are partially overlapping.     

Solution structure of the PWWP domain of the hepatoma-derived growth factor family

Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded beta-barrel with a PWWP-specific long loop connecting beta2 and beta3 (PR-loop), followed by a helical region including two alpha-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the beta1/beta2 loop (beta-beta arch) and the beta3/beta4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.     

Localization of the heparin binding exosite of factor IXa

Factor IXa (FIXa) is known to have a binding site for heparin that has not been mapped by a mutagenesis study. By homology modeling based on structural data, we identified eight basic residues in the catalytic domain of FIXa that can potentially bind to heparin. These residues, Lys(98), Lys(126), Arg(165), Arg(170), Lys(173), Lys(230), Arg(233), and Lys(239) (chymotrypsin numbering) were substituted with Ala in separate constructs in Gla-domainless forms. Following activation, it was found that all FIXa derivatives cleaved the chromogenic substrate CBS 31.39 with near normal catalytic efficiencies. Similarly, antithrombin inactivated FIXa derivatives with a similar second-order association rate constant (k(2)) in both the absence and presence of pentasaccharide. In the presence of a full-length heparin, however, k(2) values were dramatically impaired with certain mutants. Direct binding studies revealed that the same mutants lost their affinities for binding to heparin-Sepharose. Both kinetic and direct binding data indicated that five basic residues of FIXa in the following order of importance, Arg(233) > Arg(165) > Lys(230) > Lys(126) > Arg(170) are critical for binding to heparin. Consistent with these results, examination of the crystal structure of the catalytic domain of FIXa indicated that all five basic residues are spatially aligned in a manner optimal for interaction with heparin.     

Insights into the structure and molecular basis of ligand docking to the G protein-coupled secretin receptor using charge-modified amino-terminal agonist probes

The amino terminus and third loop regions of class B G protein-coupled receptors play critical roles in ligand docking and action. For the prototypic secretin receptor, the hormone amino terminus is spatially approximated with receptor region high in transmembrane segment 6 (TM6), whereas residues ranging from position 6 through 26 label the amino terminus. Here, we focus on the role of charge of the secretin amino terminus, using a series of full-agonist, acetylated probes. Sites of covalent labeling were examined using sequential purification, chemical and enzymatic cleavage, and Edman degradation. High-affinity amino-terminally-blocked probes labeled the distal amino-terminal tail, rather than TM6, while adding a basic residue, again labeled TM6. These data suggest that the secretin amino terminus docks between the amino terminus and TM6 of the receptor, with this region of secretin likely interacting with an acidic residue within the receptor TM6 and the third extracellular loop. To explore this, candidate acidic residues were mutated to Ala (E341A, D342A, E345A, E351A). The E351A mutant markedly interfered with binding, biological activity, and internalization, whereas all others bound secretin and signaled and internalized normally. This supports the possibility that there is a charge-charge interaction between this residue and the amino terminus of secretin that is critical to its normal docking.     

CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis

CXCR3 is a G-protein-coupled seven-transmembrane domain chemokine receptor that plays an important role in effector T-cell and NK cell trafficking. Three gamma interferon-inducible chemokines activate CXCR3: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Here, we identify extracellular domains of CXCR3 that are required for ligand binding and activation. We found that CXCR3 is sulfated on its N terminus and that sulfation is required for binding and activation by all three ligands. We also found that the proximal 16 amino acid residues of the N terminus are required for CXCL10 and CXCL11 binding and activation but not CXCL9 activation. In addition, we found that residue R216 in the second extracellular loop is required for CXCR3-mediated chemotaxis and calcium mobilization but is not required for ligand binding or ligand-induced CXCR3 internalization. Finally, charged residues in the extracellular loops contribute to the receptor-ligand interaction. These findings demonstrate that chemokine activation of CXCR3 involves both high-affinity ligand-binding interactions with negatively charged residues in the extracellular domains of CXCR3 and a lower-affinity receptor-activating interaction in the second extracellular loop. This lower-affinity interaction is necessary to induce chemotaxis but not ligand-induced CXCR3 internalization, further suggesting that different domains of CXCR3 mediate distinct functions.     

Basic residues in azurocidin/HBP contribute to both heparin binding and antimicrobial activity

Azurocidin/CAP37/HBP is an antimicrobial and chemotactic protein that is part of the innate defenses of human neutrophils. In addition, azurocidin is an inactive serine protease homolog with binding sites for diverse ligands including heparin and the bovine pancreatic trypsin inhibitor (BPTI). The structure of the protein reveals a highly cationic domain concentrated on one side of the molecule and responsible for its strong polarity. To investigate the role of this highly basic region, we produced three recombinant azurocidin mutant proteins that were altered in either one or both of two clusters of 4 basic residues located symmetrically on each side of a central cleft in the cationic domain. Two of the mutant proteins (Loop 3: R5Q, K6Q, R8Q, and R10Q; Loop 4: R61Q, R62Q, R63Q, and R65Q) exhibited little or no change in heparin and BPTI binding or in antimicrobial function. In contrast, the Loop 3/Loop 4 mutant (R5Q, K6Q, R8Q, R10Q, R61Q, R62Q, R63Q, and R65Q) in which all 8 basic residues were replaced showed greatly decreased ability to bind heparin and to kill Escherichia coli and Candida albicans. Thus, we report that the 8 basic residues that were altered in the Loop 3/Loop 4 mutant contribute to the ability of the wild-type azurocidin molecule to bind heparin and to kill E. coli and C. albicans. Because BPTI binding was comparable in wild-type and Loop 3/Loop 4 mutant protein, we conclude that the same 8 basic residues are not involved in the binding of BPTI to azurocidin, supporting the notion that the binding site for BPTI is distinct from the site involved in heparin binding and antimicrobial activity. Finally, we show that removal of all 4 positively charged amino acids in the 20-44 azurocidin sequence (DMC1: R23Q,H24S,H32S,R34Q), a region previously thought to contain an antimicrobial domain, does not affect the activity of the protein against E. coli, Streptococcus faecalis, and C. albicans.     

Identification of conserved residues required for the binding of a tetratricopeptide repeat domain to heat shock protein 90.

The sequential binding of heat shock protein 90 (hsp90) to a series of tetratricopeptide repeat (TPR) proteins is critical to its function as a molecular chaperone. We have used site-directed mutagenesis to clarify the structural basis for the binding of hsp90 to the TPR domain of phosphoprotein phosphatase 5 (PP5). This TPR domain was chosen for study because its three-dimensional structure is known. We examined co-immunoprecipitation of hsp90 with wild type and mutant TPR constructs from transfected cells. Only mutations located on one face of the TPR domain affected hsp90 binding. This allowed the identification of a binding groove. Three basic residues that are highly conserved in hsp90-binding TPR proteins extend prominently into this groove. Lys-97 and Arg-101 were absolutely required for hsp90 binding, while mutation of Arg-74 diminished, but did not abrogate, hsp90 binding. Mutation of Lys-32, another conserved basic residue in the binding groove, also blocked hsp90 binding. The TPR domain of PP5 bound specifically to a 12-kDa C-terminal fragment of hsp90. This binding was reduced by mutation of acidic residues in the hsp90 fragment. These data suggest conservation, among hsp90-binding TPR proteins, of a binding groove containing basic residues that interact with acidic residues near the C terminus of hsp90.     

Dual role of the beta2-adrenergic receptor C terminus for the binding of beta-arrestin and receptor internalization

Homologous desensitization of beta2-adrenergic and other G-protein-coupled receptors is a two-step process. After phosphorylation of agonist-occupied receptors by G-protein-coupled receptor kinases, they bind beta-arrestins, which triggers desensitization and internalization of the receptors. Because it is not known which regions of the receptor are recognized by beta-arrestins, we have investigated beta-arrestin interaction and internalization of a set of mutants of the human beta2-adrenergic receptor. Mutation of the four serine/threonine residues between residues 355 and 364 led to the loss of agonist-induced receptor-beta-arrestin2 interaction as revealed by fluorescence resonance energy transfer (FRET), translocation of beta-arrestin2 to the plasma membrane, and receptor internalization. Mutation of all seven serine/threonine residues distal to residue 381 did not affect agonist-induced receptor internalization and beta-arrestin2 translocation. A beta2-adrenergic receptor truncated distal to residue 381 interacted normally with beta-arrestin2, whereas its ability to internalize in an agonist-dependent manner was compromised. A similar impairment of internalization was observed when only the last eight residues of the C terminus were deleted. Our experiments show that the C terminus distal to residue 381 does not affect the initial interaction between receptor and beta-arrestin, but its last eight amino acids facilitate receptor internalization in concert with beta-arrestin2.     

Domain structure of pleiotrophin required for transformation

The pleiotrophin (PTN) gene (Ptn) is a potent proto-oncogene that is highly expressed in many primary human tumors and constitutively expressed in cell lines derived from these tumors. The product of the Ptn gene is a secreted 136-amino acid heparin binding cytokine with distinct lysine-rich clusters within both the N- and C-terminal domains. To seek domains of PTN functionally important in neoplastic transformation, we constructed a series of mutants and tested their transforming potential by four independent criteria. Our data establish that a domain within PTN residues 41 to 64 and either but not both the N- or C-terminal domains are required for transformation; deletion of both the N and C termini abolishes the transformation potential of PTN. Furthermore, deletion of two internal 5-amino acid residue repeats enhances the transformation potency of PTN 2-fold. Our data indicate that PTN residues 41-64 contain an essential domain for transformation and suggest the hypothesis that this domain requires an additional interaction of the highly basic clusters of the N or C terminus of PTN with a negatively charged "docking" site to enable the transforming domain itself to engage and initiate PTN signaling through its cognate receptor.     

Two-step mechanism of binding of apolipoprotein E to heparin: implications for the kinetics of apolipoprotein E-heparan sulfate proteoglycan complex formation on cell surfaces

The interaction of apolipoprotein E (apoE) with cell-surface heparan sulfate proteoglycans is an important step in the uptake of lipoprotein remnants by the liver. ApoE interacts predominantly with heparin through the N-terminal binding site spanning the residues around 136-150. In this work, surface plasmon resonance analysis was employed to investigate how amphipathic alpha-helix properties and basic residue organization in this region modulate binding of apoE to heparin. The apoE/heparin interaction involves a two-step process; apoE initially binds to heparin with fast association and dissociation rates, followed by a step exhibiting much slower kinetics. Circular dichroism and surface plasmon resonance experiments using a disulfide-linked mutant, in which opening of the N-terminal helix bundle was prevented, demonstrated that there is no major secondary or tertiary structural change in apoE upon heparin binding. Mutations of Lys-146, a key residue for the heparin interaction, greatly reduced the favorable free energy of binding of the first step without affecting the second step, suggesting that electrostatic interaction is involved in the first binding step. Although lipid-free apoE2 tended to bind less than apoE3 and apoE4, there were no significant differences in rate and equilibrium constants of binding among the apoE isoforms in the lipidated state. Discoidal apoE3-phospholipid complexes using a substitution mutant (K143R/K146R) showed similar binding affinity to wild type apoE3, indicating that basic residue specificity is not required for the effective binding of apoE to heparin, unlike its binding to the low density lipoprotein receptor. In addition, disruption of the alpha-helix structure in the apoE heparin binding region led to an increased favorable free energy of binding in the second step, suggesting that hydrophobic interactions contribute to the second binding step. Based on these results, it seems that cell-surface heparan sulfate proteoglycan localizes apoE-enriched remnant lipoproteins to the vicinity of receptors by fast association and dissociation.     

The oligomeric structure of vaccinia viral envelope protein A27L is essential for binding to heparin and heparan sulfates on cell surfaces: a structural and functional approach using site-specific mutagenesis

The soluble domain of the self-assembly vaccinia virus envelope protein A27L, sA27L-aa, consists of a flexible extended coil at the N terminus and a rigid hydrophobic coiled-coil region at the C terminus. In the former, a basic strip of 12 residues is responsible for binding to cell-surface heparan sulfates. Although the latter is believed to mediate self-assembly, its biological role is unclear. However, an in vitro bioassay showed that peptides comprising the 12 residue basic region alone failed to interact with heparin, suggesting that the C-terminal coiled-coil region might serve an indispensable role in biological function. To explore this structural and functional relationship, we performed site-specific mutagenesis in an attempt to specifically disrupt the hydrophobic core of the coiled coil. Three single mutants, L47A, L51A, and L54A, and one triple mutant, L47,51,54A, were expressed and purified from Escherichia coli. The physical properties of the mutants were carefully examined by gel-filtration chromatography, CD, and NMR spectroscopy, and the biological activities were assessed by an in vitro SPR bioassay and three in vivo bioassays: binding to cells, blocking virus infection and blocking cell fusion. We showed that the L47A mutant, which is similar to the parental sA27L-aa in forming a hexamer, is biologically active. L51A and L54A mutants form tetramers and are less active. Notably, in the triple mutant, the self-assembly hydrophobic core structure is uncoiled; as a consequence, the tetrameric structure is biologically inactive. Thus, we conclude that the leucine residues, in particular Leu51 and Leu54, sustain the hydrophobic core structure that is essential for the biological function of vaccinia virus envelope protein A27L, binding to cell-surface heparan sulfate.     

Primary structure of the heparin-binding site of type V collagen

The abilities of collagens, type I, II, III, IV, and V, to bind heparin were examined by heparin-affinity chromatography and binding studies with [35S]heparin. At a physiological pH and ionic strength, only type V collagen bound to heparin. Collagens type I and II showed higher affinities than types III and IV for heparin, but did not bind to a heparin column at a physiological ionic strength. The heparin binding site of type V collagen was located in a 30 kDa CNBr fragment of the alpha 1(V) chain, and the amino acid sequence of this fragment was determined. The 30 kDa fragment contained a cluster of basic amino acid residues, and enzymatic cleavage within this basic domain greatly reduced the heparin-binding activities of the resulting peptides. Thus this basic region is probably the heparin-binding site of type V collagen.     

Orientation of heparin-binding sites in native vitronectin. Analyses of ligand binding to the primary glycosaminoglycan-binding site indicate that putative secondary sites are not functional

A primary heparin-binding site in vitronectin has been localized to a cluster of cationic residues near the C terminus of the protein. More recently, secondary binding sites have been proposed. In order to investigate whether the binding site originally identified on vitronectin functions as an exclusive and independent heparin-binding domain, solution binding methods have been used in combination with NMR and recombinant approaches to evaluate ligand binding to the primary site. Evaluation of the ionic strength dependence of heparin binding to vitronectin according to classical linkage theory indicates that a single ionic bond is prominent. It had been previously shown that chemical modification of vitronectin using an arginine-reactive probe results in a significant reduction in heparin binding (Gibson, A., Baburaj, K., Day, D. E., Verhamme, I. , Shore, J. D., and Peterson, C. B. (1997) J. Biol. Chem. 272, 5112-5121). The label has now been localized to arginine residues within the cyanogen bromide fragment-(341-380) that contains the primary heparin-binding site on vitronectin. One- and two-dimensional NMR on model peptides based on this primary heparin-binding site indicate that an arginine residue participates in the ionic interaction and that other nonionic interactions may be involved in forming a complex with heparin. A recombinant polypeptide corresponding to the C-terminal 129 amino acids of vitronectin exhibits heparin-binding affinity that is comparable to that of full-length vitronectin and is equally effective at neutralizing heparin anticoagulant activity. Results from this broad experimental approach argue that the behavior of the primary site is sufficient to account for the heparin binding activity of vitronectin and support an exposed orientation for the site in the structure of the native protein.     

Mechanism of human group V phospholipase A2 (PLA2)-induced leukotriene biosynthesis in human neutrophils. A potential role of heparan sulfate binding in PLA2 internalization and degradation

Human group V phospholipase A(2) (hVPLA(2)) has been shown to have high activity to elicit leukotriene production in human neutrophils (Han, S. K., Kim, K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. 274, 11881-11888). To determine the mechanism by which hVPLA(2) interacts with cell membranes to induce leukotriene formation, we mutated surface cationic residues and a catalytic residue of hVPLA(2) and measured the interactions of mutants with model membranes, immobilized heparin, and human neutrophils. These studies showed that cationic residues, Lys(7), Lys(11), and Arg(34), constitute a part of the interfacial binding surface of hVPLA(2), which accounts for its moderate preference for anionic membranes. Additionally, hVPLA(2) binds heparin with high affinity and has a well defined heparin-binding site. The site is composed of Arg(100), Lys(101), Lys(107), Arg(108), and Arg(111), and is spatially distinct from its interfacial binding surface. Importantly, the activities of the mutants to hydrolyze cell membrane phospholipids and induce leukotriene biosynthesis, when enzymes were added exogenously to neutrophils, correlated with their activities on phosphatidylcholine membranes but not with their affinities for anionic membranes and heparin. These results indicate that hVPLA(2) acts directly on the outer plasma membranes of neutrophils to release fatty acids and lysophospholipids. Further studies suggest that products of hVPLA(2) hydrolysis trigger the cellular leukotriene production by activating cellular enzymes involved in leukotriene formation. Finally, the temporal and spatial resolution of exogenously added hVPLA(2) and mutants suggests that binding to cell surface heparan sulfate proteoglycans is important for the internalization and clearance of cell surface-bound hVPLA(2).     

Amino acid sequence homology between bovine and human platelet proteins

The complete amino acid sequence of bovine platelet factor 4 (PF-4) was determined. Comparison of the 88 residue bovine protein with its 70 residue human counterpart indicated 73% homology. There is 53% homology between this bovine protein and another human platelet protein, beta-thromboglobulin (beta-TG). These heparin binding proteins share greatest homology around a lysine-rich octa-peptide near the carboxy-terminus which is the putative heparin binding domain. Graphic comparison of these proteins suggests that a point mutation at position 55 (human PF-4 numbering) could cause a significant difference among the folding properties of these 3 proteins and might be critical for their different heparin binding properties.