The Low Affinity Neurotrophin Receptor , p 75 LNTR , Is Palmitoylated by Thioester Formation through Cysteine 279 *

The low  affinity  neurotrophin  receptor,  termed p75-, plays a role in increasing the amount of nerve growth factor that becomes bound to the tyrosine kinase receptor, trkA (Barker, P. A, and Shooter, E. M. (1994) Neuron 13: 203-215), possibly by increasing the nerve growth factor concentration in the microenvironment surrounding the trkA receptor. Because protein acylation may be a means by which ell surface receptor distribution may be regulated, we have determined the acylation status of ~ 7 5 ~ . We find that ~ 7 5 ~ ~ expressed in PC12, PCNA, or transfected COS cells is metabolically labeled with [3H]palmitic acid. This modification occurs post-translationally, and the incorporated fatty acid is removed by hydroxylamine treatment at pH 7 or 11 and by sulfhydryl reducing agents, suggesting a thioester linkage to palmitic acid. Consistent with this, ~ 7 5 ~ ~ in which the juxtamembrane cysteine present at position 279 is substituted with alanine is expressed but cannot be metabolically labeled with [3Hlpalmitic acid. Substitution of other cysteine residues present in the transmembrane or intracellular domain of the receptor has no effect on protein acylation, suggesting that only Cys2” is esterified to palmitate.

The low affinity neurotrophin receptor, termed p75-, plays a role in increasing the amount of nerve growth factor that becomes bound to the tyrosine kinase receptor, trkA (Barker, P. A, and Shooter, E. M. (1994) Neuron 13: 203-215), possibly by increasing the nerve growth factor concentration in the microenvironment surrounding the trkA receptor. Because protein acylation may be a means by which cell surface receptor distribution may be regulated, we have determined the acylation status of ~7 5~. We find that ~7 5~~ expressed in PC12, PCNA, or transfected COS cells is metabolically labeled with [3H]palmitic acid. This modification occurs post-translationally, and the incorporated fatty acid is removed by hydroxylamine treatment at pH 7 or 11 and by sulfhydryl reducing agents, suggesting a thioester linkage to palmitic acid. Consistent with this, ~7 5~~ in which the juxtamembrane cysteine present at position 279 is substituted with alanine is expressed but cannot be metabolically labeled with [3Hlpalmitic acid. Substitution of other cysteine residues present in the transmembrane or intracellular domain of the receptor has no effect on protein acylation, suggesting that only Cys2" is esterified to palmitate.
The neurotrophins are a family of proteins believed to play crucial roles in the maintenance and development of the nervous system. To date, four members of this family have been identified: nerve growth factor (NGF),' brain-derived neurotrophic factor (BDNF) (Barde et al., 1982;Leibrock et al., 1989), neurotrophin-3 (NT-3) (Ernfors et al., 1990;Hohn, 1990;Jones and Reichardt, 1990;Maisonpierre et al., 1990;Rosenthal et al., 19901, and neurotrophin-4/5 (NT-4/5) (Berkemeier et al., 1991;Hallbook et al., 1991;Ip et al., 1992), all of which share about 50% amino acid homology. The neurotrophins bind to two forms of cell surface receptors. The trk family is composed of three related receptor tyrosine kinases that recognize the neurotrophins with a relatively high degree of binding specificity: trkA preferentially binds NGF, trkB prefers BDNF and NT-4/5, and The abbreviations used are: NGF, nerve growth factor; BDNF, brainderived neurotrophic factor; NT, neurotrophin; LNTR, low affinity neu-DMEB, DMEM supplemented with bovine serum albumin; PAGE, poly-rotrophin receptor; DMEM, Dulbecco's modified Eagle's medium; acrylamide gel electrophoresis. trkC prefers NT-3 (for review see Barbacid, 1993). The importance of each of these receptors in neuronal development has been demonstrated recently by the production of mice in which the receptors have been rendered null by homologous recombination (Klein et al., 1993(Klein et al., , 1994Smeyne et al., 1994).
The other class of neurotrophin receptor contains the low affinity neurotrophin receptor p75 (~7 5~~1 , which binds each of the neurotrophins with approximately equal avidity. ~7 5 belongs to a superfamily of related receptors that includes CD27, CD30, CD40, 4-1BB, 0x40, the fas antigen, and the tumor necrosis factor receptors TNFRl and TNFR2 (for review see Bazan, 1993). Each of these proteins has two to four domains within the extracellular region which contain 6 cysteines that form disulfide bridges critical for the maintenance of the domain structure. Some receptors of this group may mediate signal transduction events directly in response to ligand binding by a pathway involving activation of a sphingomyelinase and generation of ceramide (Dressler et al., 1992;Dbaibo et al., 19931, whereas others may play an indirect role, concentrating ligand at the cell surface to enhance binding to a signal transducing receptor (Tartaglia et al., 1993). The functional role of the p7EiLNTR has remained elusive. Several studies have indicated that, together with trkA, p7EiLNTR contributes to the formation of high affinity NGF binding sites believed to be required for NGF action on neurons (Hempstead et al., 1989(Hempstead et al., , 1991Battleman et al., 1993). However, other work suggests that activation of the trkA receptor alone is sufficient to mediate survival and neuritic outgrowth responses to NGF by PC12 cells and neurons (Weskamp and Reichardt, 1991;Ibanez et al., 1992;Rovelli et al., 1993). Mice in which the p75LNTR gene has been rendered null by homologous recombination show apparently normal central nervous system development but display a progressive loss of sensory and sympathetic nerve endings from peripheral targets (Lee et al., 1992). Interestingly, when assayed for neurotrophin-mediated survival in uitro, BDNF-, NT-3-. and NT-4/5-responsive neurons isolated from the p75LNTR null mice behave identically to those isolated from wild type litter mates, but NGF-responsive neurons show a shift in their dose response, with the p7!jLNTR null mice showing decreased responsiveness at low NGF concentrations (Davies et al., 1993;Lee et al., 1994). Recent studies indicate that these disparate findings might be reconciled if p75 acts to increase the amount of NGF which ultimately becomes bound to the trkA receptor. Mahadeo et al. (1994) have shown that p75LNTR increases the rate of association into the high affinity component, and recent findings from Verdi et al. (1994) indicate that p75LNTR acts to enhance the responsiveness of MAH cells expressing trkA. We have recently provided direct biochemical data which indicate that the presence of p75iLNTR acts to enhance binding of NGF to trkA in a dose-dependent manner, being most relevant a t low NGF concentrations (Barker and Shooter, 1994). Based on these and previous data, we have suggested that ~7 5~ may act to concentrate NGF locally in the microenvironment surrounding the trk receptor and thus enhance the ability of trkA 30645 to bind and respond to NGF. Such an action would likely depend on some cell surface colocalization of the two receptors either by direct association or by concentration within a cell surface subdomain.
Many cellular proteins have been shown to be modified posttranslationally by the covalent attachment of the 14-or 16carbon saturated acyl chains, myristic acid, and palmitic acid (for review see McIlhinney (1990)). Myristoylation normally occurs by cotranslational formation of an amide bond with an amino-terminal glycine (Wilcox et al., 19871, whereas palmitoylation is a post-translational modification (Bonatti et al., 1989) generally occurring by thioester formation with one or more intracellular cysteine residues (Sefton and Buss, 1989). Unlike myristoylation, no consensus sequence for palmitoylation has yet been identified, and the cellular machinery responsible for this modification remains poorly characterized. Palmitic acid that becomes covalently linked to protein is turned over rapidly (Alverez et al., 1990;Paige et al., 19931, and alterations of the steady-state level of palmitoylation could therefore act as a regulatory mechanism of protein function. Protein palmitoylation may play several important roles within the cell, including mediating protein-membrane attachment, involvement in protein-protein interactions, and determination of cellular localization (O'Dowd et al., 1989;Skene and Virag, 1989;Shenoy-Scaria et al., 1993). Because palmitoylation has been suggested to play roles in protein localization and protein-protein contact, we have tested the possibility that p75LNTR is acyl-modified. Our results show that the ~7 5~~~ is palmitoylated post-translationally, that this modification occurs via formation of a thioester bond, and that the thioester is formed with a single cysteine residue, CYS~'~, which is conserved in rat, chick, and human forms of the receptor.
EXPERIMENTAL PROCEDURES Materials-9,10-[3HlPalmitic acid (30-60 Cilmmol), 9,10-[3Hlmyristic acid (30-60 CVmmol), and EnHance were purchased from DuPont NEN. [35SlCysteine and 35S-dATP and Enhanced Chemiluminescence reagent were from Amersham Corp. Sequenase 2.0 was from U. S. Biochemical Corp. Nitrocellulose was from Schleicher & Schueil. Bovine calf serum and equine serum were from HyClone Laboratories. 2.5 S NGF was from Bioproducts for Science. MC192, a monoclonal antibody directed against the extracellular domain of the rat p75 receptor, was produced in ascites fluid in BALB/c mice and purified using Immunopure columns (Pierce) as directed by the manufacturer. The polyclonal antiserum directed against p75LNTR, designated ap75-pl, was raised against a peptide containing amino acids 393408 of the rat ~7 5 "~~. Anti-mouse IgG, agarose, and anti-rabbit agarose were obtained from Sigma. Horseradish peroxidase-conjugated anti-rabbit antibodies were obtained from Jackson Laboratories.
Construction of ~7 5~" Expression Constructs with Intracellular Mutations and Their Expression in COS7 Cells-For expression studies, the rat ~7 5~~~ open reading frame was cloned into the cytomegalovirus promoter-based expression vector pCMX (Davis et al., 1991) to produce plasmid pPB162. Mutant p75-isoforms in which each of 4 cysteine residues located in the transmembrane or intracellular domain were individually changed to alanine were constructed by polymerase chain reaction-based overlap extension mutagenesis using specific synthetic oligonucleotides (PCR-Mate, Applied Biosystems) essentially as described previously (Barker et al., 1994). The fidelity of the mutated regions was confirmed by dideoxynucleotide sequencing with Sequenase as per the manufacturer's instructions, and cDNA containing the intended mutation was cloned into the appropriate restriction sites of Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate). The lysates were scraped into prechilled microcentrifuge tubes, vortexed, and centrifuged for 10 min to remove insoluble material. Cleared lysates were incubated with 5 pg of MC192 for 2 h, then supplemented with 45 pl of agarose-conjugated goat anti-rabbit IgG (Sigma) and incubated for an additional 2 h. Beads were pelleted and washed in lysis buffer three times and then suspended in 100 pl of Laemmli sample buffer lacking reducing agents and boiled for 5 min. For experiments in which PC12 cells were treated with NGF, cells were first metabolically labeled for 4 h and then exposed to NGF at either 5 or 200 ng/ml for 1,5, or 20 min. In separate experiments, cells were pretreated with 5 ng/ml NGF in DMEB for 3 or 7 days and then metabolically labeled with [3H]palmitic acid in the presence of NGF for 4 h. Immunoblotting and Fluorography-For immunoblots, 2 0 4 aliquots of the immunoprecipitates described above were supplemented with dithiothreitol to a final concentration of 50 mM, boiled 5 min, and then separated on 10% Laemmli acrylamide gels and transferred to nitrocellulose. Blots were rinsed in TBST (10 m M Tris (pH 7.4), 150 m M NaCl, 0.2% Tween 20), blocked in Blotto (10 m M Tris (pH 7.4), 150 m M NaCl, 5% dry skim milk powder) containing 0.2% Tween 20 for 1 h and then incubated with ap75-pl at a dilution of 1:1,000 for 90 min. Blots were washed three times in TBST and then incubated in Blotto containing a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit antibody for 45 min. Following four washes in TBST, reactive bands were detected using Enhanced Chemiluminescence according to the manufacturer's instructions. For fluorography, 2 0 4 aliquots were separated on 10% Laemmli acrylamide gels, and the gels were fixed with EnHance for 30 min, dried, and exposed to XAR x-ray film for 7-21 days. For hydroxylamine treatment, identical aliquots of labeled, immunoprecipitated material were separated on a 10% Laemmli acrylamide gel, and the gel was cut with a razor to separate individual lanes. Gel slices were partially fxed in 10% methanol, 10% acetic acid, rinsed in water, and then soaked in either 1 M hydroxylamine (pH 7.0), 1 M hydroxylamine (pH ll.O), 1 M Tris (pH 8.0), or 0.2 M NaOH overnight.  Gels were then prepared for fluorography, dried, and exposed to x-ray film as described above.

RESULTS
To determine if p75LNTR is acyl-modified, PC12 cells were metabolically labeled with [3H]palmitic acid, lysed with detergent-containing buffer, and ~7 5~~~ was immunoprecipitated using MC192, a monoclonal antibody directed against the receptor's extracellular domain. Fig. 1 shows that MC192 immunoprecipitates a labeled protein from PC12 cells which comigrates with a 66-kDa molecular mass standard. This molecular mass value for nonreduced p7EiLNTR is consistent with previous reports. Similar analyses performed on PCNA cells, a fibroblast cell line stably transfected with the rat p7FiLNTR gene (Radeke et al., 1986) also showed specific labeling of a 66-kDa protein (Fig. 1). To confirm that the labeled 66-kDa protein was in fact p75LNTR, COS7 cells transiently transfected with either a plasmid driving expression of rat p751ANTR or with control plasmid were metabolically labeled with [3H]palmitic acid and then analyzed by immunoprecipitation followed by fluorography.  Fig. 1 shows that the 66-kDa protein detected by fluorography was immunoprecipitated by MC192 only from cells transfected with the p7EiLNTR expression plasmid, indicating that the MC192-immunoreactive 66-kDa protein that incorporates [3Hlpalmitic acid is indeed ~7 5~~~~.
Biochemical analyses were performed to determine the nature of the acylation linkage. Palmitoylation occurs most commonly via a thioester bond with cysteine and less often by formation of a hydroxyester with serine (Kaufman et al., 1984). The thioester bond is susceptible to cleavage with hydroxyla-mine treatment at neutral or basic pH or with reducing agents such as dithiothreitol and P-mercaptoethanol. In contrast, a hydroxyester formed through serine is labile only to hydroxylamine treatment at basic pH. To examine the linkage to p7EiLNTR, PCNA cells were metabolically labeled with [3H]palmitic acid, and ~7 5 "~~ was immunoprecipitated using MC192. Aliquots were then run in separate lanes of SDS-PAGE gels which were subsequently separated and exposed to hydroxylamine at either basic or neutral pH. Fig. 2A shows that treatment of samples with hydroxylamine leads to a substantial reduction in the amount of incorporated [3Hlpalmitic acid detected by fluorography regardless of the pH, indicating that the linkage of the acyl moiety to the protein was unlikely to occur via a hydroxyester. To test directly the possibility that the fatty acid linkage was due to a thioester, labeled p75LNTR derived from PCNA cells was separated into aliquots that were heated either in the absence of reducing agents or with dithiothreitol or P-mercaptoethanol. Fluorography of SDS-PAGE gels containing these samples shows that treatment of labeled p75LNTR with either of these reducing agents leads to virtually a complete loss of the incorporated radioactivity ( Fig. 2 B ) , indicating that its covalent attachment to the protein is mediated by formation of a thioester with cysteine. Fig. 3A shows the positions of the 4 cysteine residues located within the transmembrane and intracellular domain of the rat ~7 5~~~. To identify the specific residue(s) required for this acyl modification, the transmembrane and intracellular cysteines were individually mutated to alanine. Each of the receptor isoforms containing these mutations was then expressed in COS7 cells and subjected to metabolic labeling with [3H]palmitic acid. Mutation of Cys379 or Cys4"j to alanine did not affect labeling of the ~7 5~~~ (Fig. 3B), and replacing transmembrane residue 257 with cysteine similarly did not affect the ability of the receptor to become labeled with [3Hlpalmitic acid. However, replacing CYS"~ with alanine completely abolished the incorporation of [3H]palmitic acid into ~7 5~~. Together with the finding that the attachment of an acyl moiety to p75LNTR occurs via a thioester bond, these results indicate that the palmitic acid moiety present on ~7 5~~ is attached to the protein by esterification of the intracellular cysteine at position 279.
Several studies have indicated that myristoylation is mainly a cotranslational event, whereas palmitoylation occurs mainly post-translationally. To determine if the incorporation of fatty acid into ~7 5~"~ is a post-translational process, COS7 cells were transfected with the p7EiLNTR expression plasmid and metabolically labeled with either [3H]palmitic acid or [35Slcysteine in the presence or absence of cycloheximide. Fig. 4 shows that whereas cycloheximide treatment almost completely blocked the incorporation of [35S]cysteine into p7EiLNTR, it had no effect on [3Hlpalmitic acid incorporation. This indicates that the incorporation of [3Hlpalmitic acid into p7!jLNTR is a posttranslational, rather than a cotranslational, modification.
Although some conversion of [3H]palmitic acid to [3Hlmyristic acid might occur by P-oxidation during the course of these experiments, the fact that the addition of fatty acid is both post-translational and labile to sulfhydryl reducing agents indicates that the fatty acid incorporated into the protein is palmitic rather than myristic acid. To test this directly, COS7 cells were transfected with expression plasmids for either wild-type p7EiLNTR or for p75LNTR containing the C279A mutation and metabolically labeled with equivalent molar concentrations and specific activities of [3H]palmitic acid or [3Hlmyristic acid. Fig. 5 shows that metabolic labeling with [3Hlmyristic acid results in low levels of acyl incorporation to p75LNTR relative to that observed with [3H]palmitic acid and that incorporation [3H]myristic acid can be removed from p75LNTR by sulfnydryl reducing agents, indicating attachment is via a thioester through Cys279. Incorporation of myristic acid via a thioester is atypical; myristoylation normally occurs via cotranslational amide formation with an amino-terminal glycine. Thus, the low level of myristic acid incorporation into p7!jLNTR observed in these experiments likely reflects that the palmitoyltransferase responsible for the post-translational addition of fatty acid into p75LNTR is relatively nondiscriminating with respect to acyl chain length, consistent with previous observations (Schmidt et al., 1979;Wedegaertner et al., 1993). DISCUSSION This study establishes that p7EiLNTR becomes modified posttranslationally by the addition of palmitic acid. This covalent modification of the receptor does not depend on a specific cell context, as it can be observed in PC12 cells, in stably transfected mouse fibroblasts, in humanA875 cells (data not shown), or in COS7 cells transiently transfected with p7EiLNTR expression constructs. Treatment of the labeled protein with hydroxylamine under neutral or basic pH or with p-mercaptoethanol or dithiothreitol results in loss of the acyl group from the receptor, suggesting that its incorporation is dependent upon formation of a thioester. Site-directed mutagenesis of transmembrane or intracellular cysteine residues indicates that CYS'~~, located 6 amino acids within the intracellular domain, is the sole palmitoylation site within the receptor. Substitution of alanine for cysteine at this position reduces labeling of the receptor to undetectable levels. The conservation of Cys279 among rat, human, and chick forms of the receptor (Radeke et al., 1987;Johnson et al., 1986;Large et al., 1989;Heuer et al., 1990) suggests that this palmitoylation event may be shared among species. Some conversion of labeled fatty acids may occur during the course of metabolic labeling, and the possibility that the incorporated acyl group is myristic rather than palmitic acid must be considered. Under physiological conditions, myristoylation is a co-translational event that occurs via amide formation through an amino-terminal glycine (Resh, 1994). In contrast, we have shown that the addition of the acyl moiety to p7FiLNTR occurs post-translationally by formation of a thioester, typical of palmitoylation. Therefore, although p7EiLNTR does show a low amount of labeling with [3H]myristic acid, this most likely occurs via thioester formation through CYS~'~, indicating either that the palmitoyltransferase is relatively nondiscriminating with respect to acyl chain length, as noted previously by Wedegaertner and Bourne (19941,  Palmitoylation has been suggested to play diverse functional roles within the cell. Palmitoylation of the neuronal growth cone protein GAP-43 reduces its ability to catalyze nucleotide exchange on a G protein (Sudo et at., 1992) and may play a key role in directing targeting of this protein to the growth cone (Liu et al., 1993). Several G protein-coupled receptors appear to be palmitoylated and in the case of the P-adrenergic receptor, palmitoylation appears to play a role in coupling to G,, (O'Dowd et al., 1989). The a subunits of G proteins are palmitoylated, and several recent reports indicate that their palmitoylation is involved both in membrane attachment as well as in modulating signaling capability (Linder et al., 1993;Grassie et al., 1993;Wedegaertner et al., 1993). This may prove to be important physiologically since the level of palmitoylation of some of these proteins is regulated by ligand binding to the cognate receptor (Degtyarev et al., 1993;Mumby et al., 1994;Wedegaertner and Bourne, 1994). Exposure of PC12 cells to NGF at either 5 or 200 ng/ml does not appear to alter the level of palmitoylation of p75LmR, however (data not shown). The src family of kinases is modified by the addition of acyl moieties (Resh, 1994). An interesting example of the importance of palmitoylation of these molecules has recently been demonstrated for signaling via the glycosyl phosphatidylinositol-linked protein CD59. CD59 normally transduces an activating signal to the src-like kinases p56Ick and p59' Yn, both of which are palmitoylated and myristoylated. Interestingly, when these proteins are specifically mutated to lack palmitoylation but retain myristoylation, they no longer become activated following CD59 stimulation (Shenoy-Scaria et al., 1993). "he mechanism(s) by which palmitoylation contributes to these diverse processes remains unclear. The acyl chain might play a role in protein-protein interactions, either by providing a crucial structural constraint or by directly contributing to a binding interface. An attractive alternative possibility is that palmitoylation may play a role in directing the cellular localization of various proteins, either by directing vectorial transport or by directing proteins to particular membrane subdomains. This latter notion has been supported by recent studies which indicate that palmitoylation of ~5 9 '~" directs the protein to caveoli (Shenoy-Scaria et al., 1994).
Biophysical analysis of the potential association between trkA and p75 have shown that the lateral mobility of pELNTR is decreased when it is coexpressed with trkA (Ross et al., 1993). It will be very interesting to determine the contribution of p75LNTR palmitoylation to this effect, especially given previous data showing that the lateral diffusion rate of a mutated nonpalmitoylated form of vesicular stomatitus virus G protein is %fold higher than its wild-type palmitoylated counterpart (Scullion et al., 1987). Using a fibroblast cotransformation assay, Hantzopoulos et al. (1994) have recently demonstrated a functional collaboration between a mutated form of p75 and the trk receptors; interestingly, the juxtamembrane region of p75 receptor which contains the palmitoylation site identified here appears to a play critical role in regulating this functional collaboration. Further studies will determine if palmitoylation plays a specific role in mediating associations between ~7 5 and trk.