Proc. Natl. Acad. Sci. USA Vol. 88, pp. 159-163, January 1991 Neurobiology
Nerve growth factor binding domain of the nerve growth factor receptor (neurotrophic factors/mutagenesis/cross-linking)
ANDREW A. WELCHER*, CATHERINE M. BITLER, MONTE J. RADEKEt, AND ERIC M. SHOOTER Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305
Communicated by Robert L. Baldwin, September 25, 1990
A structural analysis of the rat low-affinity ABSTRACT nerve growth factor (NGF) receptor was undertaken to define the NGF binding domain. Mutant NGF receptor DNA constructs were expressed in mouse fibroblasts or COS cells, and the ability of the mutant receptors to bind NGF was assayed. In the first mutant, all but 16 amino acid residues of the intracellular domain of the receptor were removed. This receptor bound NGF with a Kd comparable to that of the wild-type receptor. A second mutant contained only the four cysteine-rich sequences from the extraceflular portion of the protein. This mutant was expressed in COS cells and the resultant protein was a secreted soluble form of the receptor that was able to bind NGF. Two N-terinal deletions, in which either the first cysteine-rich sequence or the first and part of the second cysteine-rich sequences were removed, bound NGF. However, a mutant lacking all four cysteine-rich sequences was unable to bind NGF. These results show that the four cysteinerich sequences of the NGF receptor contain the NGF binding domain.
The role of nerve growth factor (NGF) in the development and survival of peripheral sensory and sympathetic neurons has been well characterized (1). Studies have also suggested a role for NGF in the development and maintenance of cholinergic neurons in the mammalian forebrain (2). The first step in the mechanism of action of NGF is the interaction of NGF with its specific receptor [the NGF receptor (NGFR)] on responsive cells or cell lines. There are two types or states of the NGFR as determined by binding studies (3). On sensory and sympathetic neurons, these receptors are described as high-affinity (Kd = 10 pM) and low-affinity (Kd = 1 nM) NGFRs, respectively. Although the NGFRs in PC12 cells differ less in their Kd values, they share many features of the receptors on primary neurons, including a slower rate of dissociation of NGF from the high- compared to the low-affinity receptor (4). The biological response of NGF in neuronal cells correlates with occupancy of the high-afflinity receptor. Indirect evidence suggests that the high-affinity NGFR comprises the low-affinity receptor complexed with an additional cytoplasmic or membraneassociated protein or proteins (5, 6). The low-affinity receptor has been cloned in rat (7), chicken (8), and human (9), and the three receptors share considerable homology. Recent studies have shown that Schwann cells in rat sciatic nerve express high levels of the low-affinity NGFR after nerve crush or transection (10, 11). This expression is repressed once contact is reestablished between the Schwann cells and the regenerating axons. These low-affinity receptors may bind the NGF that is synthesized by nonneuronal cells distal to a peripheral nerve injury and create a localized
source of NGF on the surface of the Schwann cells over which the advancing neuronal growth cone migrates (12). In addition, a truncated form of the low-affinity receptor is secreted into the extracellular medium of cultured Schwann cells, PC12 cells, and superior cervical ganglia neurons, as well as the plasma and urine of normal rats (13). It is clear that a structural analysis of the low-affinity receptor is critical to understanding the binding and subsequent mode of action of NGF. In this report we have utilized a transient expression system to produce and analyze mutant NGFRs. The structural analysis of these mutants has delineated regions of the receptor critical for NGF binding.
MATERIALS AND METHODS Construction of Mutants. NGFRt256 was constructed by linearizing the vector pcNGFR' (7) with the restriction enzyme Nar I, inserting the universal translation terminator (Pharmacia), and recircularizing the vector with T4 DNA ligase (Fig. 1). Ligation to the terminator added the peptide Gly-Leu-Ile-Asn to the C terminus of the protein. The mutant NGFRt168 was constructed as follows. The vector pNGFR.1 was digested with the restriction enzyme BamHI, and the 720-base-pair insert fragment was purified (14). The vector pBJ5, same as vector pJFE14 (15), was digested with the restriction enzyme BamHI and ligated to the receptor fragment using T4 DNA ligase. An in-frame stop codon was generated by ligation to the vector, resulting in the addition of the dipeptide Gln-Thr to the protein. The antisense NGFRt168 construct contained the same sequence but was cloned into pBJ5 in the opposite orientation. Mutants NHD1, NHD2, and NHD3 were made using exonuclease III. The vector pNGFR.1 was linearized with EcoRI, and deletions were made using the Erase-A-Base kit (Promega Biotec). The deletion-containing sequences were digested with HindIII and subcloned into a modified version of pNGFR.1 from which all but the first 221 base pairs of the NGFR cDNA had been removed by digestion with Stu I and HindIII. The first 221 base pairs were left intact to maintain the wild-type signal peptide. Clones were sequenced using the dideoxynucleotide sequencing method (16). The in-frame mutants were digested with the restriction enzyme HincII and subcloned into the EcoRI site of pBJ5. The enzyme HincII was used to remove a portion of the 3' untranslated DNA that was unstable in eukaryotic vectors grown in several strains of bacteria (data not shown). All enzymes were from BRL. DNA Transfection, Metabolic Labeling, and Immunoprecipitation. Mouse LTK- cells were grown, transfected, and sorted by a fluorescence-activated cell sorter as described (7). COS cells (17) were grown and transfected using the Abbreviations: NGF, nerve growth factor; NGFR, NGF receptor. *To whom reprint requests should be addressed. tPresent address: Neuroscience Research Institute, University of California, Santa Barbara, CA 93106.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Proc. Natl. Acad. Sci. USA 88 (1991)
C-Terminal Deletions NGFRt256
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DEAE-dextran procedure (18) on 100-mm tissue culture plates. Cells were metabolically labeled by growing the cells in medium (7) lacking cysteine (GIBCO) but supplemented with [35S]cysteine (Amersham; 50 ACi/ml; 1 Ci = 37 GBq) for 4-24 hr. The medium was removed, and normal cysteinecontaining medium was added for the remainder of the experiment. The receptor was immunoprecipitated as follows. For the secreted receptor, COS cells were harvested 72 hr after transfection. The antibody MC192 (19) was added to the conditioned medium supernatant (final concentration, 2.5 ,ug/ml) and incubated overnight at 40C. Tachisorb (Calbiochem; 100 ,.L) was added, and the incubation continued for another 1 hr. The Staphylococcus aurelus bacteria were pelleted by centrifugation in a microcentrifuge at 9550 x g for 3 min, and bound antibody-antigen complexes were eluted from the bacteria by incubation in 100 jul of protein sample buffer (14) at 34WC for 30 min. To precipitate cellular proteins, the transfected cells were removed from the plate using phosphate-buffered saline containing 1 mM EDTA (PBS/ EDTA) and lysed in 10 mM Tris-HCl, pH 8.0/150 mM NaCl/10 mM KCl/1 mM EDTA/1% Nonidet P-40/1% sodium deoxycholate/0.1% SDS (RIPA). The lysed cells were then immunoprecipitated as described for the conditioned medium supernatant. Protein samples were separated by SDS/PAGE; the gels were fixed, enhanced, and autoradiographed as described (14). Immunoblot Analysis. After the proteins were transferred to nitrocellulose, the nitrocellulose was incubated with MC192 (hybridoma-conditioned medium) or the purified antibody (final concentration, 200 ng/ml) raised against a peptide sequence in the intracellular domain of the the receptor [anti-peptide antibody (20)]. The blots were incubated with horseradish peroxidase-conjugated anti-IgG (Cappel Laboratories) for 1 hr at room temperature, and bands were visualized using diaminobenzidine (Sigma). NGF Binding Analysis and Cross-Linking. NGF iodination and Scatchard analysis were done as described (4). The construct NGFRt168 was analyzed as follows. The MC192 antibody was coupled to Affi-Gel HZ (Bio-Rad) according to the manufacturer's instructions. Conditioned medium supernatant from transfected COS cells was incubated with the MC192-coupled beads at 40C overnight. The beads were pelleted by centrifugation at 600 x g for 5 min and washed
twice in NGF binding buffer (4). The beads were then used in the standard binding assay (4). For cross-linking, the COS cells were removed from the plate 72 hr after transfection by using PBS/EDTA, pelleted by centrifugation at 3000 rpm as above for 3 min at 40C, and then resuspended in NGF binding buffer. The cells were incubated with 1 nM 1251I-labeled NGF for 1 hr at 0°C. Bound NGF was cross-linked to the receptors by incubating the cells with 2 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Sigma) for 30 min at room temperature. Unbound NGF was removed by washing the cells three times with NGF binding buffer. The cells were then lysed in protein sample buffer and analyzed by SDS/PAGE.
RESULTS The Extracellular Portion of the Receptor Contains the NGF Binding Domain. Previous experiments showed that PC12 cells treated with trypsin were unable to bind NGF (21), indicating that the extracellular region of the NGFR was needed for NGF binding. To test whether the intracellular region is important for NGF binding, for example, by stabilizing the extracellular domain, the first receptor mutation was devised to remove the intracellular region. The mutant pNGFRt256 (Fig. 1) retained only 16 amino acid residues of the intracellular domain. This construct was transfected into mouse LTK- cells. To confirm expression of the truncated receptor, the transfected cells were metabolically labeled with [35S]cysteine, lysed in Nonidet P 40, and immunoprecipitated with the MC192 antibody, which is specific for the rat NGFR (19). The precipitated proteins were separated by SDS/PAGE, and [35S]cysteine-labeled proteins were visualized by fluorography. Cells that were transfected with the full-length receptor cDNA (wt-NGFR) expressed a receptor protein with an apparent molecular weight of 81,000, whereas cells transfected with the truncated cDNA clone expressed a receptor with an apparent molecular weight of 67,000 (data not shown). Scatchard analysis of the binding of NGF to the truncated receptor is shown in Fig. 2. Not only did NGF bind to the truncated form of the receptor (-45,000 NGFRs per cell) but also it had exactly the same binding constant (=1 nM) as the full-length receptor. Therefore, it seems that all or at least most of the essential components for binding of NGF to the NGFR are contained within the first 256 amino acid
Neurobiology: Welcher et aL
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residues ofthe protein. Similar results have been reported for the human NGFR (22, 23). The Secreted Form of the Receptor Can Bind NGF. A secreted C-terminally truncated form of the NGFR has been shown to bind NGF (24). However, this receptor contained an additional 51 amino acid residues beyond the end of the cysteine-rich sequences. To test if the region containing the four cysteine-rich sequences in the extracellular portion of the receptor alone was able to bind NGF, a truncated secreted form of the receptor was constructed. This construct (NGFRtl68) encodes the first 168 amino acid residues of the receptor (Fig. 1). The receptor constructs were transfected into COS cells, the cells were metabolically labeled, and the cells and the medium ofthe transfected cells were assayed 72 hr after transfection. Proteins from the lysed cells or proteins secreted into the conditioned medium supernatant were immunoprecipitated with the MC192 antibody and analyzed by SDS/PAGE, followed by fluorography. The expression of the cellular and secreted forms of the receptor is shown in Fig. 3. Lanes 2 and 5 show the NGFR present in the cells and conditioned medium supernatant of COS cells transfected with the NGFR construct. The cells contain the normal membrane form with a molecular weight of 80,000, whereas the conditioned medium supernatant contains the nonmembrane form of the receptor with an apparent molecular weight of 58,000. Naturally occurring soluble forms of the wild-type receptor are present in the medium of other cells grown in tissue culture and may represent cell-bound forms of the receptor that are released from the membrane by the action of proteinases (13). Lanes 3 and 6 show the NGFRt168 receptor associated with the cells and conditioned medium supernatant, respectively, of COS cells transfected with the NGFRt168 construct. The secreted form of this receptor, found in the conditioned medium supernatant, has an apparent molecular weight of 48,000. Based on migration through a denaturing gel, it appears that this secreted truncated mutant receptor contains fewer amino acids than the naturally occurring soluble form of the receptor. Longer chase times led to more of the NGFRt168 being secreted from the cells into the medium (data not shown). No receptor protein was precipitated from the cells of conditioned medium supernatant of cells transfected with an antisense construct (lanes 4 and 7). Secretion of these truncated receptors prevented the measurement of NGF binding by conventional means. To overcome this, MC192 antibodies were coupled to Sepharose beads and tested for their ability to bind the truncated
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FIG. 3. Expression of NGFRt168. Transfected COS cells were metabolically labeled, and the proteins from cells or conditioned medium supernatants were immunoprecipitated with the MC192 antibody. The labeled proteins were subjected to SDS/PAGE and fluorographed. Lanes 2-4 contain proteins from transfected cells. Lanes 5-7 contain proteins from the conditioned medium supernatants of transfected cells. COS cells were transfected with the following constructs: wt-NGFR (lanes 2 and 5), NGFRt168 (lanes 3 and 6), or antisense NGFRt168 (lanes 4 and 7). Molecular weight markers are in lane 1. The bands at the top of the gel represent material trapped at the stacking gel interface. Molecular weights (x10-3) are shown to the left.
secreted forms of the receptor. Experiments utilizing the metabolically labeled proteins, from cells transfected with the various constructs, indicated that the receptor proteins were the only proteins that bound to the MC192-beads (data not shown). Therefore, the MC192-beads were used for the binding assay. Conditioned medium supernatants from cells transfected with the various NGFR constructs were incubated with the MC192-Sepharose beads and binding of 125ilabeled NGF to the receptor-bead complexes was measured. Nonspecific binding was determined by including a 500-fold excess of unlabeled NGF in parallel binding reactions. Fig. 4 10-
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shows that the secreted form of the receptor is able to bind NGF. Medium, conditioned from cells transfected with the secreted truncated mutant receptor or the non-membranebound form of the wild-type receptor, contained NGF binding activity, as judged by the difference between total (hatched bars) and nonspecific (solid bars) binding. The difference in binding between the secreted and wild-type receptor probably is due to a difference in the amounts of each receptor in the conditioned medium. No binding was detected in medium conditioned from cells transfected with the pBJ5 vector alone. These experiments show that the region containing the four cysteine-rich sequences are capable of binding NGF. NGF Binding Sites Within Four Cysteine-Rich Sequences. To determine whether there are subdomains within this region that are responsible for NGF binding, a series of deletions were made in the NGFR cDNA sequence. The sizes of three of these mutated NGFR sequences is shown in Fig. 1. The deletions were constructed such that the signal peptide sequence remained intact for proper processing to occur. Each of the deletions start at amino acid 8 and removed from 27 to 160 amino acid residues. These three particular mutants were chosen for further study because they resulted in the deletion of various portions of the cysteine-rich sequences. The three mutant NGFR sequences were subcloned into the transient expression vector pBJ5 and transfected into COS cells. Initial experiments were aimed at determining if the mutant receptors were expressed. The proteins in lysates from transfected COS cells were separated by SDS/PAGE, transferred to nitrocellulose, and incubated with the antipeptide antibody. Fig. SA shows that the wt-NGFR (lane 1), NHD1 (lane 2), NHD2 (lane 3), and NHD3 (lane 4) bound the anti-peptide antibody (20). The deletion mutants migrated faster than the wild-type receptor and the difference is consistent with the number of amino acid residues removed from each mutant receptor. No proteins bound the antipeptide antibody from COS cells transfected with the pBJ5 vector only (lane 5). Based on the immunoblot staining, the deletion mutants were all expressed at comparable levels in the COS cells. When a similar nitrocellulose filter was probed with the MC192 antibody, only the wild-type receptor protein was able to bind the antibody (data not shown). Next, the mutants were tested for their ability to bind NGF. Transfected COS cells were cross-linked with 1251_ labeled NGF, the cellular proteins were separated by SDS/ PAGE, and proteins that bound '25I-labeled NGF were visualized by autoradiography. The results of a typical crosslinking are shown in Fig. SB. Cells transfected with the wild-type construct expressed the receptor and NGF bound to this receptor, resulting in a combined molecular weight of 100,000 (Fig. SB, lane 2). NGF bound to the receptor mutant NHD1 (Fig. SB, lane 3) and to receptor mutant NHD2 (Fig. SB, lane 4) but not to NHD3 (Fig. 5B, lane 5). The extent of cross-linking of 1251-labeled NGF to mutants NHD1 and NHD2 were much less than to the wild-type receptor. COS cells transfected only with pBJ vector failed to produce any proteins that bound NGF (Fig. SB, lane 6). Lanes 1, 2, and 3 also contain a higher molecular weight band at -200,000. This is an oligomer of the NGFR (25) and is seen in all cross-linking experiments. The band appears to migrate faster in lanes 2 and 3, suggesting that the smaller mutant receptors can also form the oligomer.
DISCUSSION In this report, we have shown that removal of most of the intracellular portion of the low-affinity NGFR has no effect on NGF binding to the extracellular domain of the receptor. Furthermore, within the extracellular domain, the four cysteine-rich sequences when expressed alone are capable of
Proc. Natl. Acad. Sci. USA 88 (1991)
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FIG. 5. N-terminal deletion mutants of NGFR are expressed and bind NGF. (A) Immunoblot of lysates from transfected COS cells. Equivalent amounts of proteins were subjected to SDS/PAGE, electroblotted to nitrocellulose, and incubated with anti-peptide antibody. COS cells were transfected with the following constructs: wt-NGFR (lane 1), NHD1 (lane 2), NHD2 (lane 3), NHD3 (lane 4), or antisense NGFR (lane 5). Lane 6 contains molecular weight markers (x10-3). (B) Cross-linking of 125I-labeled NGF to wt-NGFR and the N-terminal deletion mutants of NGFR. 125I-labeled NGF was bound and cross-linked to transfected COS cells using 1-ethyl-3-(3dimethylaminopropyl)carbodiimide. Cells were lysed and proteins were subjected to SDS/PAGE. The gel was dried and autoradiographed. COS cells were transfected with the following constructs: wt-NGFR (lane 2), NHD1 (lane 3), NHD2 (lane 4), NHD3 (lane 5), or pBJ5 vector only (lane 6). Molecular weight markers are in lane 1 and positions are indicated (x10-3) in A and B. The bands at the bottom of the gel represent uncross-linked 125I-labeled NGF.
binding NGF. Attempts to define the NGF binding domains further by mutational analysis within the four cysteine-rich sequences are probably not possible with the present approach. The data indicate that the third and fourth cysteinerich sequences plus part of the second cysteine-rich sequence are required for NGF binding. If the reduced cross-linking of 1251-labeled NGF to the mutants lacking the first cysteine-rich
Neurobiology: Welcher et al. sequence and the first and part of the second cysteine-rich sequences, respectively, accurately reflects their lowered affinities for NGF, then these sequences also contribute to NGF binding. However, the reduced cross-linking could also result from a decreased efficiency of cross-linking to the mutant receptors or to improper folding of the receptor lacking their full complement of disulfide bridges in the cysteine-rich sequences. The measurement of the actual Kd values of the mutant receptors to resolve these issues is probably not possible in the present system, particularly if they are significantly higher than the Kd of the wild-type receptor. Sehgal et al. (26) also showed that deletion of -40 amino acid residues from the N terminus of the human NGFR reduced NGF binding compared to the wild-type receptor. Given the extensive homology in the cysteine-rich sequences of the receptor among rat, human, and chicken (8), it is likely that all three receptors have similar NGF binding sites. The structural motif characterized by repeats of homologous cysteine-rich sequences in the extracellular domain is found in a number of receptors that bind proteins or large peptides. Of particular interest with respect to the NGFR is the family of receptor molecules that share significant homology in three or four cysteine-rich sequences. These include CD40 (27), OX40 (28), and the tumor necrosis factor receptor (29). These homologies suggest a common evolutionary precursor based on the repeating cysteine residues around which residues appropriate to the binding of basic (NGF) or more acidic (tumor necrosis factor) ligands have evolved (29). The tertiary structure generated by this general motif in the NGFR is even more intriguing because it has now been shown that this receptor binds not only NGF but also the homologous neurotrophic factor, brain-derived neurotrophic factor (30). How one receptor can mediate the different neuronal specificities of two neurotrophic factors remains to be determined. A comparison of the antibody binding capacities of some of the mutant receptors helps define the epitope for the MC192 rat NGFR antibody. Since MC192 binds to NGFRt168, which contains only the four cysteine-rich sequences, this region must contain the MC192 epitope. On the other hand, the receptor with the smallest N-terminal deletion (NHD1) fails to bind the MC192 antibody. Therefore, either this deleted region, amino acid residues 8-35, contains the MC192 epitope or the tertiary structure of the region is important for MC192 binding to another site. The fact that NHD1 still binds NGF suggests that its tertiary structure is not grossly changed and that this particular amino acid sequence may well contain the MC192 epitope. It follows that the NGF binding site and the MC192 epitope are in reasonable proximity, and this fits well with the observation (20) that MC192 enhances the ability of the low-affinity NGFR to bind NGF. This work was supported by grants from the National Institute of Neurological Disorders and Stroke (NS04270), the American Cancer Society (BE-47J), the Isabelle M. Niemella Trust, and a gift from Mrs. Anita Hecht. A.W. was supported by a National Research Service Award fellowship (NS08443). C.B. was supported by a Bank of America Giannini foundation grant and a National Research Service Award fellowship (NS08232).
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Nerve growth factor binding domain of the nerve growth factor receptor (neurotrophic factors/mutagenesis/cross-linking)
ANDREW A. WELCHER*, CATHERINE M. BITLER, MONTE J. RADEKEt, AND ERIC M. SHOOTER Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305
Communicated by Robert L. Baldwin, September 25, 1990
A structural analysis of the rat low-affinity ABSTRACT nerve growth factor (NGF) receptor was undertaken to define the NGF binding domain. Mutant NGF receptor DNA constructs were expressed in mouse fibroblasts or COS cells, and the ability of the mutant receptors to bind NGF was assayed. In the first mutant, all but 16 amino acid residues of the intracellular domain of the receptor were removed. This receptor bound NGF with a Kd comparable to that of the wild-type receptor. A second mutant contained only the four cysteine-rich sequences from the extraceflular portion of the protein. This mutant was expressed in COS cells and the resultant protein was a secreted soluble form of the receptor that was able to bind NGF. Two N-terinal deletions, in which either the first cysteine-rich sequence or the first and part of the second cysteine-rich sequences were removed, bound NGF. However, a mutant lacking all four cysteine-rich sequences was unable to bind NGF. These results show that the four cysteinerich sequences of the NGF receptor contain the NGF binding domain.
The role of nerve growth factor (NGF) in the development and survival of peripheral sensory and sympathetic neurons has been well characterized (1). Studies have also suggested a role for NGF in the development and maintenance of cholinergic neurons in the mammalian forebrain (2). The first step in the mechanism of action of NGF is the interaction of NGF with its specific receptor [the NGF receptor (NGFR)] on responsive cells or cell lines. There are two types or states of the NGFR as determined by binding studies (3). On sensory and sympathetic neurons, these receptors are described as high-affinity (Kd = 10 pM) and low-affinity (Kd = 1 nM) NGFRs, respectively. Although the NGFRs in PC12 cells differ less in their Kd values, they share many features of the receptors on primary neurons, including a slower rate of dissociation of NGF from the high- compared to the low-affinity receptor (4). The biological response of NGF in neuronal cells correlates with occupancy of the high-afflinity receptor. Indirect evidence suggests that the high-affinity NGFR comprises the low-affinity receptor complexed with an additional cytoplasmic or membraneassociated protein or proteins (5, 6). The low-affinity receptor has been cloned in rat (7), chicken (8), and human (9), and the three receptors share considerable homology. Recent studies have shown that Schwann cells in rat sciatic nerve express high levels of the low-affinity NGFR after nerve crush or transection (10, 11). This expression is repressed once contact is reestablished between the Schwann cells and the regenerating axons. These low-affinity receptors may bind the NGF that is synthesized by nonneuronal cells distal to a peripheral nerve injury and create a localized
source of NGF on the surface of the Schwann cells over which the advancing neuronal growth cone migrates (12). In addition, a truncated form of the low-affinity receptor is secreted into the extracellular medium of cultured Schwann cells, PC12 cells, and superior cervical ganglia neurons, as well as the plasma and urine of normal rats (13). It is clear that a structural analysis of the low-affinity receptor is critical to understanding the binding and subsequent mode of action of NGF. In this report we have utilized a transient expression system to produce and analyze mutant NGFRs. The structural analysis of these mutants has delineated regions of the receptor critical for NGF binding.
MATERIALS AND METHODS Construction of Mutants. NGFRt256 was constructed by linearizing the vector pcNGFR' (7) with the restriction enzyme Nar I, inserting the universal translation terminator (Pharmacia), and recircularizing the vector with T4 DNA ligase (Fig. 1). Ligation to the terminator added the peptide Gly-Leu-Ile-Asn to the C terminus of the protein. The mutant NGFRt168 was constructed as follows. The vector pNGFR.1 was digested with the restriction enzyme BamHI, and the 720-base-pair insert fragment was purified (14). The vector pBJ5, same as vector pJFE14 (15), was digested with the restriction enzyme BamHI and ligated to the receptor fragment using T4 DNA ligase. An in-frame stop codon was generated by ligation to the vector, resulting in the addition of the dipeptide Gln-Thr to the protein. The antisense NGFRt168 construct contained the same sequence but was cloned into pBJ5 in the opposite orientation. Mutants NHD1, NHD2, and NHD3 were made using exonuclease III. The vector pNGFR.1 was linearized with EcoRI, and deletions were made using the Erase-A-Base kit (Promega Biotec). The deletion-containing sequences were digested with HindIII and subcloned into a modified version of pNGFR.1 from which all but the first 221 base pairs of the NGFR cDNA had been removed by digestion with Stu I and HindIII. The first 221 base pairs were left intact to maintain the wild-type signal peptide. Clones were sequenced using the dideoxynucleotide sequencing method (16). The in-frame mutants were digested with the restriction enzyme HincII and subcloned into the EcoRI site of pBJ5. The enzyme HincII was used to remove a portion of the 3' untranslated DNA that was unstable in eukaryotic vectors grown in several strains of bacteria (data not shown). All enzymes were from BRL. DNA Transfection, Metabolic Labeling, and Immunoprecipitation. Mouse LTK- cells were grown, transfected, and sorted by a fluorescence-activated cell sorter as described (7). COS cells (17) were grown and transfected using the Abbreviations: NGF, nerve growth factor; NGFR, NGF receptor. *To whom reprint requests should be addressed. tPresent address: Neuroscience Research Institute, University of California, Santa Barbara, CA 93106.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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C-Terminal Deletions NGFRt256
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FIG. 1. Structure of the NGFR variants.
DEAE-dextran procedure (18) on 100-mm tissue culture plates. Cells were metabolically labeled by growing the cells in medium (7) lacking cysteine (GIBCO) but supplemented with [35S]cysteine (Amersham; 50 ACi/ml; 1 Ci = 37 GBq) for 4-24 hr. The medium was removed, and normal cysteinecontaining medium was added for the remainder of the experiment. The receptor was immunoprecipitated as follows. For the secreted receptor, COS cells were harvested 72 hr after transfection. The antibody MC192 (19) was added to the conditioned medium supernatant (final concentration, 2.5 ,ug/ml) and incubated overnight at 40C. Tachisorb (Calbiochem; 100 ,.L) was added, and the incubation continued for another 1 hr. The Staphylococcus aurelus bacteria were pelleted by centrifugation in a microcentrifuge at 9550 x g for 3 min, and bound antibody-antigen complexes were eluted from the bacteria by incubation in 100 jul of protein sample buffer (14) at 34WC for 30 min. To precipitate cellular proteins, the transfected cells were removed from the plate using phosphate-buffered saline containing 1 mM EDTA (PBS/ EDTA) and lysed in 10 mM Tris-HCl, pH 8.0/150 mM NaCl/10 mM KCl/1 mM EDTA/1% Nonidet P-40/1% sodium deoxycholate/0.1% SDS (RIPA). The lysed cells were then immunoprecipitated as described for the conditioned medium supernatant. Protein samples were separated by SDS/PAGE; the gels were fixed, enhanced, and autoradiographed as described (14). Immunoblot Analysis. After the proteins were transferred to nitrocellulose, the nitrocellulose was incubated with MC192 (hybridoma-conditioned medium) or the purified antibody (final concentration, 200 ng/ml) raised against a peptide sequence in the intracellular domain of the the receptor [anti-peptide antibody (20)]. The blots were incubated with horseradish peroxidase-conjugated anti-IgG (Cappel Laboratories) for 1 hr at room temperature, and bands were visualized using diaminobenzidine (Sigma). NGF Binding Analysis and Cross-Linking. NGF iodination and Scatchard analysis were done as described (4). The construct NGFRt168 was analyzed as follows. The MC192 antibody was coupled to Affi-Gel HZ (Bio-Rad) according to the manufacturer's instructions. Conditioned medium supernatant from transfected COS cells was incubated with the MC192-coupled beads at 40C overnight. The beads were pelleted by centrifugation at 600 x g for 5 min and washed
twice in NGF binding buffer (4). The beads were then used in the standard binding assay (4). For cross-linking, the COS cells were removed from the plate 72 hr after transfection by using PBS/EDTA, pelleted by centrifugation at 3000 rpm as above for 3 min at 40C, and then resuspended in NGF binding buffer. The cells were incubated with 1 nM 1251I-labeled NGF for 1 hr at 0°C. Bound NGF was cross-linked to the receptors by incubating the cells with 2 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (Sigma) for 30 min at room temperature. Unbound NGF was removed by washing the cells three times with NGF binding buffer. The cells were then lysed in protein sample buffer and analyzed by SDS/PAGE.
RESULTS The Extracellular Portion of the Receptor Contains the NGF Binding Domain. Previous experiments showed that PC12 cells treated with trypsin were unable to bind NGF (21), indicating that the extracellular region of the NGFR was needed for NGF binding. To test whether the intracellular region is important for NGF binding, for example, by stabilizing the extracellular domain, the first receptor mutation was devised to remove the intracellular region. The mutant pNGFRt256 (Fig. 1) retained only 16 amino acid residues of the intracellular domain. This construct was transfected into mouse LTK- cells. To confirm expression of the truncated receptor, the transfected cells were metabolically labeled with [35S]cysteine, lysed in Nonidet P 40, and immunoprecipitated with the MC192 antibody, which is specific for the rat NGFR (19). The precipitated proteins were separated by SDS/PAGE, and [35S]cysteine-labeled proteins were visualized by fluorography. Cells that were transfected with the full-length receptor cDNA (wt-NGFR) expressed a receptor protein with an apparent molecular weight of 81,000, whereas cells transfected with the truncated cDNA clone expressed a receptor with an apparent molecular weight of 67,000 (data not shown). Scatchard analysis of the binding of NGF to the truncated receptor is shown in Fig. 2. Not only did NGF bind to the truncated form of the receptor (-45,000 NGFRs per cell) but also it had exactly the same binding constant (=1 nM) as the full-length receptor. Therefore, it seems that all or at least most of the essential components for binding of NGF to the NGFR are contained within the first 256 amino acid
Neurobiology: Welcher et aL
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residues ofthe protein. Similar results have been reported for the human NGFR (22, 23). The Secreted Form of the Receptor Can Bind NGF. A secreted C-terminally truncated form of the NGFR has been shown to bind NGF (24). However, this receptor contained an additional 51 amino acid residues beyond the end of the cysteine-rich sequences. To test if the region containing the four cysteine-rich sequences in the extracellular portion of the receptor alone was able to bind NGF, a truncated secreted form of the receptor was constructed. This construct (NGFRtl68) encodes the first 168 amino acid residues of the receptor (Fig. 1). The receptor constructs were transfected into COS cells, the cells were metabolically labeled, and the cells and the medium ofthe transfected cells were assayed 72 hr after transfection. Proteins from the lysed cells or proteins secreted into the conditioned medium supernatant were immunoprecipitated with the MC192 antibody and analyzed by SDS/PAGE, followed by fluorography. The expression of the cellular and secreted forms of the receptor is shown in Fig. 3. Lanes 2 and 5 show the NGFR present in the cells and conditioned medium supernatant of COS cells transfected with the NGFR construct. The cells contain the normal membrane form with a molecular weight of 80,000, whereas the conditioned medium supernatant contains the nonmembrane form of the receptor with an apparent molecular weight of 58,000. Naturally occurring soluble forms of the wild-type receptor are present in the medium of other cells grown in tissue culture and may represent cell-bound forms of the receptor that are released from the membrane by the action of proteinases (13). Lanes 3 and 6 show the NGFRt168 receptor associated with the cells and conditioned medium supernatant, respectively, of COS cells transfected with the NGFRt168 construct. The secreted form of this receptor, found in the conditioned medium supernatant, has an apparent molecular weight of 48,000. Based on migration through a denaturing gel, it appears that this secreted truncated mutant receptor contains fewer amino acids than the naturally occurring soluble form of the receptor. Longer chase times led to more of the NGFRt168 being secreted from the cells into the medium (data not shown). No receptor protein was precipitated from the cells of conditioned medium supernatant of cells transfected with an antisense construct (lanes 4 and 7). Secretion of these truncated receptors prevented the measurement of NGF binding by conventional means. To overcome this, MC192 antibodies were coupled to Sepharose beads and tested for their ability to bind the truncated
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FIG. 3. Expression of NGFRt168. Transfected COS cells were metabolically labeled, and the proteins from cells or conditioned medium supernatants were immunoprecipitated with the MC192 antibody. The labeled proteins were subjected to SDS/PAGE and fluorographed. Lanes 2-4 contain proteins from transfected cells. Lanes 5-7 contain proteins from the conditioned medium supernatants of transfected cells. COS cells were transfected with the following constructs: wt-NGFR (lanes 2 and 5), NGFRt168 (lanes 3 and 6), or antisense NGFRt168 (lanes 4 and 7). Molecular weight markers are in lane 1. The bands at the top of the gel represent material trapped at the stacking gel interface. Molecular weights (x10-3) are shown to the left.
secreted forms of the receptor. Experiments utilizing the metabolically labeled proteins, from cells transfected with the various constructs, indicated that the receptor proteins were the only proteins that bound to the MC192-beads (data not shown). Therefore, the MC192-beads were used for the binding assay. Conditioned medium supernatants from cells transfected with the various NGFR constructs were incubated with the MC192-Sepharose beads and binding of 125ilabeled NGF to the receptor-bead complexes was measured. Nonspecific binding was determined by including a 500-fold excess of unlabeled NGF in parallel binding reactions. Fig. 4 10-
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shows that the secreted form of the receptor is able to bind NGF. Medium, conditioned from cells transfected with the secreted truncated mutant receptor or the non-membranebound form of the wild-type receptor, contained NGF binding activity, as judged by the difference between total (hatched bars) and nonspecific (solid bars) binding. The difference in binding between the secreted and wild-type receptor probably is due to a difference in the amounts of each receptor in the conditioned medium. No binding was detected in medium conditioned from cells transfected with the pBJ5 vector alone. These experiments show that the region containing the four cysteine-rich sequences are capable of binding NGF. NGF Binding Sites Within Four Cysteine-Rich Sequences. To determine whether there are subdomains within this region that are responsible for NGF binding, a series of deletions were made in the NGFR cDNA sequence. The sizes of three of these mutated NGFR sequences is shown in Fig. 1. The deletions were constructed such that the signal peptide sequence remained intact for proper processing to occur. Each of the deletions start at amino acid 8 and removed from 27 to 160 amino acid residues. These three particular mutants were chosen for further study because they resulted in the deletion of various portions of the cysteine-rich sequences. The three mutant NGFR sequences were subcloned into the transient expression vector pBJ5 and transfected into COS cells. Initial experiments were aimed at determining if the mutant receptors were expressed. The proteins in lysates from transfected COS cells were separated by SDS/PAGE, transferred to nitrocellulose, and incubated with the antipeptide antibody. Fig. SA shows that the wt-NGFR (lane 1), NHD1 (lane 2), NHD2 (lane 3), and NHD3 (lane 4) bound the anti-peptide antibody (20). The deletion mutants migrated faster than the wild-type receptor and the difference is consistent with the number of amino acid residues removed from each mutant receptor. No proteins bound the antipeptide antibody from COS cells transfected with the pBJ5 vector only (lane 5). Based on the immunoblot staining, the deletion mutants were all expressed at comparable levels in the COS cells. When a similar nitrocellulose filter was probed with the MC192 antibody, only the wild-type receptor protein was able to bind the antibody (data not shown). Next, the mutants were tested for their ability to bind NGF. Transfected COS cells were cross-linked with 1251_ labeled NGF, the cellular proteins were separated by SDS/ PAGE, and proteins that bound '25I-labeled NGF were visualized by autoradiography. The results of a typical crosslinking are shown in Fig. SB. Cells transfected with the wild-type construct expressed the receptor and NGF bound to this receptor, resulting in a combined molecular weight of 100,000 (Fig. SB, lane 2). NGF bound to the receptor mutant NHD1 (Fig. SB, lane 3) and to receptor mutant NHD2 (Fig. SB, lane 4) but not to NHD3 (Fig. 5B, lane 5). The extent of cross-linking of 1251-labeled NGF to mutants NHD1 and NHD2 were much less than to the wild-type receptor. COS cells transfected only with pBJ vector failed to produce any proteins that bound NGF (Fig. SB, lane 6). Lanes 1, 2, and 3 also contain a higher molecular weight band at -200,000. This is an oligomer of the NGFR (25) and is seen in all cross-linking experiments. The band appears to migrate faster in lanes 2 and 3, suggesting that the smaller mutant receptors can also form the oligomer.
DISCUSSION In this report, we have shown that removal of most of the intracellular portion of the low-affinity NGFR has no effect on NGF binding to the extracellular domain of the receptor. Furthermore, within the extracellular domain, the four cysteine-rich sequences when expressed alone are capable of
Proc. Natl. Acad. Sci. USA 88 (1991)
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FIG. 5. N-terminal deletion mutants of NGFR are expressed and bind NGF. (A) Immunoblot of lysates from transfected COS cells. Equivalent amounts of proteins were subjected to SDS/PAGE, electroblotted to nitrocellulose, and incubated with anti-peptide antibody. COS cells were transfected with the following constructs: wt-NGFR (lane 1), NHD1 (lane 2), NHD2 (lane 3), NHD3 (lane 4), or antisense NGFR (lane 5). Lane 6 contains molecular weight markers (x10-3). (B) Cross-linking of 125I-labeled NGF to wt-NGFR and the N-terminal deletion mutants of NGFR. 125I-labeled NGF was bound and cross-linked to transfected COS cells using 1-ethyl-3-(3dimethylaminopropyl)carbodiimide. Cells were lysed and proteins were subjected to SDS/PAGE. The gel was dried and autoradiographed. COS cells were transfected with the following constructs: wt-NGFR (lane 2), NHD1 (lane 3), NHD2 (lane 4), NHD3 (lane 5), or pBJ5 vector only (lane 6). Molecular weight markers are in lane 1 and positions are indicated (x10-3) in A and B. The bands at the bottom of the gel represent uncross-linked 125I-labeled NGF.
binding NGF. Attempts to define the NGF binding domains further by mutational analysis within the four cysteine-rich sequences are probably not possible with the present approach. The data indicate that the third and fourth cysteinerich sequences plus part of the second cysteine-rich sequence are required for NGF binding. If the reduced cross-linking of 1251-labeled NGF to the mutants lacking the first cysteine-rich
Neurobiology: Welcher et al. sequence and the first and part of the second cysteine-rich sequences, respectively, accurately reflects their lowered affinities for NGF, then these sequences also contribute to NGF binding. However, the reduced cross-linking could also result from a decreased efficiency of cross-linking to the mutant receptors or to improper folding of the receptor lacking their full complement of disulfide bridges in the cysteine-rich sequences. The measurement of the actual Kd values of the mutant receptors to resolve these issues is probably not possible in the present system, particularly if they are significantly higher than the Kd of the wild-type receptor. Sehgal et al. (26) also showed that deletion of -40 amino acid residues from the N terminus of the human NGFR reduced NGF binding compared to the wild-type receptor. Given the extensive homology in the cysteine-rich sequences of the receptor among rat, human, and chicken (8), it is likely that all three receptors have similar NGF binding sites. The structural motif characterized by repeats of homologous cysteine-rich sequences in the extracellular domain is found in a number of receptors that bind proteins or large peptides. Of particular interest with respect to the NGFR is the family of receptor molecules that share significant homology in three or four cysteine-rich sequences. These include CD40 (27), OX40 (28), and the tumor necrosis factor receptor (29). These homologies suggest a common evolutionary precursor based on the repeating cysteine residues around which residues appropriate to the binding of basic (NGF) or more acidic (tumor necrosis factor) ligands have evolved (29). The tertiary structure generated by this general motif in the NGFR is even more intriguing because it has now been shown that this receptor binds not only NGF but also the homologous neurotrophic factor, brain-derived neurotrophic factor (30). How one receptor can mediate the different neuronal specificities of two neurotrophic factors remains to be determined. A comparison of the antibody binding capacities of some of the mutant receptors helps define the epitope for the MC192 rat NGFR antibody. Since MC192 binds to NGFRt168, which contains only the four cysteine-rich sequences, this region must contain the MC192 epitope. On the other hand, the receptor with the smallest N-terminal deletion (NHD1) fails to bind the MC192 antibody. Therefore, either this deleted region, amino acid residues 8-35, contains the MC192 epitope or the tertiary structure of the region is important for MC192 binding to another site. The fact that NHD1 still binds NGF suggests that its tertiary structure is not grossly changed and that this particular amino acid sequence may well contain the MC192 epitope. It follows that the NGF binding site and the MC192 epitope are in reasonable proximity, and this fits well with the observation (20) that MC192 enhances the ability of the low-affinity NGFR to bind NGF. This work was supported by grants from the National Institute of Neurological Disorders and Stroke (NS04270), the American Cancer Society (BE-47J), the Isabelle M. Niemella Trust, and a gift from Mrs. Anita Hecht. A.W. was supported by a National Research Service Award fellowship (NS08443). C.B. was supported by a Bank of America Giannini foundation grant and a National Research Service Award fellowship (NS08232).
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