A biosynthetic pathway for anandamide
Jie Liu*†, Lei Wang*, Judith Harvey-White*, Douglas Osei-Hyiaman*, Raj Razdan‡, Qian Gong§, Andrew C. Chan§,
Zhifeng Zhou¶, Bill X. Huang?, Hee-Yong Kim?, and George Kunos*†
Laboratories of *Physiologic Studies,¶Neurogenetics, and?Molecular Signaling, National Institute on Alcohol Abuse and Alcoholism, National Institutes of
Health, Bethesda, MD 20892;‡Organix, Inc., Woburn, MA 01801; and§Genentech, Inc., South San Francisco, CA 94080
Edited by Tomas Ho ¨kfelt, Karolinska Institutet, Stockholm, Sweden, and approved July 18, 2006 (received for review March 6, 2006)
The endocannabinoid arachidonoyl ethanolamine (anandamide) is
a lipid transmitter synthesized and released ‘‘on demand’’ by
neurons in the brain. Anandamide is also generated by macro-
phages where its endotoxin (LPS)-induced synthesis has been
implicated in the hypotension of septic shock and advanced liver
cirrhosis. Anandamide can be generated from its membrane
through cleavage by a phospholipase D (NAPE–PLD). Here we
document a biosynthetic pathway for anandamide in mouse brain
and RAW264.7 macrophages that involves the phospholipase C
(PLC)-catalyzed cleavage of NAPE to generate a lipid, phospho-
anandamide, which is subsequently dephosphorylated by phos-
phatases, including PTPN22, previously described as a protein
tyrosine phosphatase. Bacterial endotoxin (LPS)-induced synthesis
of anandamide in macrophages is mediated exclusively by the
PLC?phosphatase pathway, which is up-regulated by LPS, whereas
NAPE–PLD is down-regulated by LPS and functions as a salvage
pathway of anandamide synthesis when the PLC?phosphatase
pathway is compromised. Both PTPN22 and endocannabinoids
have been implicated in autoimmune diseases, suggesting that the
PLC?phosphatase pathway of anandamide synthesis may be a
biosynthesis ? phosphatase ? phospholipase C ? phosphoanandamide
‘‘on demand’’ by neurons in the brain (1). AEA is also generated
by macrophages (2), where its bacterial endotoxin (LPS)-
induced synthesis has been implicated in the hypotension of
septic shock (3, 4) and liver cirrhosis (5, 6). Macrophage-derived
AEA has been also implicated in antiinflammatory effects both
in the periphery (7) and in the central nervous system (8, 9).
AEA is thought to be generated from its membrane precursor,
N-arachidonoyl phosphatidylethanolamine (NAPE), through
cleavage by a phospholipase D (NAPE–PLD) (10, 11), up-
regulation of which can result in increased tissue levels of AEA
(12). We earlier reported that LPS potently stimulates AEA
synthesis in RAW264.7 mouse macrophages, in which it in-
creases both the generation of NAPE from [14C]diarachidonoyl
phosphatidylcholine and the conversion of NAPE to AEA (4).
Because these effects could be prevented by inhibitors of RNA
transcription or protein synthesis (4), we hypothesized that LPS
induces the expression of proteins involved in the biosynthesis of
AEA, and a subtraction cloning strategy using resting and
LPS-treated macrophages may help identifying such proteins.
Although a specific N-acyltransferase (NAT) involved in the
generation of NAPE has not yet been discovered, a NAPE-
specific PLD has been identified and its ability to generate AEA
from NAPE has been established (11). The results presented
here indicate that, unexpectedly, NAPE–PLD is not involved in
the stimulated synthesis of AEA in RAW264.7 macrophages.
Instead, we identified the lipid phosphoanandamide (pAEA),
which is also present in brain, and is generated from NAPE by
phospholipase C (PLC). We also identified PTPN22, previously
described as a protein tyrosine phosphatase (13, 14), as one of
he endocannabinoid N-arachidonoyl ethanolamine (anan-
damide, AEA) is a lipid transmitter synthesized and released
the enzymes responsible for the generation of AEA from its
NAPE–PLD Is Not Involved in LPS-Stimulated AEA Synthesis. To
confirm the feasibility of the subtraction cloning strategy men-
tioned above, we first tested whether NAPE–PLD expression in
RAW264.7 cells is induced by LPS. Surprisingly, the dramatic
increase in cellular AEA levels induced by LPS was associated by
a marked decrease rather than increase in NAPE–PLD gene
expression (Fig. 1a), suggesting that the increased conversion of
NAPE to AEA may involve an alternative pathway. This was
further indicated by the finding that siRNA knockdown of
NAPE–PLD expression did not influence the basal level of AEA
or its increase by LPS treatment, the latter being even greater
than in mock-transfected controls (Fig. 1b).
Identification of Proteins Involved in LPS-Induced AEA Synthesis. To
find proteins involved in the biosynthesis of AEA, we generated
differentially expressed cDNAs in LPS-stimulated versus control
RAW264.7 cells, using the PCR-Select method of cDNA sub-
traction. The differentially expressed cDNAs were labeled with
[?-32P]dCTP and used as probes to screen a commercial cDNA
library prepared from LPS-treated RAW264.7 cells. The DNA
targets were sequenced and the identities of the corresponding
genes were established by Blast search of the GenBank database.
To find out which of these genes might be involved in the
regulation of AEA synthesis, full-length cDNAs were transiently
transfected into RAW264.7 cells, and the cellular levels of
AEA were measured by liquid chromatography (LC)?MS.
Transfection of 21 of the ?150 LPS-induced genes resulted in a
significant (?50%) increase in the cellular level of AEA, and
RAW264.7 cell lines stably transfected with these genes have
LPS Induces the Expression of the Protein Tyrosine Phosphatase
PTPN22 in RAW264.7 Cells. One of the genes, whose stable over-
expression resulted in a 2-fold increase in AEA levels in
RAW264.7 cells, encodes PTPN22 (Lyp, PEP, PTPN8), a
nonreceptor protein tyrosine phosphatase predominantly ex-
pressed in lymphoid tissues (13, 14). Exposure of wild-type
RAW264.7 cells to 10 ng?ml LPS for 90 min resulted in a 3.2 ?
0.5-fold increase in the expression of PTPN22, as verified by
real-time PCR (n ? 3). The possibility suggested by these
findings that AEA may arise by dephosphorylation of a precur-
sor, generated from NAPE by a PLC, had been earlier contem-
plated (10, 14), but not proved because pAEA, once produced
in the body, is quickly modified by phosphatases to AEA (15).
Conflict of interest statement: No conflicts declared.
Freely available online through the PNAS open access option.
Abbreviations: AEA, anandamide; pAEA, phosphoanandamide; NAPE, N-arachidonoyl
phosphatidylethanolamine; PLD, phospholipase D; SRM, selected reaction monitoring; LC,
†To whom correspondence may be addressed. E-mail: email@example.com or gkunos@
© 2006 by The National Academy of Sciences of the USA
September 5, 2006 ?
vol. 103 ?
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Identification of pAEA in RAW264.7 Cells and in Mouse Brain. To test
whether pAEA may be formed in vivo, we incubated homoge-
nates of RAW264.7 cells with [14C]NAPE in the absence or
presence of the nonselective tyrosine phosphatase inhibitor
sodium orthovanadate (NaVO3, 1 mM), and analyzed the prod-
ucts by thin layer chromatography. A band comigrating with
synthetic pAEA could be observed in the presence, but not in the
absence, of NaVO3, in extracts of both control RAW264.7 cells
and RAW264.7 cells stably transfected with PTPN22
(RAW264.7-Lyp cells, not shown). To definitively identify
pAEA, extracts of both RAW264.7 cells and mouse brain
incubated either with or without NaVO3 were analyzed by
high-performance liquid chromatography?in line electrospray
ionization tandem mass spectrometry (HPLC?ESI-MS?MS),
using [2H4]AEA as internal standard. PAEA could be clearly
identified along with AEA in both RAW264.7 cells and mouse
brain extracts by selected reaction monitoring (SRM) using the
transition of molecular ion at m?z 428 to m?z 330, the major
fragment formed by dephosphorylation under collision-induced
dissociation condition (Fig. 2b). In brain tissue incubated with
NaVO3, the concentrations of pAEA increased up to 15-fold
compared with controls (Fig. 2c).
Interaction of PAEA with CB1 Receptors. The ability of pAEA to
directly interact with CB1 receptors was tested in radioligand
binding displacement assays using 0.5 nM [3H]CP-55,940 as the
labeled ligand. PAEA bound to mouse brain CB1receptors with
an apparent Kdof 46.7 nM compared with a Kdof 13.2 nM for
AEA, which is comparable to earlier findings (15). However, in
the presence of NaVO3, the affinity of pAEA was reduced
?10-fold to 432.1 nM, whereas the affinity of AEA remained
unchanged (16.3 nM), indicating that pAEA itself is not func-
tional at CB1receptors and its apparent potency is due to its
conversion to AEA in the assay mixture.
In Vivo Conversion of PAEA to AEA by Phosphatases. To determine
whether pAEA is enzymatically converted to AEA in vivo,
synthetic pAEA was incubated with extracts of control
RAW264.7 cells or RAW264.7-Lyp cells in the presence of the
fatty acid amidohydrolase inhibitor URB597 (16) to prevent
by LC?MS. The formation of AEA was time and protein
concentration-dependent (Fig. 3a), and it was about twice
greater in RAW264.7-Lyp than in control RAW264.7 cells (Fig.
3b). Furthermore, preincubation of both cell types with 10 ng?ml
LPS for 90 min significantly increased the conversion of pAEA
to AEA, which could be blocked by boiling the cell extracts or
by the presence of NaVO3 in the medium (Fig. 3b). Similar
results were obtained by using mouse brain extracts (Fig. 4b),
suggesting that PTPN22 is present in brain, which was then
verified by Western and Northern blotting and immunohisto-
chemistry (Fig. 5).
C LPS C LPS
v i t a l e
5 / l o
) s l l e
ng?ml for 90 min) reduces NAPE–PLD mRNA and increases AEA levels in
RAW264.7 cells. (b) siRNA knockdown of NAPE–PLD expression is associated
with unchanged basal AEA and increased LPS-stimulated AEA levels. NAPE–
as described in Materials and Methods. Means ? SE from three to four
experiments are shown. Asterisks indicate significant difference (*, P ? 0.05;
**, P ? 0.005) from values in control (C) cells or from value in mock-
transfected, LPS-stimulated cells (#).
v i t a l e
m/z348 m/z287 m/z348 m/z287
m/z352 m/z287 m/z352 m/z287
m/z428 m/z330 m/z428 m/z330
A re a
o i t a
Mouse Brain Tissue Extract
pAEA, and d4AEA, the internal standard. (b) Representative ion chromato-
grams for pAEA and AEA obtained from ?400 fmol standards (Upper) and
RAW 264.7 (Lower) cells treated with NaVO3 and LPS by using SRM. The
transitions of m?z 348 3 287, m?z 352 3 287, and m?z 428 3 330 were
selected for AEA, d4AEA, and pAEA, respectively. (c) Effect of NaVO3on pAEA
levels in brain tissue extract. (Left) Representative SRM ion chromatograms
obtained from the control brain sample. The presence of pAEA peak at 4 min
was further confirmed by spiking with standard pAEA. (Right) The peak area
ratio of pAEA to d4AEA in brain samples incubated with or without NaVO3
(mean ? SE, n ? 3).
pAEA is an intermediate of AEA synthesis. (a) MS?MS spectra of AEA,
www.pnas.org?cgi?doi?10.1073?pnas.0601832103 Liu et al.
Contribution of PTPN22 to the Generation of AEA in Vivo. To test
whether PTPN22 is involved in the dephosphorylation of pAEA
in vivo, we generated recombinant PTPN22 in a wheat germ
cell-free protein translation system by using PTPN22 cDNA-
pIVEX1.4WG construct as template. Possibly due to the rela-
tively large size of the protein (105 kDa), only low level expres-
sion was achieved, which was verified by Western blotting.
Nevertheless, when recombinant PTPN22 was incubated with 5
nmol of synthetic pAEA, AEA was generated in a time-
(Fig. 3c). The role of PTPN22 in the generation of AEA in vivo
was further tested in two ways. First, graded siRNA knockdown
of PTPN22 in RAW264.7 cells caused a progressive reduction in
LPS-induced increase in AEA production, although the degree
of the reduction was less than the reduction in PTPN22 message
at any level of knockdown (Fig. 4a). Second, heat- and NaVO3-
sensitive conversion of synthetic pAEA to AEA was 38% lower
in brain extracts from PTPN22 knockout compared with wild-
type mice (Fig. 4b). These findings indicate that PTPN22 does
contribute to the dephosphorylation of pAEA in vivo, but is not
the only phosphatase that can do so.
LPS-Induced Generation of AEA from NAPE Is Mediated via the
PLC?Phosphatase Pathway. The above findings strongly suggest
that NAPE is converted to AEA in vivo via a two-step process
involving its cleavage by a PLC to yield pAEA, which is then
dephosphorylated by phosphatases including PTPN22. To fur-
ther test the possible role of this pathway in LPS-induced AEA
synthesis, RAW264.7 cells were preincubated with the PLC
inhibitor neomycin or a phosphatase inhibitor mixture before
their exposure to vehicle or 10 ng?ml LPS for 90 min, after which
cellular AEA levels were quantified by LC?MS. As shown in Fig.
6a, LPS treatment failed to increase AEA levels under these
conditions, which indicates that the LPS-induced increase in
AEA synthesis proceeds through the PLC?phosphatase path-
way. Blocking this pathway by neomycin resulted in a parallel
increase in the basal levels of AEA and NAPE (Fig. 6b).
It has been generally accepted that the endogenous cannabinoid
AEA is produced through a single-step, phosphodiesterase-
mediated cleavage of its membrane precursor NAPE (10). The
) l o
/ n i e t
) l o
Generation of AEA from synthetic pAEA is time- and protein concentration-
dependent. Homogenates of RAW264.7 cells with different protein concen-
homogenates (10 ?g of protein) pretreated with vehicle (C) or 10 ng?ml LPS
for 90 min (LPS) were incubated for 20 s with 5 pmol of synthetic pAEA (open
columns). Parallel aliquots were tested after boiling for 5 min (shaded col-
are shown. Asterisk indicates significant difference (P ? 0.01) from AEA levels
in the same treatment group (C, control). Pound sign indicates significant
AEA from synthetic pAEA by recombinant PTPN22 expressed in wheat germ
extracts. Aliquots of wheat germ extract containing no construct (control) or
pAEA for the indicated times, and the amount of AEA generated was mea-
sured by LC?MS. Values shown represent AEA generated over the amount
detected in controls, which was abolished by boiling the extracts, as tested at
1 min (filled square). Points are means of two to three separate experiments.
Phosphatase activity involved in conversion of pAEA to AEA. (a)
/ n i e t
% decrease in PTPN22 mRNA
n i -
00 2020 40 4060 60 8080
24 h24 h
thesis and the conversion of pAEA to AEA. (a) Graded siRNA knockdown of
LPS-induced AEA synthesis. The degree of knockdown was verified by real-
time PCR for each batch of cells. (b) Enzymatic conversion of pAEA to AEA by
brain extracts from wild-type and PTPN22 knockout mice. Aliquots of brain
extracts (10 ?g of protein) were incubated with 5 nmol of synthetic pAEA for
1 min at 37°C, and the AEA generated was measured by LC?MS. Columns and
bars represent means ? SE from three separate brains in each group.
Knockdown or knockout of PTPN22 reduces LPS-induced AEA syn-
Liu et al.
September 5, 2006 ?
vol. 103 ?
no. 36 ?
present findings document the existence in both brain and
macrophages of a parallel pathway through which AEA is
generated from NAPE by a two-step process involving the
PLC-catalyzed cleavage of NAPE to yield pAEA, which is
subsequently dephosphorylated by phosphatases, including
PTPN22, originally described as a protein tyrosine phosphatase
(13, 14). The present findings also indicate that the regulated
(i.e., LPS-induced) synthesis of AEA in macrophages proceeds
is considered to be a constitutively active rather than a regulated
enzyme (17), may function as a salvage pathway of AEA
synthesis when the PLC?phosphatase pathway is compromised.
The nonexclusive role of NAPE–PLD in the conversion of
NAPE to AEA is clearly indicated by the unchanged brain levels
of AEA in NAPE–PLD knockout mice, as documented in a
recent paper that appeared after the present manuscript had
been submitted for publication (18).
The existence of a regulated PLC?phosphatase pathway is
strongly suggested by several lines of evidence. First, a pAEA
intermediate has been identified in both brain tissue and mac-
rophages, and its quantity is markedly increased when its deg-
radation is blocked by nonselective inhibition of phosphatase
activity; this may explain why in the absence of phosphatase
inhibition it escaped detection with earlier, less sensitive meth-
ods (14). Conclusive identification of pAEA in both brain tissue
and macrophages was made possible through the application of
the highly selective SRM monitoring mode, using LC?ES-MS?
MS, which enables molecule-selective detection with high sen-
sitivity for quantification (19). Second, macrophage or brain
tissue extracts rapidly convert synthetic pAEA to AEA in a heat-
and phosphatase inhibitor-sensitive manner. Third, incubation
of macrophages with either the PLC inhibitor neomycin or a
mixture of phosphatase inhibitors prevents the LPS-induced
increase in cellular AEA levels. These treatments also lead to the
cellular accumulation of NAPE, suggesting that the parallel
modest increase in cellular AEA, which is likely mediated by
constitutively active NAPE–PLD, is due to increased substrate
availability, although a modest increase in enzyme expression is
Several lines of evidence suggest the involvement of PTPN22
in the generation of AEA. LPS induces the expression of
PTPN22 in RAW264.7 cells, and overexpression of PTPN22
results in increased conversion of pAEA to AEA by extracts of
such cells. More importantly, recombinant PTPN22 expressed in
a wheat germ system can dephosphorylate pAEA. The finding
that this reaction was reduced but not eliminated in the absence
of PTPN22 indicates that additional as yet unidentified phos-
phatases are also involved. Given that the levels of PTPN22 are
much lower in brain than in immune cells (see http://
symatlas.gnf.org/SymAtlas/), other phosphatases may play a
more dominant role in anandamide synthesis in the nervous
system. However, the relative contribution of such phosphatases
to AEA synthesis may be overestimated by the present findings
in PTPN22 knockout mice, in which such phosphatases may be
overexpressed to compensate for the life-long loss of PTPN22.
PTPN22 and its mouse homolog, PEST domain-enriched
tyrosine phosphatase (PEP) are predominantly expressed in
lymphoid and hematopoietic tissues (13, 14, 20), and have been
implicated in susceptibility to various autoimmune disorders (14,
21). For example, mice deficient in PEP have enhanced effector?
hippocampal fimbriaCA3/CA2 region
10 µg 5 µg
double staining providing a blue color, whereas neuron-specific (Left) and
in mouse brain by Northern blotting. (c) Western blot of 105-kDa PTPN22
protein in a mouse brain extract (Left) is eliminated by the presence of a
blocking peptide (Right).
PTPN22 is present in the mouse brain. (a) Immunohistochemical
n i d l o
n i d l o
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) s l l e
CC Neo Neo
basal, AEA synthesis. (a) RAW264.7 cells were incubated for 2 h with 3 mM
neomycin or with a mixture of tyrosine phosphatase inhibitors containing 2
mM imidazole, 1 mM NaF, 1.15 mM sodium molybdate, 1 mM NaVO3, and 4
mM sodium tartrate dihydrate. AEA was then quantified in lipid extracts of
vehicle (open columns) or LPS-treated (hatched columns) cells as described in
Materials and Methods. Values represent means ? SE from two to four
control cells. (b) Incubation of RAW264.7 cells with neomycin (NEO) increases
both NAPE and AEA levels.
Blocking PLC or tyrosine phosphatases prevents LPS-induced, but not
www.pnas.org?cgi?doi?10.1073?pnas.0601832103Liu et al.
memory T cell functions, which can lead to the development of
autoimmunity (14). We now find that PTPN22 is also present in
the brain, and is involved in the inducible generation of the lipid
messenger AEA. Thus, PTPN22 may act as a lipid phosphatase,
even though it had earlier been characterized as a protein
the tumor suppressor PTEN phosphatase can act on both
polypeptide and phosphoinositide substrates (22).
There is evidence that an LPS-induced increase in AEA
production in circulating macrophages mediates the early hypo-
tensive phase of septic shock (3, 4), and also plays a key role in
the vasodilated state of advanced liver cirrhosis (5, 6), a condi-
tion that contributes to potentially fatal complications such as
ascites and variceal rupture. The observed exclusive role of the
PLC?phosphatase pathway in LPS-induced AEA synthesis may
offer therapeutic targets for the treatment of these conditions.
Furthermore, cannabinoids have immunosuppressive effects in
autoimmune models of multiple sclerosis (23) and diabetes (24),
and mice deficient in CB1receptors show increased susceptibility
to neuronal damage found in autoimmune encephalitis (25).
Also, AEA limits immune responses after primary CNS damage
in multiple sclerosis in humans (9). It is tempting to speculate
that the robust link between PTPN22 and autoimmunity may be
related, at least in part, to the role of PTPN22 in the regulated
synthesis of the endocannabinoid AEA.
Materials and Methods
Gene Screening and Plasmid Transfection.AcustomLPS-stimulated
RAW264.7 cell ZAP express EcoRI?XhoI cDNA library was
mid vectors from the ZAP Express vector were released by in
vivo excision following the manufacturer’s protocol. Positive
clones were screened with [?-32P]dCTP-labeled cDNAs differ-
entially expressed in response to LPS, as generated by using a
PCR-Select cDNA subtraction kit (Clontech, Palo Alto, CA).
Individual positive clones in pBK-CMV phagemid vectors were
transiently transfected into RAW264.7 cells. Using an initial
seeding density of 104cells per ml, RAW264.7 cells were ready
for transfection at 18–24 h after seeding. For each 75-cm2flask
of cells to be transfected, 60 ?l of Lipofactamine 2000 reagent
and 5 ?g of DNA were diluted separately in 1,500 ?l of
combined, gently mixed, and incubated for 20 min at room
temperature. The DNA–lipid complexes were added subse-
quently to each flask containing 6 ml of normal medium and
mixed gently. Transfected cells were harvested for AEA mea-
surement or real-time PCR after 24 h. Stable transformants were
selected by using growth medium containing 1 mg?ml G418.
siRNA Knockdown and Real-Time PCR. Transfection of RAW264.7
cells with 300 pmol of siRNA for NAPE–PLD was as described
above. The degree of reduction of NAPE–PLD mRNA at 48 h
after transfection was verified by real-time PCR, using the
NAPE–PLD probe 6FAM-GGTTCCAAAGAGGAACT-
MGB, forward primer ATGGCTGATAATGGAGAAGAAT-
CAC, and reverse primer CGTCTTCAGGGTCACTGA-
CAAA. Probe and primer mix for real-time PCR of PTPN22
were from Applied Biosystems (Foster City, CA). For siRNA
knockdown of PTPN22, predesigned siRNAs 1–7 were pur-
chased from Qiagen (Valencia, CA), and the degree of knock-
down was established by real-time PCR for each probe set.
TLC. To detect pAEA, 1 mg of RAW264.7 cell homogenate was
incubated with 5 ? 105dpm [14C]NAPE at 37°C for 1 h.
[14C]NAPE was synthesized as described (26). Lipids were
extracted and separated by TLC with chloroform?methanol?
NH4OH (80:20:1) as the mobile phase. Radioactivity of the lipid
spots was quantified by PhosphorImaging (Typhoon 8600). The
MeOH?H2O (0.5:0.5:1). Organic layers were collected, dried
under nitrogen flow, and reconstituted with MeOH after pre-
cipitating proteins with ice-cold acetone. For measuring AEA
without simultaneously measuring pAEA, LC?MS atmospheric
standard, as described (27). For simultaneous measurement of
AEA and pAEA, the reconstituted samples were subjected to
HPLC?electrospray ionization-MS?MS analysis using a Finni-
gan TSQ Quantum Ultra triple stage quadrupole mass spec-
trometer (San Jose, CA) equipped with an Agilent 1100 series
microflow HPLC system. Separation was performed on a BDS
Hypersil C18 Pioneer column (2.1 ? 50 mm; Thermo Electron,
Waltham, MA). The flow rate was set at 400 ?l?min. Elution
solvent A consisted of 0.1% formic acid in water, and solvent B
was methanol. As solvent C, 100 mM ammonium acetate was
used to assist in removing the carryover. The solvent composi-
tion was changed from 70% A?30% B to 0% A?100% B in 1 min
returned to 70% A?30% B and maintained until 5 min. Subse-
quently, the column was washed with 100% C for 1 min, and the
solvent composition returned to the initial composition of 70%
A?30% B. The TSQ Quantum Ultra was operated with electro-
spray voltage set at 4,500 V and ion transfer tube temperature
at 350°C. The sheath and auxiliary gas pressures were 39 and 5
psi, respectively. Collision-induced dissociation (CID) was per-
formed by using argon as the collision gas at 1.5 mTorr.
Quantitation was performed by SRM in the positive ion mode
using [2H4]AEA as the internal standard. Recorded reactions
were as follows: m?z 428 3 330 for pAEA, m?z 348 3 287 for
AEA, and m?z 352 3 287 for the internal standard, [2H4]AEA.
Radioligand Binding. The binding of pAEA and AEA to mouse
brain cannabinoid receptors was assessed as described (28),
except that the membranes were treated with PMSF (28). The
membranes were subsequently treated with or without 1 mM
NaVO3for 1 h on ice before the initiation of the assay. Binding
eight concentrations (10 nM to 30 ?M) of the displacing ligands.
IC50values were obtained by nonlinear regression of log con-
centration percent displacement data and then converted to KD
values by using Prism 3.03 software.
Phosphatase Activity. RAW264.7 cell or mouse brain homoge-
nates (10 ?g of protein unless indicated otherwise) were incu-
bated with 5 pmol to 5 nmol of synthetic pAEA in 50 mM Tris
buffer with no added calcium or magnesium, pH 8.0, containing
1 ?M URB597 with or without 1 mM NaVO3for the indicated
time. Enzymatic reactions were stopped by adding two volumes
internal standard, and the AEA generated was measured as
above. The reaction was linear between 20 s and 5 min. Endog-
enous AEA in the samples was ?1% of the AEA generated from
5 nmol of synthetic pAEA.
Wheat Germ Cell-Free Protein Translation. Full-length mouse
PTPN22 cDNA was obtained by RT-PCR using LPS stimulated
RAW264.7 cell mRNA as template. An NcoI and an SmaI
restriction site were generated at the two ends of the amplicon,
which was then inserted into a wheat germ his6-tag vector
pIVEX1.4WG. Mouse PTPN22 protein was translated from the
mPTPN22-pIVEX1.4WG construct by using RTS 100 wheat
germ CECF kit, following the manufacturer’s instructions
(Roche Diagnostics, Indianapolis, IN).
Liu et al.
September 5, 2006 ?
vol. 103 ?
no. 36 ?
Northern Blot Hybridization. Northern blot hybridization was per- Download full-text
formed as described (29). A 342-bp PTPN22 cDNA probe was
generated with plasmid mPTPN22-pIVEX1.4WG as a template
for amplification by PCR with forward primer ATAGCAAC-
CCACACGACTCC and reverse primer GTGGAGGAGAAC-
CATCCTGA. The cDNA probe was labeled with alkaline
phosphatase by using the AlkPhos Direct kit (Amersham
Pharmacia, Piscataway, NJ) according to the manufacturer’s
Western Blotting and Immunohistochemistry. Native PTPN22 in
mouse brain homogenate was detected by Western immunoblot-
ting using a rabbit polyclonal antibody against PTPN22 (Novus
Biologicals, Littleton, CO), as described (30). The same antibody
was used in immunohistochemical localization of PTPN22 in
mouse brain sections. Double staining using the PTPN22 anti-
body and a rabbit anti GFAP antibody (Chemicon, Temecula,
CA) or the neuron-specific mouse anti-MAP-2 antibody
(Chemicon) was done as described (31). For blocking?
competition, PTPN22 antibody was combined with a 5-fold
excess of blocking peptide in 500 ?l of 1? PBS and incubated
overnight at 4°C before being diluted into blocking buffer.
quantified by the amount of AEA released through its digestion
with Streptomyces chromofuscus PLD, as described (4, 26).
PTPN22 Knockout Mice. PTPN22?/?mice and their wild-type
controls were developed and maintained as described (14). The
brain was removed by decapitation and frozen on dry ice until
used. Procedures have been approved by the Institutional Ani-
mal Use and Care Committee.
We thank R. L. Veech for critically reading the manuscript. This work
was supported by intramural funds of the National Institutes of Health.
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