Two Restricted Sites on the Surface of the Nerve Growth Factor Molecule Independently Determine Specific TrkA Receptor Binding and Activation*

(Received for publication, December 11, 1996)

Klas Kullander Dagger , David Kaplan § and Ted Ebendal

From the Department of Developmental Neuroscience, Biomedical Centre, Uppsala University, Uppsala S-751 23, Sweden, and the § Montreal Neurological Institute, McGill University, Montreal PQ H3A 2B4, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Nerve growth factor (NGF) and neurotrophin-3 (NT-3) mediate activities such as survival, differentiation, and proliferation in various subsets of neurons. In this report, we define precisely the residues in human NGF responsible for NGF biological activity and binding specificity to the neurotrophin receptor TrkA. In earlier studies we defined five amino acid residues of NGF which confer NGF-like activity to NT-3 when replacing corresponding residues in the 120-amino acid long NT-3 molecule. Using this gain-of-function strategy we report the further dissection of this functional epitope. We also define another motif separated topographically in the NGF dimer and determined to be independently responsible for NGF specificity. The first of the two motifs determined to elicit NGF specificity is defined by the residues Val-48, Pro-49, and Gln-96, which are situated in the two top beta -loops of NGF. The second motif is represented by residues Pro-5 and Phe-7 situated in the proximal part of the NH2 terminus. Both motifs contain structurally important residues revealing a novel principle, where specificity for neurotrophin ligand-receptor interactions could be determined by variable residues modifying the conformation of the neurotrophin backbone. These findings will enhance further the possibility of mimicking NGF with low molecular weight compounds.


INTRODUCTION

Nerve growth factor (NGF),1 brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4; NT-5 is the mammalian homolog to NT-4) are members of a family comprising structurally related proteins termed neurotrophins. These proteins promote growth and survival of neurons in the nervous system (for review, see Ref. 1). Each of these proteins binds to at least two membrane receptors. The p75 neurotrophin receptor, which binds all members of the neurotrophin family, enhances the NGF binding affinity and ligand binding specificity together with the TrkA receptor. Moreover, p75 neurotrophin receptor has the ability to signal through the ceramide pathway in response to NGF (2), activate nuclear factor-kappa B, and mediate apoptosis in neural cells (3, 4). The other known receptors for neurotrophins are tyrosine kinases that show different specificities for binding to different neurotrophins; the TrkA receptor preferentially binds NGF, the related TrkB receptor binds BDNF and NT-4, and TrkC binds NT-3. Although data on the cross-binding of NT-3 to all Trk receptors has been published, these studies have been made using cell lines expressing ectopic receptors and with neurotrophin concentrations above those expected to be present in vivo. At physiological concentrations of NT-3, the preferred receptor is TrkC (5).

The roles of NGF and NT-3 and their receptors in neural development have been studied extensively by gene targeting in mice (6-10). Data from these and many other types of studies have established that these neurotrophins are crucial for the development of the peripheral nervous system; their roles in the central nervous system are less well defined. In the peripheral nervous system NGF is a survival factor for sympathetic neurons and small sensory neurons associated with nociception. In the central nervous system, NGF mediates the differentiation and survival of basal forebrain cholinergic neurons, a population that deteriorates in Alzheimer's disease (9, 11, 12). NT-3 has been shown to be a survival factor for the proprioceptive neurons in the peripheral nervous system (7, 13), facial motor neurons, and noradrenergic neurons of the locus coeruleus (14). Thus, NGF and NT-3 have major roles in the development of the peripheral nervous system as well as several defined effects on populations of neurons in the central nervous system.

This family of closely related polypeptides has many appealing features that make them suitable for mutational analysis. The neurotrophins are small (usually around 120 amino acids) structurally related molecules with clusters of conserved or variable residues. The three-dimensional structure has been determined for NGF (15, 16) and a BDNF·NT-3 heterodimer (17), which revealed a flat molecule organized as a symmetrical dimer with protruding beta -loops. The examination of structural determinants for specificity among the neurotrophins can provide insight into how ligand-receptor interactions have evolved in the complex nervous system. In addition, precise knowledge of the residues in the neurotrophins which bind their receptors will provide clues for identification of low molecular neurotrophic antagonists and agonists that may have therapeutic potentials for the treatment of neurodegenerative diseases such as Alzheimer's and Parkinson's or in stimulating or blocking neurotrophin actions in peripheral neuropathies and pain conditions.

Several approaches have been used to elucidate which sequences of the neurotrophins are required to specifically bind and activate their cognate receptors (18). A first approach used chemical modification of NGF where residues receptive for different agents could be blocked and the effect analyzed (for review, see Ref. 19). Enzymatic cleavage of NGF has identified the NH2 terminus to be critical for binding to TrkA (20-22). Establishment of heterodimers between neurotrophins has brought further insight regarding specificities, where a BDNF·NT-3 heterodimer was 10-fold less biologically active than a mixture of BDNF and NT-3 homodimers (23). Deletions of the NGF coding sequence by site-directed mutagenesis have identified the NH2 and COOH termini as necessary for binding to TrkA (24, 25). Furthermore, by exchanging blocks of amino acid residues among the neurotrophins, it has been established that the NH2 terminus and the variable beta -loops are involved in neurotrophin specificity (26-28). When blocks of residues from BDNF variable regions I, III, and V were substituted into the corresponding segments of NGF, there was no loss of NGF activity, implying that the remaining beta -loops II and V or the NH2 and COOH termini, are involved in NGF specificity (26). Thus, the understanding of neurotrophin specificity and neurotrophin binding is emerging from several studies, although thus far most studies have focused upon NGF. Using a gain-of-function strategy, we have shown previously that exchanges of NGF amino acids in NT-3 allowed NT-3 to act with a NGF-like activity (28), thus defining a motif involved in NGF specificity situated in the two top beta -loops. Here we report the further identification of the amino acids necessary for determining NGF specificity in the two top beta -loops as well as in the NH2 terminus, as assessed by biological effects on NGF-responsive neurons and activation of TrkA.


EXPERIMENTAL PROCEDURES

Cells, Neurotrophins, and Mutants

Human NGF and NT-3 were cloned by polymerase chain reaction using a cDNA library constructed from primary astrocyte-like cells derived from a 9-week-old human fetal brain (clinical abortion). For a thorough description of the principle for this strategy of mutagenesis, see Ref. 28. Briefly, single mutants were produced first, and a secondary polymerase chain reaction using the single mutants as template produced the combinatorial mutants shown in Fig. 1A. In this work mutants were given their names according to the NGF amino acids exchanged into the NT-3 backbone. VF-QAA and parts thereof were amino acids in NGF (see Fig. 1A) residue positions 48, 49, and 96-98. N-term and parts thereof designate the amino terminus of NGF corresponding to the first seven amino acids in the mature protein SSSHPIF. For clarity, residues or position numbers in Roman type refer to human mature NGF, and residues or position numbers in italics refer to human mature NT-3. As seen in Fig. 1, residue position numbers in NGF and NT-3 do not match because of gaps inserted in the alignment for best fit of the protein sequences. The fragments were inserted by direct cloning into the pBSKS+ cloning vector (Stratagene), cleaved with EcoRV, and subsequently incubated with Taq polymerase and 2 mM dTTP at 72 °C for 2 h. After sequencing the fragments were subcloned to the expression vector pXM (Genetics Institute), and 30 mg was electroporated (Porator, InVitrogen) into 1 × 106 COS cells in 0.6 ml of phosphate-buffered saline at 400 V and 250 microfarads at 22 °C. After 1 day of incubation in DMEM with 10% fetal calf serum (FCS) the medium was changed to DMEM + 0.5% FCS for harvest after another 3 days. Parallel porations were metabolically labeled by incubation for 5 h in 200 ml of cysteine/methionine free DMEM supplemented with [35S]cysteine/methionine. 50 ml of this labeled medium was boiled in SDS sample buffer and run on a 15% SDS-polyacrylamide gel. To equalize the concentrations of the produced proteins in the conditioned media, the gels were dried down and exposed using a PhosphorImager, and the resulting bands were quantified. The medium giving rise to the NGF band was measured in an enzyme immunoassay for NGF (29) to obtain an absolute concentration value corresponding to the density of the NGF band.


Fig. 1. Analysis of residues in two defined motifs. Panel A, alignment of the amino acid residues in human NGF and human NT-3. Mutated sites are shown in boxes. The two shaded boxes represent the NGF-specific VF-QAA motif also seen in Fig. 6, A-D, as three-dimensional models. Also, between shaded boxes the various mutants produced are shown. The open box in the beginning of the sequence represents the NH2 terminus and the mutants produced in this region. The position of the NH2 terminus is shown in Fig. 6D; a model of the peptide structure comprising the six NH2-terminal residues can be seen in Fig. 6, E and F. Dots indicate identity; dashes indicate gaps inserted for best alignment. The variable regions in the three beta -loops are marked in the figure and referred to as regions I, II, and V (as defined in Ref. 26) in the text. Panels B and D, TrkA tyrosine phosphorylation measured on 3T3 cells expressing TrkA after a 5-min ligand stimulation correlates well with panels C and E, the relative ratio of fiber outgrowth activity from the sympathetic ganglia versus the Remak's ganglion. The fiber outgrowth was assayed on whole ganglia explants that were stimulated for 2 days in culture.
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Fiber Outgrowth and Survival Assay

Sympathetic and Remak's ganglia were dissected from E9 chicken and embedded in a gel of native collagen fibrils (30). The cultures were incubated for 2 days under standard tissue culture conditions (Eagle's basal medium, 1% FCS) together with neurotrophins and then examined in dark-field illumination through an inverted microscope. The fiber outgrowth activity was determined on a scale from 0 to 1 (increments of 0.1 unit), where 1 is a full outgrowth with a dense halo of fibers originating from the ganglia (30). To check for survival effects, sympathetic ganglia dissected from E9 chicken were dissociated in 0.25% trypsin and plated in a thin collagen gel for incubation for 2 days. Neurotrophins or mutants were added immediately at different concentrations ranging from 0.5 to 100 ng/ml, and surviving neurons were counted in a phase-contrast microscope.

Binding Studies

Rat pheochromocytoma PC12 cells were grown in DMEM supplemented with 10% horse serum and 5% FCS and resuspended in DMEM + 0.5% FCS to a concentration of 1 × 107 cells/ml. 3T3-TrkA cells were harvested with 1% EDTA in phosphate-buffered saline and also used at a concentration of 1 × 107 cells/ml. Incubation was performed for 1 h with agitation at 4 °C with 100 ml of cell suspension mixed with 100 ml of medium containing different concentrations of purified mutant or wild type protein. After blocking, iodinated mouse 2.5 S NGF (Amersham Corp., 1,500 Ci/mmol) was added to a final concentration of 4 × 10-11 M for PC12 cells and 5 × 10-10 M for 3T3-TrkA cells and incubated for another h. To separate unbound NGF from bound NGF the cells were centrifuged through a sucrose gradient consisting of a 0.3 M sucrose bottom layer with 0.15 M sucrose overlay in a microcentrifuge at 13,000 rpm for 10 min (31). The tubes were frozen in liquid nitrogen, and the bottoms with the cell pellet were cut off and counted in a LKB 1275 mini-gamma counter. To measure the unspecific binding a 100-fold excess of cold mouse beta -NGF was added and the obtained value (never more than 10% of signal) subtracted from the measurements. Experiments were repeated three or four times, and the ratio of binding was calculated as cpm bound/(cpm bound + cpm free).

In Vitro Tyrosine Kinase Assay

3T3-TrkA expressing cells at a density of 2 × 107 cells/ml were stimulated with conditioned media or purified protein (100 ng/ml) for 5 min at +37 °C, washed with TBS buffer (20 mM Tris, pH 7.5, 137 mM NaCl), and then lysed in Nonidet P-40 buffer (20 mM Tris, pH 7.5, 137 mM NaCl, 500 mM Na3VO4, 10% glycerol, 1% Nonidet P-40, 1 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). The cells were centrifuged to pellet cell debris and incubated with an anti-Trk (C-14, Santa Cruz Biotechnology) antibody at a concentration of 1 µg/ml for 1 h. Protein A-Sepharose beads (Pharmacia Biotech Inc.) were added and the mixture incubated for an additional 30 min. All procedures were done at 4 °C. The beads were centrifuged and washed three times in Nonidet P-40 buffer and once in distilled water. The samples were subsequently boiled in SDS-gel loading buffer, run on a 7.5% acrylamide gel, transferred to nitrocellulose. and detected with 4G10 anti-phosphotyrosine antibody (Upstate Biotechnology). A secondary antibody conjugated with horseradish peroxidase was used together with chemiluminescence to detect phosphorylated tyrosine residues.

PC12 Cell Differentiation Assay

PC12 cells or PC12 6-24 cells were grown in DMEM supplemented with 5% FCS and 10% horse serum. Neurotrophins or mutants were added to the cultures, and after 6 days (PC12) or 2 days (PC12 6-24) the ratio of cells with fibers longer than two cell diameters was counted. Three different plates were counted for each concentration, and at least 200 cells were analyzed in each plate.

Purification of Mutated Neurotrophins

Constructs with NT-3, the P-F mutant, or the VF-QAA mutant were electroporated into dihydrofolate reductase negative (dhfr-) Chinese hamster ovary cells. After selection in growth medium without nucleoside analogs supplemented with 10% dialyzed FCS, positive clones were tested for their ability to produce neurotrophin mutants with neurite outgrowth activity on Remak's ganglia. Such clones were amplified for higher expression levels of desired protein by using successively higher concentrations of methotrexate in the growth media (32). Chinese hamster ovary cells that after amplification produced ~1,000 ng/ml of desired neurotrophin were grown in triple layered culture flasks, and 2 liters of medium was collected for further purification. Serum proteins in the medium were precipitated away using 15% saturation of ammonium sulfate. Remaining proteins in the supernatant were then precipitated by increasing the ammonium sulfate concentration to 80% of saturation. The protein pellet was resuspended and dialyzed against 50 mM Tris-HCl, pH 9.0. The sample was loaded on an equilibrated (50 mM Tris-Cl, pH 9.0) CM-Sepharose ion exchange column (Pharmacia). The column was washed with 3 column volumes of equilibration buffer before being eluted with 0.4 M NaCl in 50 mM Tris-HCl, pH 9.0. Fractions of 5 ml were collected of the eluate, tested for biological activity in the ganglion assay, and analyzed on a 10-20% SDS-polyacrylamide gradient gel. Typically this process yielded about 1.5 mg of active neurotrophin.


RESULTS

By using polymerase chain reaction-based in vitro mutagenesis we have produced various NT-3 mutants containing different exchanges of NGF residues (Fig. 1). Mutants are produced in COS cells and metabolically labeled conditioned media are used to verify that the mutants are expressed at similar levels, ranging from 100 to 300 ng/ml. All mutants presented here (Table I) were found to be expressed within this range, and they were biologically active and capable of phosphorylating the TrkC receptor. As shown, the specific NGF-like activity varied considerably depending on which residues were introduced into the NT-3 backbone.

Table I.

Fiber outgrowth activity and tyrosine phosphorylation of wild type and mutant neurotrophic proteins


NGF residues replaced in NT-3a Fiber outgrowth activityb
Tyrosine phosphorylationc
Symp Rem TrkA TrkC

NT-3 + +++  - +++
N-term 1-7 +++ +++ +++ +++
SSS 1-3 + +++  - +++
PIF 5-7 +++ +++ +++ +++
P 5 + +++  - +++
IF 6-7 + +++  - +++
P-F 5, 7 +++ +++ +++ +++
N-term-VF-QAA +++ +++ +++ +++
VF-QAA 48-49, 96-98 +++ +++ +++ +++
VF 48-49 +(+) +++ +(+) +++
QAA 96-98 + +++  -/+ +++
V-QAA 48, 96-98 + +++ + +++
F-QAA 49, 96-98 + +++ + +++
VF-Q 48-49, 96 +++ +++ +++ +++
VF-A 48-49, 97 +++ +++ +++ +++
VF-A 48-49, 98 +++ +++ +++ +++
VF-AAA 48-49, 96-98 + +++ ND ND
AAA-AA-AAA, 43-45, 48 49, 96-98 + +++ ND ND
NGF +++  - +++  -
BDNF  - ND ND ND
B3 (NIN-VF-QAA)d + ND ND ND

a Mutants designated by the residues from NGF which replaced the original NT-3 residues; NGF residue positions given.
b Data from at least three independent dose-response experiments where neurite outgrowth was determined after stimulation for 2 days in culture. ND, not determined; Symp, sympathetic ganglia; Rem, Remak's ganglion.
c Measured on TrkA- or TrkC-3T3 cells stimulated by conditioned media with approximately 50 ng of neurotrophin/ml. Results were obtained from triplicate experiments.
d NGF residues replacing corresponding residues in BDNF.

To analyze the function of the NH2 terminus of NGF, we produced a NT-3 mutant with the six NH2-terminal residues replaced with the corresponding seven NGF NH2-terminal residues (protein sequences are shown in Fig. 1A). Confirming previous reports (20-22, 25, 33), the NH2-terminal mutant showed robust NGF-like responses (Fig. 1, B and C), as judged by both tyrosine phosphorylation of the TrkA receptor in 3T3 cells (Fig. 1B) and fiber outgrowth from chick E9 sympathetic ganglia (Fig. 1C). This prompted us to dissect the NH2 terminus further to establish the individual residues responsible for this effect. Two mutants were produced in which the NH2 terminus of NT-3 was replaced with either the three most distal NH2-terminal residues (SSS replacing YAE; residues or position numbers in Roman type refer to amino acids in human mature NGF, and residues or position numbers in italics refer to amino acids in human mature NT-3) or the three proximal residues following the shared His-4 (PIF replacing KS, Fig. 1, B and C). The fiber outgrowth response and tyrosine phosphorylation of the TrkA receptor (Fig. 1, B and C) indicated that NGF-like activity was achieved only when the PIF motif in NGF replaced the KS motif in NT-3. Further analysis of the PIF motif, where Pro-5 replaced Lys-5 in one mutant and Ile-6 and Phe-7 replaced Ser-6 in another mutant, revealed that these replacements did not allow NT-3 to achieve NGF-like activity (Fig. 1, B and C). However, a NT-3 mutant where Pro-5 replaced Lys-5 and Phe-7 was inserted after Ser-6, was produced, and this mutant displayed substantial NGF-like activity (Fig. 1, B and C). This mutant also promoted survival in dissociated sympathetic neurons as seen in Fig. 2. Thus, Pro-5 and Phe-7 are two residues important for NGF specificity.


Fig. 2. Survival of sympathetic neurons. Mutants identified as potent TrkA activators could also enhance survival of dissociated sympathetic nerve cells. As shown, the VF-QAA and the P-F mutant are both considerably more efficient compared with NT-3 or the NH2-terminal mutant SSS. B3 is a mutant with NGF residues NIN(43-45), VF(48-49), and QAA(96-98) replacing corresponding BDNF residues. Note that all values are in percentage of wild type NGF activity.
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The motif VF-QAA (Fig. 1) was established earlier as a potent activator of NGF-like activity when these residues were inserted in the corresponding positions in the NT-3 (28). To establish further which of these five amino acids are involved in the NGF-like response, a set of five mutants was produced: V-QAA, F-QAA, VF-Q, VF-A(97), and VF-A(98). Our earlier results showed that the VF or QAA motif by itself did not achieve NGF-like responses (Fig. 1, D and E). Two NT-3 mutants were generated to evaluate the VF motif, V(48)-QAA and F(49)-QAA. These mutants did not exhibit NGF-like activity assessed by tyrosine phosphorylation of TrkA (Fig. 1D) and the lack of induced fiber outgrowth from chick E9 sympathetic ganglia (Fig. 1C). This suggests that both Val-48 and Phe-49 in the VF-QAA motif are residues necessary to achieve NGF-like activity. Phe-49 is placed like a shovel in the groove formed by the top beta -loop (II) and the proximal beta -loop from the other protomer (V) and could thereby be available for a hydrophobic interaction with the TrkA receptor (see Fig. 6, A-D). In contrast, three mutants in the QAA motif, VF-Q(96), VF-A(97), and VF-A(98), all yielded NGF-like activity (Fig. 1, D and E), suggesting that the residues Gln-96, Ala-97, and Ala-98 each independently contributes to the NGF-like activity seen in the NT-3 mutants when combined with the VF motif. To establish further whether the interactions from these residues in the two top beta -loops determine specificity by attraction or repulsion, alanines were instead exchanged at the corresponding place in the NT-3 backbone. A mutant with the NGF residues VF replacing PV (residues 48 and 49) in combination with alanines exchanging LVG (residues 96, 97, and 98) was analyzed as well as a mutant where alanines replaced KTG-PV-LVG (residues 42-44, 47-48, 96-98). Interestingly, none of these mutants showed any significant NGF-like activity, although they all displayed normal NT-3 like activity (Table I). These results suggest that Gln-96 is actively contributing to NGF specificity, and its presence overrides Val-97 and Gly-98. In turn, Val-97 and Gly-98 may suppress NGF-like activity of NT-3 by weak repulsive actions directed toward TrkA. This is shown by the results where replacing one of these residues with alanine results in an NGF-like activity (Fig. 1, B and D). Also, we have used a BDNF triple mutant, B3 (NGF residues NIN 43-45, VF 48-49, and QAA 96-98 replacing corresponding BDNF residues) to analyze the VF-QAA motif when put in a neurotrophin backbone other than NT-3. The B3 mutant is somewhat more efficient in promoting survival of sympathetic neurons than wild type BDNF (Fig. 2) but not as efficient as comparable mutants using the NT-3 backbone as a framework (Table I), indicating differences among the neurotrophins as to their specific receptor determinants.


Fig. 6. Three-dimensional representation of the NGF-dimer with models of the mutants examined. Panel A-C, the top part of NGF consisting of two beta -loops from each protomer with the mutated sites in red (residues QAA) and blue (residues VF). The two protomers are shown in green and yellow, respectively, as seen from three angles in a space-filling model of the protein including side chains. In panel A the view is from the arrowhead in panel D. In panel B, the molecule is rotated 90° counterclockwise along the axis of symmetry compared with panel A. In panel C a further 20° rotation is shown. The VF motif from one protomer and the QAA motif from the other protomer are closer and more likely to form a patch interacting with the receptor than the two motifs within each protomer. Panel D, a three-dimensional representation of the peptide backbone of the NGF dimer (the two protomers shown in green and yellow. The three cystine bridges forming the cystine knot (typical for neurotrophins and some other families of growth factors) are seen in blue in the lower part of the protein. panel E The NH2 terminus of mature human NT-3 modeled as a peptide with the first amino acid (Y) to the right. Note that the conformation of the first 11 amino acid residues of NGF is unknown. The NT-3 amino acid residues are shown in panel E; panel F shows the conformational changes predicted in the model when a lysine is replaced by a proline, and a phenylalanine is inserted in the gap at position 7 as in the P-F mutant examined here. This alteration profoundly disrupts the wild type peptide backbone structure and may explain the effective gain of NGF-like activity despite the minute changes of residues.
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To analyze further the motifs identified above to be important in determining NGF specificity we purified the VF-QAA mutant, the P-F mutant, and wild type NT-3. Transfected Chinese hamster ovary cells that expressed the desired mutants were selected, and the expression was amplified to a yield a production of the neurotrophin mutants of ~1,000 ng/ml. The conditioned media were subjected to fractionated precipitation with ammonium sulfate and further fractionated by ion exchange chromatography (Fig. 3A). Following this, the neurotrophin mutants were obtained at a concentration of 300 µg/ml and appeared as a single band with an apparent molecular mass of 14 kDa on a 10-20% gradient gel (Fig. 3B). The purified mutants were tested again for biological activity to confirm data from transient transfections. The analysis showed that the NH2-terminal mutant had a higher NGF-like activity than the VF-QAA mutant and that the purified mutants displayed the expected biological activities (Table I and Figs. 4 and 5). Examples of survival and fiber outgrowth assays using purified proteins are shown in Fig. 4, A and B. In survival assays using sympathetic neurons the effects from three different protein concentrations (20, 5, and 2 ng/ml) were analyzed and compared with the effects of equivalent concentrations of NGF (Fig. 4). At 20 ng/ml there were no differences between the mutants and NGF (Fig. 4C). Also, when testing lower concentrations, the potency of the mutants compared with NGF was approximately 60-80%, with the P-F mutant being somewhat more potent than the VF-QAA mutant. Similar results were obtained using a PC12 differentiation assay (Fig. 5, A and B), with the neurotrophins assayed at a concentration of 20 ng/ml.


Fig. 3. Purification of the two most efficient mutants as well as NT-3. Panel A, three fractions eluted from the CM-Sepharose column (one-step elution by 0.4 M NaCl at pH 9.0, 0.2 M Tris-HCl) run out on a 10-20% SDS-polyacrylamide gradient gel. Fraction 7 holds many contaminating proteins, but fraction 8 is almost devoid of detectable proteins. Fraction 9 contains the desired mutant neurotrophin as a single band. Panel B, three purified neurotrophins (wild type NT-3, VF-QAA, and P-F) run out on a 10-20% SDS-polyacrylamide gradient gel compared with murine NGF (40). The three neurotrophins with NT-3 backbones migrate as 14-kDa proteins; NGF migrates as a slightly smaller molecule.
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Fig. 4. Activity of purified neurotrophins. The purified neurotrophin mutants were assayed for their ability to evoke fiber outgrowth response from chick E9 sympathetic ganglia tested at 10 ng/ml (dark-field microscopy) (panel A) and to rescue dissociated sympathetic neurons (phase-contrast microscopy) (panel B). In panel C the survival activities of wild type NT-3 and of the VF-QAA and P-F mutants are represented as percentage of NGF activity measured at three different concentrations (n = 4) compared with background survival in Eagle's basal medium (BME). Cultures were incubated for 2 days. Scale bar is 500 µm in panel A and 100 µm in panel B.
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Fig. 5. Activation and binding to the TrkA receptor. The purified mutant neurotrophins were tested for their ability to induce differentiation in PC12 cells (panel A) as well as cell line PC12 6-24-overexpressing TrkA receptors (panel B). The mutants were also potent NGF agonists in this assay. Panel C, phosphorylation of the TrkA receptor expressed on 3T3 fibroblasts after stimulation with 100 ng/ml of the indicated protein. Panels D and E, capacity of NT-3, NGF, and the purified mutants to displace iodinated NGF from either (panel D) PC12 cells (n = 4) or (panel E) 3T3 fibroblasts expressing TrkA (n = 3). Error bars represent S.E.
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Finally, we examined the abilities of the neurotrophin mutants to induce differentiation in PC12 cells or PC12 6-24 cells that overexpress human TrkA 20-fold compared with endogenous rat TrkA (34). As shown in Fig. 5, A and B, the relative potencies of the mutants are comparable in both cell types, although the responses to the mutants were approximately 15% higher in the 6-24 cells. Furthermore, we confirmed that the purified mutants activated the TrkA receptor, as assessed in 3T3-TrkA cells (Fig. 5C). Competitive binding assays revealed differences between the two mutants when comparing displacement of iodinated NGF from either PC12 or 3T3-TrkA cells (Fig. 5, C and D). The P-F mutant was 10 times less efficient in displacing 50% of labeled NGF than NGF itself, whereas the VF-QAA mutant was 30 times less efficient. However, on TrkA receptors in 3T3 cells (not expressing p75), the P-F mutant was only five times less efficient and the VF-QAA mutant 10 times less efficient. These differences result from the presence of p75 neurotrophin receptor on PC12 cells and not on 3T3-TrkA cells. In summary, the P-F mutant is somewhat more NGF-like than the VF-QAA mutant when comparing biological activity, TrkA receptor activation, and NGF receptor binding.


DISCUSSION

In this study, NT-3 served as a framework in which potential residues defining NGF specificity replaced the corresponding NT-3 residues (Fig. 1A). The assay for neurotrophin specificity is based on two experimental systems, one biological and one biochemical. First, we use a robust biological assay measuring fiber outgrowth from explanted embryonic chicken ganglia (30). For the analysis of NGF fiber outgrowth activity we use the sympathetic ganglia that express TrkA mRNA (35) and only weakly respond to NT-3 (36, 37). To measure NT-3 fiber outgrowth activity we use Remak's ganglia, which do not respond to NGF and only express TrkC mRNA (38). Second, we examined ligand-induced receptor tyrosine phosphorylation using 3T3-TrkA fibroblasts expressing TrkA but not TrkC and 3T3-TrkC fibroblasts that express TrkC but not TrkA. Hence the two systems for analyzing mutants are dual, i.e. they analyze in comparative fashion the NGF and NT-3 activity in primary neurons and in cell lines. The ratios are less dependent on the concentration used and therefore a more reliable way to estimate the potential NGF-like activity of the mutants.

We establish that replacement of the NH2 terminus of NT-3 with corresponding residues of NGF confers NGF-like activity independent of our previously defined VF-QAA motif in variable regions II and V of neurotrophins (28). A further dissection of the NH2 terminus showed that the inner part of this region determines NGF specificity. More precisely, when Lys-5 was exchanged for Pro-5 together with an insertion of Phe-7 in the corresponding place in NT-3 (between Ser-6 and His-7, see Fig. 1A) this NT-3 molecule was nearly as potent as NGF (Figs. 1, B and C, 4, and 5). When NGF NH2-terminal residues 3-9 were replaced by the corresponding BDNF residues, different results were obtained: one group reported only 5-fold decreased binding on TrkA receptors (33), whereas another group reported a 2-4-fold effect on bioactivity but no decrease in binding to PC12 cells (39). These results are interesting since the BDNF residues include a proline and also lack a residue where NGF has Phe-7. It is thus tempting to argue that these amino acids account for the differences seen when using BDNF and NGF rather than NGF and NT-3 for the homolog scanning. The NH2 and COOH termini are not resolved in the available crystal structures (Fig. 6) of the neurotrophins (15, 17). Recently, the bioactive conformation of the NGF amino and carboxyl termini has been predicted by molecular modeling (41). These studies demonstrate that the conserved residues His-4, Pro-5, His-8, Glu-11, and Arg-118' cause a split of the amino-carboxyl termini complex into two distinct moieties: a ring-like rigid region (residues 9-11 of one protomer, and residues 112'-118' of the second protomer), and a flexible loop (residues 1-8). Although only the rigid region seems to be necessary for TrkA-mediated activity, the flexible loop might be directly involved in those NGF-receptor interactions that reinforce geometric selectivity, primarily because of the exposed beta -sheet motif provided Phe-7 through Arg-9.

Apart from the NH2 terminus, another surface introducing NGF activity to NT-3 is comprised of amino acid residues VF-QAA of variable regions II and V (26, 28). These loop regions are structurally ideal candidates for specific interactions with receptor molecules since they can accommodate changes without affecting the overall structure (42). The combinatorial effect of the amino acid exchanges into NT-3 in the VF-QAA motif is crucial to achieve an NGF-like effect. The exchange of VF alone into the NT-3 framework did not yield NGF-like activity, but when exchanged in combination with the QAA motif this NT-3 mutant was nearly as potent as NGF (Fig. 1, D and E). When considering the three-dimensional model of NGF, the importance of this combination can be interpreted as an interaction between the two protomers. We proposed previously (28) that the VF motif from one protomer forms a patch with the QAA motif from the other protomer rather than with the QAA motif from the same protomer (Fig. 6, A-C). Similar conclusions regarding cooperation between neurotrophin protomers to achieve their specialized function have been reported by investigators using different approaches (17, 43). When replacements of alanines in the LVG (96-98) motif were carried out one at the time in combination with the VF exchange, we saw surprisingly that each of these mutants gave robust NGF-like activity (Fig. 1, D and E). In contrast, a mutant replacing the LVG residues with alanines, again in combination with VF, failed to result in NGF-like activity (Table I). This mutant only differed by Gln-96 compared with the VF-QAA mutant, and this suggests that the Gln-96 is necessary for NGF conversion. For Val-97 and Gly-98 in the QAA motif it must be considered that the effect from the alanine exchanges is of a negative nature because alanines are not likely to have a role in selection toward a receptor. More plausible is that the effect of taking away Val-97 and Gly-98 from NT-3 allows for interaction to the NGF receptor. Interestingly, when region V (containing the QAA motif in NGF) from BDNF was inserted into a NT-3 chimera (43) the interaction with TrkA was reduced, further emphasizing that this loop region contains determinants for specific TrkA binding.

Both motifs found here to determine specificity include residues known to be critical for conformation of a peptide backbone. When Pro-47 from the NT-3 wild type protein is replaced by Val-48 the peptide backbone is allowed to move more freely. If simultaneously exchanging Val-48 for Phe-49, a sterical effect to reposition the loop is likely. Also in beta -loop V, a residue known to be a structural determinant (Gly-98) is involved in the specificity, but this effect is not as radical as in the two other motifs since the change of the glycine is not necessary for the conversion to NGF-like activity in the QAA motif (Fig. 1, D and E). The flexibility of beta -loops has been apparent when analyzing several different crystals of NGF, and the differences in the positioning of beta -loops extended in some cases to 6 Å (16). Furthermore, in the BDNF·NT-3 crystal, beta -loop II resolved with higher temperature, indicating flexibility, and the superpositioning of the NT-3 structure over the NGF structure reveals conformational differences in this beta -loop region as well as in beta -loop V (17). In the NH2 terminus a proline and a phenylalanine are involved in the conversion of specificity. In NT-3 Lys-5, which is a long positively charged residue, is replaced by Pro-5, but an insertion of Phe-7 is also necessary to achieve the NGF-like activation (Figs. 2, 4, and 5). When modeling the amino terminus as a peptide, the effect on the backbone is a profound bend determined by the inserted Pro-5, and it is likely that this effect also perturbs the solvent accessible NH2 terminus of the NT-3 mutant (Fig. 6, E and F). Considering that the NH2 terminus and the three beta -loops are highly flexible in the three-dimensional structure (16, 17) it is plausible that the positioning of the NH2 terminus and the beta -loops contributes to the differences in specificity of NGF and NT-3.

Reports regarding the binding areas for neurotrophins on the Trk receptors have revealed two receptor surfaces as potential interaction sites. The IgC2-like domains, especially the second IgC2-like domain closest to the membrane, have been shown to be critical for binding of neurotrophins (44, 45). In addition, the hinge region between the IgC2-like domain and the transmembrane domain has been shown to be well conserved among TrkA for different species but markedly different compared with TrkB or TrkC (35). Deletions in this region abolish TrkA binding, indicating a possible role in neurotrophin specificity (46). Another extracellular TrkA domain, the second leucine repeat motif, has also been identified to be a major binding domain for neurotrophins (47, 48). However, these authors did not find evidence for the IgC2-like domain to be responsible for neurotrophin interaction. Interestingly, the exposed beta -sheet motif (Phe-7 through Arg-9) of the flexible loop of the predicted bioactive conformation of the NGF amino and carboxyl termini (41) has been suggested to be involved in NGF-TrkA interactions.2 Phe-7 has thus been predicted to show a stereochemical fit to a domain of a leucine-rich motif within TrkA. Together with the findings presented here, clearly indicating two independent areas conveying NGF specificity, an intriguing possibility of specificity being determined by two sites on both the receptor and ligand opens up. Moreover, a third site representing binding to p75 must be taken into account in such a model (49-51).

Even though we have defined two sites in purified mutants which are found to confer NGF-like activity to NT-3 we have not seen the reverse effect, i.e. that NT-3 loses its activity due to the mutational alterations. This is not because of the dual independent actions of the two motifs studied here since we have also analyzed a mutant with both motifs replaced (Table I). This remarkable effect has been reported in earlier studies where NGF and BDNF (26) or NT-3 (52) activities were analyzed in chimeric molecules and indicates that the different neurotrophins may each depend on different variable residues for its specificity. This is also compatible with the findings (27) showing that variable beta -loop II can determine NGF specificity in the NT-3 backbone but not in the NT-4 backbone. Also in line with these results are studies on NT-3 where mutants with alanine exchanges of Arg-31 and His-33 were shown to determine the binding site of NT-3 to its nonpreferred receptors TrkA and TrkB. These NT-3 mutants also remained active on the TrkC receptor (53) as is the case with the mutants presented in this study.

This study aimed at providing an answer to the question of what residues determine NGF specificity. It is required to define the areas responsible for specific receptor interaction when modeling how the neurotrophins work and to be able to design agonists or antagonists as potential drugs. The results presented in this work evidently point out that small areas are enough to determine specificity within the neurotrophin family. Similar findings have been gained regarding the growth hormone-receptor complex (54). From the three-dimensional models (Fig. 6) it can be concluded that the areas defined here are altered in a manner accounting for the effects seen in this study. Conclusively, adding to the information regarding specificity of NGF toward its cognate receptor we have determined Gln-96 in cooperation with positioning of beta -loop II through Val-48 and Phe-49 to distinguish NGF from NT-3. Separated from this site but of equal, if not greater, importance are residues Pro-5 and Phe-7 situated in the NH2 terminus.

Comparisons of the studies recently published on the erythropoietin ligand-receptor complex (55, 56) with data currently available for neurotrophin structure/function opens up exciting possibilities synthesizing agonists or antagonists for a protein-protein interaction. A challenging prospect for future studies is to compile available data for rational drug design, an approach already tried in the neurotrophin field (57, 58) but with the aid of phage expression cloning. One problem experienced in these studies was the conformation of beta -loops; and strikingly, in the studies of erythropoietin, a peptide sequence with no resemblance to the original ligand forms a loop region that interacts with the erythropoietin receptor.


FOOTNOTES

*   This work was supported by Swedish Natural Research Council Grant B-AA/BU 04024-317 and Swedish Medical Research Council Grant B95-13R-11098.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Developmental Neuroscience, Box 587, Biomedical Centre, Uppsala University, S-751 23 Uppsala, Sweden. Tel.: 46-18-174-386; Fax: 46-18-559-017; E-mail: Klas{at}bmc.uu.se.
1   The abbreviations used are: NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; NT, neurotrophin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.
2   I. Shamovsky, G. Ross, R. Riopelle, and D. Weaver, personal communication.

ACKNOWLEDGEMENTS

We thank Annika Kylberg and Helena Vretman for excellent technical assistance and Rick Riopelle and Carlos Ibáñez for discussions and critical reading of the manuscript. We are grateful to Kristina Luthman for helping to generate computer models of NGF and NT-3. The Genetics Institute (Boston) provided the pXM expression vector.


REFERENCES

  1. Ebendal, T. (1992) J. Neurosci. Res. 32, 461-470 [Medline] [Order article via Infotrieve]
  2. Chao, M. V. (1994) J. Neurobiol. 25, 1373-1385 [Medline] [Order article via Infotrieve]
  3. Barrett, G. L., and Bartlett, P. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6501-6505 [Abstract]
  4. Carter, B. D., Kaltschmidt, C., Kaltschmidt, B., Offenhauser, N., Bohm Matthaei, R., Baeuerle, P. A., and Barde, Y. A. (1996) Science 272, 542-545 [Abstract]
  5. Ip, N. Y., Stitt, T. N., Tapley, P., Klein, R., Glass, D. J., Fandl, J., Greene, L. A., Barbacid, M., and Yancopoulos, G. D. (1993) Neuron 10, 137-149 [Medline] [Order article via Infotrieve]
  6. Crowley, C., Spencer, S. D., Nishimura, M. C., Chen, K. S., Pitts Meek, S., Armanini, M. P., Ling, L. H., MacMahon, S. B., Shelton, D. L., Levinson, A. D., and Phillips, H. S. (1994) Cell 76, 1001-1011 [Medline] [Order article via Infotrieve]
  7. Ernfors, P., Lee, K. F., Kucera, J., and Jaenisch, R. (1994) Cell 77, 503-512 [Medline] [Order article via Infotrieve]
  8. Klein, R., Silos Santiago, I., Smeyne, R. J., Lira, S. A., Brambilla, R., Bryant, S., Zhang, L., Snider, W. D., and Barbacid, M. (1994) Nature 368, 249-251 [CrossRef][Medline] [Order article via Infotrieve]
  9. Smeyne, R. J., Klein, R., Schnapp, A., Long, L. K., Bryant, S., Lewin, A., Lira, S. A., and Barbacid, M. (1994) Nature 368, 246-249 [CrossRef][Medline] [Order article via Infotrieve]
  10. Tessarollo, L., Vogel, K. S., Palko, M. E., Reid, S. W., and Parada, L. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11844-11848 [Abstract/Free Full Text]
  11. Price, D. L., Struble, R. G., Clark, A. W., Coyle, J. T., and Delon, M. R. (1982) Science 215, 1237-1239 [Medline] [Order article via Infotrieve]
  12. Hefti, F., and Will, B. (1987) J. Neural. Transm. 24, (suppl.) 309-315
  13. Farinas, I., Jones, K. R., Backus, C., Wang, X. Y., and Reichardt, L. F. (1994) Nature 369, 658-661 [CrossRef][Medline] [Order article via Infotrieve]
  14. Arenas, E., and Persson, H. (1994) Nature 367, 368-371 [CrossRef][Medline] [Order article via Infotrieve]
  15. McDonald, N., Lapatto, R., Murray-Rust, J., Gunning, J., Wlodawer, A., and Blundell, T. (1991) Nature 354, 411-414 [CrossRef][Medline] [Order article via Infotrieve]
  16. Holland, D. R., Cousens, L. S., Meng, W., and Matthews, B. W. (1994) J. Mol. Biol. 239, 385-400 [CrossRef][Medline] [Order article via Infotrieve]
  17. Robinson, R. C., Radziejewski, C., Stuart, D. I., and Jones, E. Y. (1995) Biochemistry 34, 4139-4146 [Medline] [Order article via Infotrieve]
  18. Ibáñez, C. F. (1995) Trends Biochem. Sci. 13, 217-227 [CrossRef]
  19. Bradshaw, R. A., Murray Rust, J., Ibáñez, C. F., McDonald, N. Q., Lapatto, R., and Blundell, T. L. (1994) Protein Sci. 3, 1901-1913 [Abstract/Free Full Text]
  20. Burton, L. E., Schmelzer, C. H., Szonyi, E., Yedinak, C., and Gorrell, A. (1992) J. Neurochem. 59, 1937-1945 [Medline] [Order article via Infotrieve]
  21. Kahle, P., Burton, L. E., Schmelzer, C. H., and Hertel, C. (1992) J. Biol. Chem. 267, 22707-22710 [Abstract/Free Full Text]
  22. Shih, A., Laramee, G. R., Schmelzer, C. H., Burton, L. E., and Winslow, J. W. (1994) J. Biol. Chem. 269, 27679-27686 [Abstract/Free Full Text]
  23. Jungbluth, S., Bailey, K., and Barde, Y. A. (1994) Eur. J. Biochem. 221, 677-685 [Abstract]
  24. Drinkwater, C. C., Barker, P. A., Suter, U., and Shooter, E. M. (1993) J. Biol. Chem. 268, 23202-23207 [Abstract/Free Full Text]
  25. Woo, S. B., Timm, D. E., and Neet, K. E. (1995) J. Biol. Chem. 270, 6278-6285 [Abstract/Free Full Text]
  26. Ibáñez, C. F., Ebendal, T., and Persson, H. (1991) EMBO J. 10, 2105-2110 [Abstract]
  27. Ilag, L. L., Lönnerberg, P., Persson, H., and Ibáñez, C. F. (1994) J. Biol. Chem. 269, 19941-19946 [Abstract/Free Full Text]
  28. Kullander, K., and Ebendal, T. (1994) J. Neurosci. Res. 39, 195-210 [Medline] [Order article via Infotrieve]
  29. Söderström, S., Hallböök, F., Ibáñez, C. F., Persson, H., and Ebendal, T. (1990) J. Neurosci. Res. 27, 665-677 [Medline] [Order article via Infotrieve]
  30. Ebendal, T. (1989) in Nerve Growth Factors (Rush, R. A., ed), pp. 81-93, John Wiley & Sons, New York
  31. Herrup, K., and Shooter, E. M. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, 3884-3888 [Abstract]
  32. Alt, F. W., Kellems, R. E., Bertino, J. R., and Schimke, R. T. (1978) J. Biol. Chem. 253, 1357-1370 [Medline] [Order article via Infotrieve]
  33. Ibáñez, C. F., Ilag, L. I., Murray-Rust, J., and Persson, H. (1993) EMBO J. 12, 2281-2293 [Abstract]
  34. Hempstead, B. L., Rabin, S. J., Kaplan, L., Reid, S., Parada, L. F., and Kaplan, D. R. (1992) Neuron 9, 883-896 [Medline] [Order article via Infotrieve]
  35. Bäckström, A., Söderström, S., Kylberg, A., and Ebendal, T. (1996) J. Neurosci. Res. 46, 67-81 [CrossRef][Medline] [Order article via Infotrieve]
  36. Ernfors, P., Ibáñez, C. F., Ebendal, T., Olson, L., and Persson, H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5454-5458 [Abstract]
  37. Hallböök, F., Bäckström, A., Kullander, K., Kylberg, A., Williams, R., and Ebendal, T. (1995) Int. J. Dev. Biol. 39, 855-868 [Medline] [Order article via Infotrieve]
  38. Williams, R., Bäckström, A., Ebendal, T., and Hallböök, F. (1993) Dev. Brain Res. 75, 235-252 [Medline] [Order article via Infotrieve]
  39. Suter, U., Angst, C., Tien, C. L., Drinkwater, C. C., Lindsay, R. M., and Shooter, E. M. (1992) J. Neurosci. 12, 306-318 [Abstract]
  40. Ebendal, T., Olson, L., Seiger, Å., and Belew, M. (1984) in Cellular and Molecular Biology of Neuronal Development (Black, I. B., ed), pp. 231-242, Plenum Press, New York
  41. Shamovsky, I. L., Ross, G. M., Riopelle, R. J., and Weaver, D. F. (1996) J. Am. Chem. Soc. 118, 9743-9749 [CrossRef]
  42. McDonald, N. Q., and Chao, M. V. (1995) J. Biol. Chem. 270, 19669-19672 [Free Full Text]
  43. Ibáñez, C. F., Ilag, L. L., Murray Rust, J., and Persson, H. (1993) EMBO J. 12, 2281-2293 [Abstract]
  44. Perez, P., Coll, P. M., Hempstead, B. L., Martin Zanca, D., and Chao, M. V. (1995) Mol. Cell. Neurosci. 6, 97-105 [CrossRef][Medline] [Order article via Infotrieve]
  45. Urfer, R., Tsoulfas, P., O'Connell, L., Shelton, D. L., Parada, L. F., and Presta, L. G. (1995) EMBO J. 14, 2795-2805 [Abstract]
  46. MacDonald, J. I. S., and Meakin, S. O. (1996) Mol. Cell. Neurosci. 7, 371-390 [CrossRef][Medline] [Order article via Infotrieve]
  47. Windisch, J. M., Marksteiner, R., Lang, M. E., Auer, B., and Schneider, R. (1995) Biochemistry 34, 11256-11263 [Medline] [Order article via Infotrieve]
  48. Windisch, J. M., Auer, B., Marksteiner, R., Lang, M. E., and Schneider, R. (1995) FEBS Lett. 374, 125-129 [CrossRef][Medline] [Order article via Infotrieve]
  49. Yan, H., and Chao, M. V. (1991) J. Biol. Chem. 266, 12099-12104 [Abstract/Free Full Text]
  50. Ibáñez, C. F., Ebendal, T., Barbany, G., Murray-Rust, J., Blundell, T. L., and Persson, H. (1992) Cell 69, 329-341 [Medline] [Order article via Infotrieve]
  51. Ryden, M., Murray Rust, J., Glass, D., Ilag, L. L., Trupp, M., Yancopoulos, G. D., McDonald, N. Q., and Ibáñez, C. F. (1995) EMBO J. 14, 1979-1990 [Abstract]
  52. Urfer, R., Tsoulfas, P., Soppet, D., Escandon, E., Parada, L. F., and Presta, L. G. (1994) EMBO J. 13, 5896-5909 [Abstract]
  53. Ryden, M., and Ibáñez, C. F. (1996) J. Biol. Chem. 271, 5623-5627 [Abstract/Free Full Text]
  54. Wells, J. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1-6 [Abstract/Free Full Text]
  55. Wrighton, N. C., Farrell, F. X., Chang, R., Kashyap, A. K., Barbone, F. P., Mulcahy, L. S., Johnson, D. L., Barrett, R. W., Jolliffe, L. K., and Dower, W. J. (1996) Science 273, 458-463 [Abstract]
  56. Livnah, O., Stura, E. A., Johnson, D. L., Middleton, S. A., Mulcahy, L. S., Wrighton, N. C., Dower, W. J., Jolliffe, L. K., and Wilson, I. A. (1996) Science 273, 464-471 [Abstract]
  57. LeSauteur, L., Wei, L., Gibbs, B. F., and Saragovi, H. U. (1995) J. Biol. Chem. 270, 6564-6569 [Abstract/Free Full Text]
  58. Estenne-Bouhtou, G., Kullander, K., Karlsson, M., Ebendal, T., Hacksell, U., and Luthman, K. (1996) Intl. J. Pept. Protein Res. 48, 337-346 [Medline] [Order article via Infotrieve]

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