Research ArticleCell Biology

Heparin is an activating ligand of the orphan receptor tyrosine kinase ALK

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Science Signaling  20 Jan 2015:
Vol. 8, Issue 360, pp. ra6
DOI: 10.1126/scisignal.2005916

Abstract

Anaplastic lymphoma kinase (ALK) is one of the few remaining “orphan” receptor tyrosine kinases (RTKs) in which the ligands are unknown. Ligand-mediated activation of RTKs is important throughout development. ALK is particularly relevant to the development of the nervous system. Increased activation of RTKs by mutation, genetic amplification, or signals from the stroma contributes to disease progression and acquired drug resistance in cancer. Aberrant activation of ALK occurs in subsets of lung adenocarcinoma, neuroblastoma, and other cancers. We found that heparin is a ligand that binds specifically to the ALK extracellular domain. Whereas heparins with short chain lengths bound to ALK in a monovalent manner and did not activate the receptor, longer heparin chains induced ALK dimerization and activation in cultured neuroblastoma cells. Heparin lacking N- and O-linked sulfate groups or other glycosaminoglycans with sulfation patterns different than heparin failed to activate ALK. Moreover, antibodies that bound to the extracellular domain of ALK interfered with heparin binding and prevented heparin-mediated activation of ALK. Thus, heparin and perhaps related glycosaminoglycans function as ligands for ALK, revealing a potential mechanism for the regulation of ALK activity in vivo and suggesting an approach for developing ALK-targeted therapies for cancer.

INTRODUCTION

Receptor tyrosine kinases (RTKs) and their respective ligands play important regulatory roles throughout development and stimulate many critical cellular processes. Aberrant signaling by these molecules is implicated in various diseases, especially cancer. Very few RTKs in mammals have no known ligand and are considered “orphan” RTKs. One such receptor is anaplastic lymphoma kinase (ALK), which is present almost exclusively in the nervous system, primarily during embryonic development. Moreover, the gene encoding ALK is mutated or overexpressed in several cancers (13). Genetic fusions that result in the joining the kinase domain of ALK with various other proteins (for example, EML4-ALK and NMP-ALK) are the main drivers of a subset of non–small cell lung carcinoma and anaplastic large cell lymphoma cases (2), whereas acquired somatic or inherited germline mutations in ALK, or ALK overexpression, are implicated in neuroblastoma and anaplastic thyroid cancer (1, 3).

Pleiotrophin (PTN) and Midkine (MK), which are two related heparin-binding extracellular molecules, were reported to be physiological ligands for ALK (4, 5). However, subsequent studies were unable to confirm these results (610). Ligand interactions with RTKs induce autophosphorylation, receptor endocytosis, and activation of downstream signaling such as signaling pathways involving the kinases AKT and extracellular signal–regulated kinase 1/2 (ERK 1/2) (11). To date, no ligand has been identified for ALK that meets these criteria (610).

The extracellular domain (ECD) of mammalian ALK consists of two MAM (meprin/A5/protein tyrosine phosphatase Mu) domains, which flank an LDL-A (low-density lipoprotein class A) domain and are N-terminal to a glycine-rich region and a putative epidermal growth factor (EGF)–like domain (11, 12). The ECD is unique among RTKs, sharing high sequence similarity only with leukocyte tyrosine kinase (LTK) in the glycine-rich region and EGF domain. Like ALK, LTK is an orphan RTK (2, 11). There is a conserved, highly basic 249–amino acid N-terminal region (NTR) in mature vertebrate ALKs. The NTR of ALK has no reported function and is not conserved among invertebrate ALKs or any other protein. Moreover, ligands that bind invertebrate ALKs (Jeb in Drosophila melanogaster and HEN1 in Caenorhabditis elegans) are not conserved in mammals (2).

Here, we found that heparin is a specific, high-affinity ligand that directly binds the NTR of ALK. Short heparin chains bound monovalently to ALK ECD and antagonized ALK activation; longer heparin chains induced ALK dimerization, activation, and downstream signaling in cultured neuroblastoma cells. We developed monoclonal antibodies (mAbs) that bound to the NTR of ALK, inhibited heparin binding, and prevented heparin-induced ALK activation. Thus, heparin and perhaps other sulfated proteoglycans can function as ALK ligands or co-ligands, suggesting a mechanism similar to binding and activation of fibroblast growth factor receptors (FGFRs) by heparin and FGF (11).

RESULTS

Heparin activates ALK

The heparin-binding molecules PTN and MK are putative ligands for ALK. However, there are discrepancies among various reports regarding this role for PTN and MK (410). To investigate the ability of PTN and MK to activate ALK, we tested whether PTN or MK could activate endogenous ALK in cultured neuroblastoma cells (NB1) in the presence or absence of heparin. Similar to other RTKs, ALK autophosphorylation at several tyrosine residues can be used to monitor activation using antibodies against phosphorylated tyrosine or phosphorylation-specific antibodies targeting ALK (11). Exposing cells to mixtures of heparin and PTN or heparin and MK stimulated robust autophosphorylation of ALK (Fig. 1A). However, heparin alone was sufficient to induce ALK autophosphorylation to a similar degree as heparin mixed with PTN or heparin mixed with MK (Fig. 1A), indicating that PTN and MK were entirely dispensable for ALK activation. Moreover, heparin-induced ALK autophosphorylation and phosphorylation of downstream signaling proteins AKT and ERK 1/2 were concentration-dependent (Fig. 1B and fig. S1). Similar to ligand-mediated stimulation of other RTKs (11), heparin stimulation of NB1 cells induced ALK internalization (Fig. 1C and fig. S1).

Fig. 1 Heparin activates ALK in NB1 cells.

(A and B) Western blot for Tyr1604 phosphorylated ALK (pALK), Ser473 phosphorylated AKT (pAKT), and Thr202 or Thr204 phosphorylated ERK1 and ERK2 (pERK 1/2) using lysates from NB1 cells. In (A), cells were exposed to PTN (100 nM), MK (100 nM), heparin (HEP, 10 μg/ml), αALK2 (10 nM), or αALK1 (10 nM) alone or in the indicated combinations for 10 min. In (B), cells were exposed to heparin at different concentrations (100 to 0.01 μg/ml) for 10 min. MW, molecular weight. (C) Western blot for endogenous ALK using isolated cell surface–biotinylated proteins or lysates of NB1 cells exposed to heparin (10 μg/ml) or αALK1 (10 nM) for 6 hours. For (A) to (C), “-” indicates unstimulated negative control cells, and the blots are representative of at least two independent experiments.

We developed mAbs directed against the ECD of ALK and tested their ability to activate or inhibit ALK. The mAb αALK1 stimulated phosphorylation of ALK and ERK 1/2 (Fig. 1A) and internalization of ALK (Fig. 1C and fig. S1) in NB1 cells. In contrast, a different mAb, αALK2, inhibited the ability of heparin to induce phosphorylation of ALK and ERK 1/2 (Fig. 1A).

Heparin is a specific, high-affinity ligand for ALK

The above results suggested that heparin is a ligand or co-ligand for ALK, similar to the role of heparin as a co-ligand for all four members of the FGFR family of RTKs (11, 13, 14). Moreover, RPTPσ (receptor protein tyrosine phosphatase σ), which also plays a role in the development of the nervous system, binds directly to glycosaminoglycans (15). Therefore, we investigated the ability of heparin to bind directly and specifically to the ALK ECD.

Analysis of the NTR of ALK revealed a putative heparin-binding motif (Fig. 2A), similar to the motif found in FGFRs that is critical for the ability of heparin to act as a co-ligand for FGFR activation (13, 14). Deletion of the NTR eliminated the ability of heparin to promote autophosphorylation of ALK in transiently transfected human embryonic kidney (HEK) 293 cells (fig. S2), indicating that the NTR is required for ALK activation by heparin.

Fig. 2 Heparin directly binds the NTR in the ECD of ALK.

(A) Amino acid alignment of the NTR of ALK (amino acids 44 to 69) with the heparin-binding motif of FGFR. (B) Coomassie-stained protein gel of elution fractions from heparin-Sepharose chromatography. Top gel: FL-ECD and ΔN-ECD. Bottom gel: FL-ECD with mutations in the putative heparin-binding motif (mutation set 1: FL-ECDR48E,R51E,K52E or mutation set 2: FL-ECDR65E,R69E). Gels are representative of two independent experiments.

We characterized the biophysical properties of heparin binding to mammalian ALK in vitro. We used the ECD from Canis familiaris (dog ALK), which shares 91.2% identity to the human ECD (16). Notably, full-length ALK ECD (FL-ECD), but not a truncation mutant of the ALK ECD lacking the NTR (ΔN-ECD), could be purified using heparin-Sepharose chromatography (Fig. 2B). Mutating the basic residues in the putative heparin-binding motif of FL-ECD to acidic residues also inhibited binding to the heparin-Sepharose beads (Fig. 2B). Thus, these data suggest that heparin directly binds to the NTR domain of ALK.

To measure the affinity and specificity of binding between heparin and ALK, we used surface plasmon resonance (SPR). We immobilized biotinylated heparin to a NeutrAvidin surface and tested binding with various concentrations of FL-ECD and ΔN-ECD. The FL-ECD bound with high affinity (KD = 151 nM) (Fig. 3A), similar to the range of KDs of heparin binding to FGFs and FGFRs (14). In contrast, ΔN-ECD bound too weakly for a KD to be estimated (Fig. 3B). Moreover, the disaccharide heparin mimetic sucrose octasulfate (SOS) inhibited binding of FL-ECD to immobilized heparin at a median inhibitory concentration (IC50) of 6.5 μM and an inhibition constant (Ki) of 2.25 μM (Fig. 3, C and D), indicating a high degree of specificity for the interaction between heparin and ALK.

Fig. 3 Heparin binds to ALK with high affinity and specificity.

(A to C) SPR sensograms where a titration of FL-ECD or ΔN-ECD or a single concentration of FL-ECD in the presence of different concentrations of SOS (10 mM to 26 nM) are injected over a heparin-coated surface. RU, response units. Graphs are traces from the same surface and representative of three independent experiments. The average binding affinity (KD) of FL-ECD for the heparin-coated surface across three experiments was calculated using a steady-state model. (D) Graph of inhibition of FL-ECD binding to the heparin surface by SOS calculated from three independent experiments as shown in (C). Data are means ± SD. The Ki was calculated to be 2.25 ± 0.70 μM.

Heparin chain length correlates with affinity for ALK and ALK oligomerization

We hypothesized that the stoichiometry of the heparin-ALK interaction would depend on heparin chain length, with increasing chain length resulting in greater degrees of ALK oligomerization. The experiments used to determine binding affinity by SPR used heparin with heterogenous chain lengths. To test if there was a correlation between heparin chain length and ALK-binding affinity and oligomerization state, we used purified heparins with various degrees of polymerization (dp), where 1 dp is equal to 1 heparin disaccharide unit. However, it is important to note that these purified heparins were pools of different lengths defined by an average chain length. We used isothermal titration calorimetry (ITC) to quantify the interaction between FL-ECD and heparins with various average chain lengths. For heparin with an average chain length of dp8–9, dp15, or dp25, the molar ratio and affinity of FL-ECD for heparin indicated approximately monovalent (KD = 505 nM), bivalent (KD = 200 nM), and tetravalent or pentavalent (KD = 80 nM) binding, respectively (Fig. 4, A to C), suggesting that the enhanced affinity of ALK oligomerized on heparin of longer chain lengths is likely due to avidity associated with this interaction.

Fig. 4 ALK-binding affinity and oligomerization depends on heparin chain length.

(A to C) Results of ITC for heparin with the indicated average chain length titrated into a solution of FL-ECD. Top: Heat evolved from injection. Bottom: Integration of the heat released in top panels as a function of heparin to FL-ECD in the cell. Titration curves are representative of two independent experiments. Molar ratio is the average ratio of heparin to FL-ECD at which heat evolved is half maximal. KD is the average binding affinity of FL-ECD for heparin. (D and E) Results of SEC-MALLS for FL-ECD (D) or ΔN-ECD (E) mixed with heparin with the indicated average chain length. Solid lines represent the normalized refractive index in arbitrary units (AU) of FL-ECD or ΔN-ECD alone or as higher-order complexes. Dotted lines represent MWs of each species measured using MALLS. The measured MWs correspond to oligomeric FL-ECD species formation upon mixing with heparin. No complex formation is observed when heparin is mixed with ΔN-ECD. Graphs are representative traces from two independent experiments.

We used the molecular weight of the ALK-heparin complexes in solution to relate the molar ratio of heparin-ALK binding to ALK stoichiometry by subjecting FL-ECD and FL-ECD–heparin complexes to size exclusion chromatography coupled to a multiangle laser light scattering (SEC-MALLS) detector. In the absence of heparin or in the presence of heparins with an average chain length ≤dp10, FL-ECD eluted from the column with an apparent molecular weight equivalent to monomers (Fig. 4D and fig. S3). In contrast, in the presence of heparin dp15, a large proportion of FL-ECD eluted as an apparent dimer (Fig. 4D). In the presence of heparin dp25, FL-ECD eluted across a broad range of apparent molecular weights greater than that of monomeric or dimeric FL-ECD (Fig. 4D), suggesting a broad range of higher-order ALK oligomerization likely due, at least in part, to the heterogeneity of chain lengths in the average dp25 heparin pool. Unlike FL-ECD, ΔN-ECD did not elute at a different apparent molecular weight in the presence of heparin dp25 (Fig. 4E), consistent with the observations that ΔN-ECD did not bind to heparin-Sepharose or to the heparin-coated SPR surface (Fig. 2B).

Heparins of specific chain length and sulfation patterns activate ALK

To relate the stoichiometry of ALK-heparin binding to physiological activation of the receptor, we stimulated NB1 cells with different concentrations of heparin with different chain lengths and measured ALK autophosphorylation by enzyme-linked immunosorbent assay (ELISA). Heparin with average chain lengths greater than dp15, but not less than dp15, induced autophosphorylation of ALK (Fig. 5A), suggesting that ALK dimerization and higher-order oligomerization promote receptor activation.

Fig. 5 Heparins of specific chain length and sulfation patterns activate ALK.

(A) Graphs represent the autophosphorylation of ALK as measured by ELISA in lysates of NB1 cells exposed to heparins with the indicated average chain lengths at various concentrations for 10 min. (B) Western blot for the indicated proteins using lysates of NB1 cells exposed to mixed chain length heparin (10 μg/ml) and SOS at various concentrations for 15 min. pMAPK, phosphorylated mitogen-activated protein kinase. (C) Graphs representing the autophosphorylation of ALK measured by ELISA in lysates of NB1 cells exposed to the indicated heparins and heparin derivatives at various concentrations for 15 min. Various desulfated heparin derivatives were used that lack sulfates on either the iduronic acid or glucosamine subunits of heparin as follows: O-linked sulfates on carbon-2 of iduronic acid; O-linked sulfates on carbon-6 of glucosamine; O-linked sulfates on carbon-2 and on carbon-6 glucosamine; N-linked sulfates of glucosamine; and O- and N-linked sulfates on iduronic acid and glucosamine. (D) Western blot for the indicated proteins using lysates of NB1 cells exposed to the indicated glycosaminoglycans (10 μg/ml). For (A) and (C), data are means ± SD of three independent experiments. For (B) and (D), blots are representative of two independent experiments.

We tested whether SOS, which is equivalent to heparin with a chain length of dp1 and thus should not induce dimerization, inhibited heparin-induced activation of ALK in NB1 cells. We found that SOS reduced the ability of mixed chain length heparin to promote autophosphorylation of ALK and phosphorylation of AKT and ERK 1/2 in a concentration-dependent manner (Fig. 5B).

Heparin is an experimental proxy for physiological ligands used to study the effects of glycosaminoglycans on signaling by RTKs (2, 13, 14, 17). Glycosaminoglycans other than heparin are likely to be the physiologically relevant ALK ligand or co-ligand in vivo. Moreover, the sulfation pattern of glycosaminoglycans can influence binding and activation state of receptors, as observed with RPTPσ (14). Therefore, we tested whether exposing NB1 cells to differentially sulfated heparin or other glycosaminoglycans with different sulfation patterns activated ALK. We found that heparin, oversulfated heparin, and dextran sulfate induced autophosphorylation of ALK, whereas chondroitin sulfate, heparan sulfate, and various other specifically desulfated heparins did not (Fig. 5, C and D).

An antibody targeting the ECD of ALK inhibits heparin binding and heparin-induced activation of ALK

mAbs that directly inhibit ligand binding or receptor dimerization are useful for treatments of various clinical indications. ALK plays a role in neuroblastoma pathogenesis (1, 2). Therefore, we tested several mAbs targeting either the N-terminal or C-terminal half of the human ALK ECD for the ability to inhibit heparin binding using an SPR competition assay. The mAb αALK3 decreased the SPR refractive index (Fig. 6A), indicating that αALK3 disrupted the interaction of FL-ECD with the heparin-coated surface (increased Koff for FL-ECD). In contrast, seven other mAbs increased the SPR refractive index (Fig. 6A), indicating that these mAbs did not compete with FL-ECD for heparin binding and presumably bound at an independent site on FL-ECD, increasing the total mass on the heparin-coated surface. In the absence of FL-ECD, αALK3 and six of the other seven mAbs did not bind to the heparin-coated surface (fig. S4), indicating very little nonspecific direct binding to the surface.

Fig. 6 The mAb αALK3 inhibits heparin binding and activation of ALK.

(A) SPR sensogram of various IgG mAbs (100 nM) injected over FL-ECD bound to a heparin-coated surface. Data are representative of three independent experiments. (B) Western blot for the indicated antibodies using lysates of NB1 cells exposed to mixed chain length heparin (10 μg/ml) and the indicated concentrations of αALK3-Fab for 15 min. The blots are representative of two independent experiments.

Immunoblot analysis indicated that αALK3 bound to the NTR of ALK (fig. S5), suggesting that αALK3 directly competes with heparin at the heparin-binding site and should prevent activation of ALK by heparin. Thus, we tested whether αALK3 inhibited the ability of heparin to induce autophosphorylation of ALK. The whole immunoglobulin G (IgG) αALK3 mAb stimulated ALK autophosphorylation in NB1 cells (fig. S6), suggesting that the antibody caused receptor dimerization, which is an established mechanism for antibodies that bind RTKs and act as agonists (11). Thus, we generated a monovalent Fab fragment derived from αALK3 (αALK3-Fab). αALK3-Fab inhibited heparin-induced autophosphorylation of ALK with an estimated IC50 <100 nM (Fig. 6B). Likewise, αALK3-Fab inhibited heparin-induced phosphorylation of AKT and ERK 1/2 (Fig. 6B).

DISCUSSION

We found that heparin bound to ALK and induced ALK tyrosine autophosphorylation, suggesting a role for sulfated glycosaminoglycans and associated proteoglycans as physiological ligands for ALK. One possible mechanism of ALK activation is that sulfated carbohydrate moieties of a proteoglycan bind to the positively charged region in the NTR of ALK and the protein core of the same proteoglycan binds to another region in ALK. Another potential mechanism is that the sulfated carbohydrate moieties of the proteoglycan could act together with a second, yet to be identified, protein ligand to stimulate ALK dimerization and activation similar to the paradigm established for heparin and FGF stimulation of FGFR (11). LTK and ALK share homology in the glycine-rich region and EGF-like repeat. The glycine-rich region and EGF-like repeat comprise the entire ECD of LTK, suggesting that these regions likely serve as the binding site for an unidentified ligand. Thus, although ALK and LTK may share identical or related ligands, ALK is likely activated by two or more co-ligands: a sulfated glycosaminoglycan-linked proteoglycan that binds the NTR and a ligand that binds to the glycine-rich and EGF-like repeat of the ECD.

The identification of heparin as a ligand or co-ligand provides insight and tools for investigating ALK biology. Little is known about the function of mammalian ALK. In Drosophila, constitutive activation of ALK by Jeb is essential for maintaining normal brain metabolism during starvation (18). In addition to its role in neurobiology, ALK signaling has been implicated in driving several types of cancer (13). Similar to other RTKs, ligand-mediated increases in ALK signaling may contribute to tumorigenesis. Thus, the discovery of heparin as a bona fide ALK ligand provides mechanistic understanding of ALK activation and potential methods to inhibit oncogenic ALK by targeting its ECD.

MATERIALS AND METHODS

Cloning, expression, and purification of FL-ECD, ΔN-ECD, and heparin-binding motif mutations

The nucleotide sequence coding for amino acids 1 to 1037 of dog ALK (encoding FL-ECD) (16) was synthesized by Blue Heron. An 8X-His tag was added to the 3′ end, followed by a stop codon. An Xho I site was added upstream of the start codon, and an Xba I site was added directly after the stop codon. The construct was subcloned into pcDNA3.3. ΔN-ECD was produced by polymerase chain reaction–mediated deletion (19) of amino acids 21 to 263 using FL-ECD as the template. The mutations in the heparin-binding motif were produced using site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis protocol developed by Stratagene (catalog no. 200523). All plasmids were verified by sequencing of the complete open reading frame.

To produce recombinant His-tagged FL-ECD and ΔN-ECD, plasmids were transiently transfected into HEK293-S cells grown in Dulbecco’s modified Eagle’s medium–F12 with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin using the Lipofectamine 2000 as per the manufacturer’s instructions (Invitrogen). The culture medium was switched to Opti-MEM just before transfection. Transfected cells were incubated in Opti-MEM for 4 days. The medium was collected and clarified by centrifugation and vacuum filtration (0.45-μm polyvinylidene difluoride filter).

To isolate His-tagged proteins, nickel-Sepharose excel beads (GE Healthcare) were added to the clarified medium and incubated overnight at 4°C with agitation. Beads were washed with 30 column volumes (CVs) of phosphate-buffered saline (PBS) and 30 CVs of 20 mM imidazole in buffer A [25 mM Hepes, 150 mM NaCl, and 10% glycerol (pH 7.4)]. Elution was performed with 250 mM imidazole in buffer A. Fractions were collected and further purified by SEC on a HiLoad Superdex 200 column preequilibrated with buffer A. Fractions containing His-tagged proteins (estimated to be >97% pure) were collected, combined, and concentrated to 10 mg/ml using a 30,000-MWCO (molecular weight cutoff) concentrator device (Sartorius Stedim). For heparin-Sepharose chromatography experiments, eluted protein from the nickel column was dialyzed against buffer A without imidazole and then incubated with heparin-Sepharose beads (GE Healthcare) overnight. Heparin-Sepharose beads were washed with 50 CVs of buffer A and eluted in 25 mM Hepes, 1 M NaCl, and 10% glycerol at pH 7.4.

Generation of mAbs and αALK3-Fab

mAbs were purified from hybridoma cultures, derived from mice immunized with the ALK ectodomain (either M1-G460 or T637-S1038, which was synthesized, cloned, and expressed with 8X-His tags as described above for the dog FL-ECD and ΔN-ECD). Conditioned hybridoma medium was passed through a protein A column and washed with PBS, and mAbs were eluted with 0.1 M glycine (pH 2.7) and immediately neutralized with 1 M tris (pH 7.5). The buffer containing the purified mAbs was changed to PBS by tangential flow filtration.

αALK3-Fab was generated from the parental αALK3 IgG antibody and purified using the Fab Preparation Kit (Thermo-Pierce, catalog no. 44985). The Fab fragment was further purified by SEC, and the buffer was changed to PBS using a HiLoad Superdex 200 26/600 column.

NTR epitope mapping

The NTR of human ALK used for the experiment shown in fig. S5 was cloned into pET28b (Novagen) and expressed in BL21-(de3) Escherichia coli cells. Inclusion bodies were isolated and solubilized in Laemmli sample buffer for use in SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Solubilized inclusion bodies containing the NTR were used for Western blot.

ALK phosphorylation and signaling assays in NB1 cells

NB1 cells obtained from the American Type Culture Collection were cultured in RPMI supplemented with 10% FBS and 1% penicillin and streptomycin. NB1 cells were incubated with the indicated concentrations of heparin for 10 min at 37°C. For experiments with mAbs or αALK3-Fab, antibodies were incubated with NB1 cells before exposure to heparin. SOS, PTN, MK, and differentially sulfated glycans were added to NB1 cells at the same time as heparin. Different chain length heparins, various glycosaminoglycans, and desulfated heparins were purchased from Neoparin (catalog nos. GT8021, GT8011, GT8086, GT8085, GT80840, GT8083, GT8082, GT6030, GT6014, GT6013, GT6012, GT6011, and GT6020). Unfractionated heparin was purchased from Sigma (catalog no. H3393-25KU).

For Western blots, cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes, and membranes were incubated with antibodies against phosphorylated and total ALK, ERK 1/2, and AKT and β-tubulin (Cell Signaling Technology, catalog nos. 3633, 3333, 4695, 4370, 9272, 4060, and 2128).

For ELISAs, cell lysates were incubated in 96-well plates coated with an ALK antibody (Cell Signaling Technology, catalog no. 3791). Plates were rinsed with PBS and then incubated with a horseradish peroxidase (HRP)–conjugated phosphotyrosine antibody (R&D Systems, catalog no. HAM1676). HRP activity was detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo-Pierce) using a plate reader (BioTek). Luminescence was normalized to the maximal observed value and plotted as a function of the log10-transformed concentration of the glycosaminoglycan.

ALK internalization assay

NB1 cells were exposed to heparin or αALK1 for 6 hours. Cells were washed with PBS three times and incubated with cell-impermeable Sulfo-NHS-LC-Biotin (0.5 mg/ml) (Thermo-Pierce, catalog no. 21435) in PBS for 1 hour. Then, cells were washed with PBS containing 0.1 M glycine and lysed with 50 mM Hepes, 150 nM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 25 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and Roche Complete Protease Inhibitor Cocktail. Biotinylated proteins were enriched with 50 μl of NeutrAvidin agarose beads (Thermo-Pierce, catalog no. 29200) in 500 μl of lysate at room temperature for 1 hour, eluted overnight with sample buffer containing β-mercaptoethanol, and resolved by SDS-PAGE. Protein was transferred to nitrocellulose, and membranes were immunoblotted with an antibody for ALK.

Surface plasmon resonance

SPR experiments were performed using a Biacore T100 instrument (GE Healthcare) at 25°C. All reagents were dialyzed into a buffer composed of 25 mM Hepes, 150 mM NaCl, and 10% glycerol at pH 7.4. Biotinylated heparin (≥97% pure, Sigma, catalog no. B9806-10MG) was further purified using PD-10 prepacked columns (GE Healthcare, catalog no. 17-0851-01) to remove free biotin. Biotinylated heparin was immobilized on an assembled NeutrAvidin (Thermo-Pierce) surface (amine coupled on a CM4 Series-S Biacore chip). Three surfaces were produced with different concentrations of biotinylated heparin by varying contact time from 48 to 240 s. Threefold dilutions of FL-ECD and ΔN-ECD were injected sequentially and in random order over a reference surface without heparin and three heparin surfaces. The surface was regenerated between cycles with 2.5 M NaCl and 5 mM acetic acid at pH 4.5. For the SOS competition assay, serial dilutions of SOS were preincubated with 0.350 μM FL-ECD and then injected over the surface. IC50 and Ki values were derived by plotting the log-transformed concentration of SOS as a function of fractional bound FL-ECD at saturation and applying the Cheng-Prusoff equation.

Isothermal titration calorimetry

ITC assays were performed using a MicroCal VP-ITC instrument (Malvern) with a 1.3-ml cell volume and 250-μl ligand syringe with 25 mM Hepes, 150 mM NaCl, and 10% glycerol at pH 7.4 at 25°C. Each macromolecule and ligand was extensively dialyzed against this buffer. Heparins of defined average length were purchased from Neoparin. For dp25 heparin binding to ALK, 1.43 ml of 8.3 μM ALK was placed in the cell. Two hundred fifty microliters of 44 μM dp25 heparin was titrated in 8-μl increments. For dp15 heparin binding to ALK, 1.43 ml of 6 μM ALK was placed in the cell. Two hundred fifty microliters of 60 μM dp25 heparin was titrated in 8-μl increments. For dp8–9 heparin binding to ALK, 1.43 ml of 10 μM ALK was placed in the cell. Two hundred fifty microliters of 150 μM dp25 heparin was titrated in 10-μl increments. Data were collected and then processed, corrected for heat of dilution, and analyzed using “Origin 5.0 with MicroCal ITC feature” software. Data were fit to a one-site model by nonlinear least squares regression from which the calculated affinities and stoichiometries were derived.

Size exclusion chromatography–multiangle laser light scattering

The light scattering data were collected using a Superose 6 column in tandem with a Superdex 75, 10/300, HR SEC column (for FL-ECD experiments) or a Superose 6 column in tandem with a second Superose 6 column (for ΔN-ECD experiments) (GE Healthcare), connected to high-performance liquid chromatography (HPLC) System (Agilent 1200, Agilent Technologies) equipped with an autosampler. The elution from SEC was monitored by a photodiode array ultraviolet (UV) and visible light detector (Agilent Technologies), differential refractometer (Optilab rEX, Wyatt Corp.), and a static and dynamic MALLS detector [HELEOS II with QELS (quasielastic light scattering) capability, Wyatt Corp.]. The SEC detection system was equilibrated in 150 mM NaCl, 25 mM Hepes (pH 7.4), and 10% glycerol at a flow rate of 0.4 ml/min. ChemStation software (Agilent Technologies) was used to control the HPLC and data collection from the multiwavelength photodiode array UV and visible light detector. ASTRA software (Wyatt Corp.) was used to collect data from the refractive index detector and the light scattering detectors, and the UV trace at 280 nm, sent from the photodiode array detector, was recorded. Average molecular masses were determined across the entire elution profile in the intervals of 1 s from static light scattering measurement using ASTRA software as previously described (20). Heparin-ALK complexes were prepared by mixing heparin (purchased from Neoparin) in excess with FL-ECD.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/8/360/ra6/DC1

Fig. S1. Quantification of heparin-induced ERK 1/2 activation and ALK internalization.

Fig. S2. Heparin induces activation of full-length ALK, but not ΔN-ALK.

Fig. S3. Heparins with short chain lengths do not induce dimerization of ALK.

Fig. S4. Background binding of mAbs to heparin-only SPR surface.

Fig. S5. Epitope mapping of αALK3 by Western blot.

Fig. S6. Activation of ALK by mAbs.

REFERENCES AND NOTES

Acknowledgments: We thank J. H. Bae, J. Mohanty, S. Lee, and N. Kucera for discussion and assistance. Funding: This work was supported by a grant from Kolltan Pharmaceuticals (I.L.). Author contributions:P.B.M. designed and performed biophysical- and cell-based experiments and analyses, prepared the figures, and wrote the manuscript. I.L., G.F.L., J.S.L., E.J.N., X.S., and D.A. designed and performed cell-based assays. A.R., E.F.-S., and M.G. helped design or perform biophysical assays. D.A. and J.S. designed the overall study, supervised the experiments, and wrote the manuscript. Competing interests: The authors declare the following competing financial interests: J.S. is the founder and consultant of Kolltan. G.F.L., J.S.L., E.J.N., and D.A. are employees of Kolltan. Data and materials availability: Data and materials are available on request from J.S.
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