, 2002 (2002);
et al. Doron C. Greenbaum,
Invasion by the Human Malaria Parasite
A Role for the Protease Falcipain 1 in Host Cell
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are the target molecules that undergo the
additional poly(A) extension by TPAP in
It has been demonstrated that the deficiency
of cytoplasmic polyadenylation element–bind-
ing protein (CPEB) results in the arrests of
oogenesis and spermatogenesis at the pachytene
stages (17). Although expression of two cyto-
plasmic polyadenylation element–containing
mRNAs coding for the synaptonemal complex
proteins SCP1 and SCP3 are unaffected by the
CPEB deficiency, the poly(A) tails are reduced
CPEB-deficient mice. In this study, the expres-
sion levels and sizes of CPEB, SCP1, and SCP3
mRNAs in Tpap–/–testes were similar to those
in Tpap?/?and Tpap?/–testes (Fig. 3B). More-
size of SCP3 mRNA in pachytene spermato-
cytes was found between Tpap?/?and Tpap–/–
mice. These data suggest that CPEB is not in-
adenylation. The null mutation of the TPAP
gene does not affect the size or expression level
of mRNAs encoding ovary-specific zona pellu-
cida 1 (ZP1) and ZP2 (6) (fig. S3), verifying the
testis-specific function of TPAP.
Westen blot analysis of cytoplasmic and
nuclear protein extracts from testicular tis-
sues revealed that the level of TAF10 was
reduced only in the nuclear fraction of
TAF13, and TRF2 (16) were equally present
in the cytoplasmic or nuclear fractions of
Tpap?/?and Tpap–/–testes (Fig. 3C). The
specific reduction of TAF10 was verified by
immunoprecipitation analysis of the nuclear
extracts with antibody to TBP (Fig. 3D).
These data demonstrate that TPAP affects the
transport of at least TAF10 into the nucleus,
possibly by additional polyadenylation-de-
pendent translational activation of dormant
mRNA encoding a transporter protein or pos-
sibly by TPAP itself. Moreover, the poly(A)
tails of TAF10, TAF12, and TAF13 mRNAs
are unlikely to contribute to stability and
translational control, because the levels of
these three mRNAs and cytoplasmic proteins
in Tpap–/–testes are comparable to those in
Tpap?/?testes, despite the incomplete elon-
gation of poly(A) tails (Fig. 3, A and C).
Several TAFs, including TAF10, as a com-
ponent of the TFIID complex have been dem-
onstrated to be dispensable for general RNA
polymerase II–mediated transcription and to be
essential for selective expression of specific
genes (14, 18–22). Because the TFIID complex
containing TAF10 is severely impaired in
Tpap–/–testes by insufficient transport of
TAF10 into the nucleus (Fig. 3, C and D), it is
conceivable that TAF10 may play an important
role in the expression of a subset of haploid-
specific genes, possibly as a CREM coactivator
(23), required for the morphogenetic program
during spermatogenesis. However, a small
amount of TAF10, which probably forms the
functional TFIID complex, seems to be still
present in the nucleus of round spermatids in
Tpap–/–mice (Fig. 3, C and D). Even if this is
so, round spermatids appear to be incapable of
producing enough mRNA to advance cell mor-
phogenesis, because gene transcription (RNA
synthesis) ceases around step-10 spermatids in
the mouse (24). Our study shows a direct link
between the deficiency of a cytoplasmic
poly(A) polymerase, TPAP, and the arrest of
mouse spermiogenesis, providing information
on the regulation of haploid-specific genes by
cytoplasmic polyadenylation in male germ
References and Notes
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12. K. C. Kleene, Development 106, 367 (1989).
13. S. Y. Han et al., Biol. Reprod. 64, 507 (2001).
14. L. Tora, Genes Dev. 16, 673 (2002).
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Biophys. Res. Commun. 173, 240 (1990).
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17. J. Tay, J. D. Richter, Dev. Cell 1, 201 (2001).
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18, 4823 (1999).
19. S. R. Albright, R. Tjian, Gene 242, 1 (2000).
20. Z. Chen, J. L. Manley, Mol. Cell. Biol. 20, 5064 (2000).
21. R. N. Freiman et al., Science 293, 2084 (2001).
22. M. A. Hiller, T.-Y. Lin, C. Wood, M. T. Fuller, Genes
Dev. 15, 1021 (2001).
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Sassone-Corsi, Mol. Cell. Endocrinol. 179, 17 (2001).
24. A. L. Kierszenbaum, L. L. Tres, J. Cell Biol. 65, 258
25. We thank L. Tora, I. Davidson, and T. Tamura for
providing antibodies. This study was partly supported
by Grant-in-Aids for Scientific Research on Priority
Areas (A) and (B), Scientific Research (A), and Explor-
atory Research from the Japan Society for the Pro-
motion of Science and the Ministry of Education,
Culture, Sports, Science and Technology in Japan.
Supporting Online Material
Materials and Methods
Figs S1 to S4
References and Notes
3 June 2002; accepted 16 October 2002
A Role for the Protease
Falcipain 1 in Host Cell Invasion
by the Human Malaria Parasite
Doron C. Greenbaum,1* Amos Baruch,2Munira Grainger,4
Zbynek Bozdech,2Katlin F. Medzihradszky,1Juan Engel,3
Joseph DeRisi,2Anthony A. Holder,4Matthew Bogyo2*
Cysteine proteases of Plasmodium falciparum are required for survival of the
malaria parasite, yet their specific cellular functions remain unclear. We used a
chemical proteomic screen with a small-molecule probe to characterize the pre-
falcipain 1, was active during the invasive merozoite stage. Falcipain 1–specific
host erythrocytes, yet had no effect on normal parasite processes such as hemo-
cell invasion and establish a potential new target for antimalarial therapeutics.
Malaria is a devastating disease that affects
300 to 500 million people and kills about 2
million people per year. Currently, quino-
lines and antifolates are the most common
drugs for disease prevention and cure.
However, multidrug resistance is a major
issue, highlighting the need for new anti-
malarial drugs to combat this parasite. Pro-
teases represent one of the largest families
of potential therapeutic targets, and cys-
teine proteases have been shown to be es-
sential for the survival of several human
parasites (1–3). Cysteine proteases have
been specifically implicated in several cel-
lular functions during the P. falciparum life
cycle, including hemoglobin degradation
(4–5), cleavage of red blood cell ankyrin to
1Department of Pharmaceutical Chemistry,2Depart-
ment of Biochemistry and Biophysics,3Department of
Pathology, Veterans Affairs Medical Center, Universi-
ty of California, San Francisco, CA 94143, USA.4Di-
vision of Parasitology, National Institute for Medical
Research, Mill Hill, London NW7 1AA, UK.
*To whom correspondence should be addressed at M.
Bogyo, University of California, San Francisco, 513
Parnassus Avenue, San Francisco, CA 94043, USA.
E-mail: email@example.com (D.C.G.) and mbogyo@
R E P O R T S
6 DECEMBER 2002VOL 298SCIENCEwww.sciencemag.org
on May 18, 2007
facilitate host cell rupture (6), and the con-
comitant release of parasites from the para-
sitophorous vacuole (7). Furthermore, cys-
teine proteases are important for host cell
invasion by P. falciparum and other related
parasites (8, 9). However, the lack of a
technically facile method to genetically
manipulate the parasite has made function-
al analysis of key proteases difficult.
P. falciparum has a complex life cycle
involving two distinct sexual and asexual
stages of growth. The human asexual eryth-
rocytic phase (blood stage) is the cause of
therefore is the focus of this study. The
blood stage begins when merozoites (ini-
tially released from the liver) invade red
blood cells. Over the next 48 hours, inter-
nalized parasites differentiate (ring stage),
metabolize hemoglobin (trophozoite stage),
and replicate (schizont stage) to produce
expanded populations of invasive merozo-
ites that are released upon rupture of the
host cell (10). Merozoites have a limited
life-span outside the host cell and must
immediately find new cells to invade and
start the cycle anew.
Initially, we used a functional proteomic
method to identify and biochemically ana-
lyze all cysteine protease activity in ex-
tracts of P. falciparum (11–13). Four pro-
tease activities were detected in whole-cell
lysates from mixed blood stages of P. fal-
ciparum parasites with the radiolabeled
cysteine protease probe125I-DCG-04 (Fig.
1A). One protease activity was enriched in
an NP-40–insoluble pellet fraction, where-
as the remaining protease activities re-
mained soluble, suggesting distinct local-
ization and/or biochemical properties of
The biotin affinity tag of DCG-04 af-
forded a single-step purification of all la-
beled proteins and their subsequent identi-
fication by mass spectrometry–based se-
quencing (Fig. 1B). Each of the proteases
identified was a member of the papain fam-
ily of cysteine proteases. Human calpain 1
was isolated from the NP-40–insoluble
fraction but is not thought to play a func-
tional role in P. falciparum (14). It is un-
likely that this protease was purified as a
result of contamination by red blood cells,
because red blood cells were lysed before
isolation of proteases with the probe. The
purified cathepsin C–like protease matched
a sequence found in the P. falciparum ge-
nome database (locus
(15–17); however, no biological function
for this enzyme has been reported. Falci-
pains 2 and 3 were also isolated from the
NP-40–soluble extracts. These proteases
reside in the food vacuole, where they play
a critical role in hemoglobin degradation
(4, 5). The remaining, detergent-insoluble
protease was identified as falcipain 1. Al-
though falcipain 1 was the first cysteine
protease gene cloned from the P. falcipa-
rum genome (18), difficulties in recombi-
nant expression (19) have prevented its de-
tailed biochemical and functional charac-
terization. Thus, falcipain 1 is an ideal
target for in situ studies with pharmacolog-
ical tools to perturb its function.
To gain insight into the functional roles
of specific cysteine proteases, we used
highly synchronized parasite populations to
profile protease activities throughout the
multiple developmental stages of the para-
site. For each stage, extracts were generat-
ed at pH 5.5, although analysis of falcipain
1, 2, and 3 activity indicated a broad pH
activity profile. Cysteine protease activity
was determined by labeling both detergent-
soluble and -insoluble lysates from each
stage with125I-DCG-04 (Fig. 1C). The ac-
tivity profiles of falcipains 1, 2, and 3
indicated that regulation of these enzymes
is highly divergent (Fig. 1D). Falcipain 2
and 3 activities peaked at the trophozoite
stage, which is consistent with previously
reported protein levels and with a role for
these enzymes in hemoglobin degradation
(4, 5). In contrast, falcipain 1 activity
peaked during the merozoite stage and was
the predominant protease activity in both
the merozoite and the ring stages (Fig. 1D).
This activity profile differs from the report-
ed expression of falcipain 1 mRNA only in
ring-stage parasites. This difference can be
explained by both translational and post-
translational mechanisms that control the
maturation of falcipain 1 throughout the
Fig. 1. Identification and biochemical characterization of active cysteine proteases within the
erythrocytic cycle of P. falciparum. (A) Asynchronous cultures of P. falciparum parasites were
isolated after sorbitol lysis of host red blood cells. Parasite pellets were lysed by addition of
0.2% NP-40, and total cellular extracts were labeled with125I-DCG-04. Homogenates were
separated on a 15% polyacrylamide gel, and labeled polypeptides were visualized with a
PhosphorImager (total lysate lane). DCG-04–labeled parasite extracts were separated into
0.2% NP-40 soluble and insoluble fractions. Fractions were separated on 15% polyacrylamide
gel and visualized by phosphoimaging. (B) Identification of affinity-labeled proteases. Parasite
culture (1 liter) was harvested, lysed, and labeled with nonradioactive DCG-04. Probe-labeled
proteases were affinity-purified by a single-step affinity purification protocol (9). Isolated
proteases were excised by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and identified
by in-gel trypsin digestion followed by mass spectrometry–based sequencing. Targets were
identified by database (PlasmoDB) matching of peptide sequences. Cat C, cathepsin C. (C)
Profiling protease activity throughout the erythrocytic life cycle. Synchronous parasites were
harvested at the ring, trophozoite (troph), schizont (schiz), and merozoite (mer) stages. Lysates
were separated into 0.2% NP-40 soluble and insoluble fractions and labeled with125I-DCG-04.
(D) Falcipain 1 and 2/3 protease bands from (C) were quantified with the ImageQuant software
(Molecular Dynamics) and plotted for each of the major blood stages. Data for falcipain 1
represent a composite of insoluble and soluble activities.
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on May 18, 2007
life cycle of the parasite. This activity pro-
file suggests a primary function for falci-
pain 1 either in red blood cell rupture or
during reinfection of new host red blood
In addition to having a distinct activity
profile, falcipain 1 also changed its subcel-
lular location during the late schizont and
merozoite stages, as indicated by the ap-
pearance of an NP-40–soluble active form
(Fig. 1C, far right lane). This soluble activ-
ity was most abundant in the merozoite
stage and absent at the ring stage. During
merozoite development, several new sub-
cellular compartments are created that com-
pose the apical organelles. These organelles
participate in the process of host cell inva-
sion through a controlled secretion of their
contents (20). Localization of falcipain 1 to
specialized compartments such as these
may explain its change in detergent solu-
bility. Furthermore, the short-lived, soluble
falcipain 1 activity in merozoites could be
explained by a mechanism in which active
falcipain 1 is secreted during invasion.
To better understand the location and
Fig. 2. Localization of falcipain 1 in merozoites by immunofluorescence imaging. Samples of
merozoites were smeared and fixed in 1% paraformaldehyde. The slides were incubated with
an antibody to falcipain 1 or with antibodies specific to the rhoptry (RopH2) and microneme
(EBA-175) proteins. Nuclei were stained with 4?,6-diamidino-2-phenylindole (DAPI). The lack
of colocalization indicates that falcipain 1 is found in compartments at the apical end of the
merozoite that are distinct from the rhoptries and micronemes.
Fig. 3. Identification of
falcipain 1–specific in-
hibitors by screening
peptide epoxide posi-
tional scanning libraries
(PSLs) in crude cell ex-
tracts. (A) Structures of
the general inhibitor
screening. A single ami-
no acid position (P2)
whereas the remaining
positions contained an
isokinetic mixture of natural amino ac-
ids (Mix positions). A set of 19 natural
and 41 nonnatural amino acids were
libraries. A second set of P2 diverse
sublibraries were generated with the
enantiomeric form (2R, 3R in place of
2S, 3S) of the epoxide building block.
(B) Colorimetric cluster display of inhi-
bition data. PSLs were screened against
P. falciparum lysates by pretreatment
of samples with individual constant P2
libraries followed by labeling with125I-
DCG-04. Labeling intensity of each tar-
get relative to the control untreated
sample was used to generate percent
competition values. The resulting data
were clustered and visualized with pro-
ray data. Proteases are arrayed along
the y axis and inhibitors are arrayed
along the x axis. Natural amino acids
are indicated by standard one-letter
codes and nonnatural amino acids are
labeled with the NN prefix [for struc-
tures and corresponding number as-
signment see (12, 13)]. Libraries generated with natural amino acids in the P2
by (R, R). (C) Competition analysis of a negative control compound [YAG-
and YAH-Eps(S,S)] identified from library screening. Compounds were added to
total parasite extract for 30 min followed by labeling with125I-DCG-04 for 1
concentration; Falc, falcipain; hCalp, human calpain.
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on May 18, 2007
cellular function of falcipain 1, we used
studies with an antibody generated with a
unique, COOH-terminal peptide sequence
found in the mature enzyme. Immunoblots
confirmed that these antibodies specifically
recognized both the mature 26-kD enzyme
and the larger proenzyme (21). The anti-
bodies were then used in immunofluores-
cence assays of synchronized cultures to
visualize the location of falcipain 1. Mero-
zoites stained with an antibody to falcipain
1 revealed punctate staining in distinct
vesicular structures at the apical end of the
parasite, opposite the nucleus (Fig. 2, left
panels). This punctate falcipain 1 staining
pattern was also observed at the schizont
stage, although it was excluded from the
area of the food vacuole, further supporting
a functional divergence from its close rel-
atives falcipains 2 and 3 (22). In addition,
co-staining was performed with antibodies
to the rhoptry protein RopH2 and to the
micronemal protein EBA-175, two of three
invasion-specific organelles present only in
merozoites (Fig. 2, middle panels). Merg-
ing of the two staining patterns indicated
that the compartments containing falcipain
1 are distinct from the rhoptries and mi-
cronemes, yet localized to the apical end of
the merozoite (Fig. 2, right panels). This
unique staining of falcipain 1 implies local-
ization to the third set of apical organelles,
the dense granules, or to a subset of these
organelles. The complement of subcellular
compartments that compose the apical or-
ganelles may be more diverse than previ-
ously thought, and falcipain 1 might be
located in a previously unidentified com-
partment. Ultimately, falcipain 1 localiza-
tion to the apical end of the merozoite
suggests a potential function in the invasion
of red blood cells.
A more definitive analysis of falcipain 1
function requires application of methods
that allow inhibition or disruption of its
enzymatic activity in live parasite cultures.
Gene ablation or knockout techniques have
proven difficult in P. falciparum, and dele-
tion of many genes from the haploid ge-
nome results in a lethal phenotype. An
alternative chemical approach with a selec-
tive inhibitor that renders a specific target
protease inactive allows dissection of the
protease’s biochemical function. Such a
“chemical genetic” approach allows pheno-
typic evaluation at specific stages within
the life cycle of the parasite.
To identify falcipain 1–specific inhibi-
tors, we screened a positional scanning li-
brary of peptidyl epoxides (12, 13) in
whole parasite lysates. We generated librar-
ies by fixing a single amino acid residue on
a tripeptide inhibitor scaffold while varying
the remaining two positions (Fig. 3A). This
method produces a series of sublibraries
made up of several hundred compounds,
each having a single different fixed amino
acid residue (Fig. 3A). Inhibitor potency
and selectivity were assessed by incubation
of lysates with each inhibitor sublibrary,
followed by reaction with the general cys-
teine protease activity–based probe
DCG-04. The potency of specific inhibitor
scaffolds was measured as a ratio of the
percent residual labeled proteases after in-
hibitor treatment relative to an untreated
control. For analysis, the inhibition data
were displayed in a colorimetric format and
clustered on the basis of similarities in
inhibitor profiles using software for mi-
croarray analysis (23) (Fig. 3B). Several
falcipain 1–specific P2 amino acid residues
were identified (Fig. 3B, black rectangles)
and were used to design a series of specific
inhibitors. These optimized inhibitors and a
control, inactive P2 glycine-containing in-
hibitor (Fig. 3B, yellow rectangle), were
assayed in parasite extracts over a range of
concentrations (Fig. 3C). The most selec-
tive of the resulting inhibitors, YA29-
Eps(S,S), showed greater than 25-fold se-
lectivity for falcipain 1 over all other cys-
teine proteases. Furthermore, at optimal
concentrations of 5 to 10 ?M, we observed
selective inhibition of falcipain 1. The
compound YAG-Eps(S,S) showed no activ-
ity toward any of the protease targets and
therefore served as a control for in situ
Effects of cysteine protease inhibition in
live cultures of P. falciparum were deter-
mined by treating parasites with the general
papain family protease inhibitor E-64d,
with the falcipain 1–specific inhibitor
YA29-Eps(S,S), or with the control inhib-
itor YAG-Eps(S,S) at 40 hours after inva-
sion. After rupture (10 to 12 hours later),
the percentage of surviving cells was cal-
culated by counting parasites. As predicted,
the control inhibitor YAG-Eps(S,S) had no
effect on parasite growth, as measured by a
constant number of schizonts and levels of
ring-stage parasites comparable to those of
dimethyl sulfoxide–treated cultures (Fig.
4A, gray bars, and 4B, left panel). Inhibi-
tion of all papain family proteases with
E-64d resulted in a marked decrease in the
number of new ring-stage parasites (Fig.
4A, maroon bars). This decrease was dose
dependent and resulted from a block of
Fig. 4. Functional evaluation of falcipain 1–specific compounds in vivo. (A) Synchronous
parasites (5% parasitemia) were treated with E-64d (maroon bars), with the falcipain 1–
specific inhibitor YA29-Eps(S,S) (blue bars), or with the control inhibitor YAG-Eps(S,S) (gray
bars) at the indicated concentrations 40 hours after invasion (late schizont stage). Smears were
prepared at 58 hours after invasion. Ring- and schizont-infected red blood cells were counted
as a percentage of total red blood cells. (B) Representative images of the parasites treated
at the highest concentration (10 ?M) of inhibitors from (A). The control compound YAG-
Eps(S,S) had no effect on the parasites, as indicated by the presence of rings. The enlarged
food vacuole of the schizonts in the E-64d–treated parasites (black arrowheads) is apparent.
Falcipain 1–specific inhibitors such as YA29-Eps(S,S) blocked invasion but not host cell
rupture, as seen by the lack of ring-stage parasites with normal release of merozoites (open
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www.sciencemag.orgSCIENCEVOL 2986 DECEMBER 2002
on May 18, 2007
parasite development at the late schizont Download full-text
stage, as measured by an increase in schi-
zonts. High concentrations of E-64d also
induced a massive enlargement of the food
vacuole within parasites, which is consis-
tent with previously reported effects of
general cysteine protease inhibitors (24)
(Fig. 4B, middle panel). In contrast, falci-
pain 1–specific inhibitors caused a similar
dose-dependent decrease in the percentage
of new ring-stage parasites (Fig. 4A, blue
bars), but did not block schizont develop-
ment and subsequent rupture as indicated
by the appearance of merozoites (Fig. 4B,
right panel). Furthermore, schizont rupture
was not affected, as assessed by the con-
stant number of schizonts at all concentra-
tions of falcipain 1–specific inhibitor (Fig.
4A) and by measurement of the release of
the parasitophorous vacuolar protein SERA
(serine repeat antigen) (25). These results
suggest that falcipain 1 is not involved in
hemoglobin degradation or red blood cell
rupture at the end of schizogony, but rather
has a specific role in the invasion of red
blood cells by extracellular merozoites.
We have shown, using a functional pro-
teomics screen combined with a chemical
ing the process of host cell invasion during the
erythrocytic cycle of P. falciparum. The prima-
ry sequence of falcipain 1 is well conserved
across the Plasmodium genus (26), making it a
potentially useful new target for design of ther-
apeutic drugs in all four plasmodial species that
cause malaria in humans. Therapeutic agents
that are able to specifically prevent or slow the
process by which merozoites infect new cells
are likely to disrupt the development cycle and
allow time for the host immune response to
destroy the extracellular parasite.
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(University of California, San Francisco) for critical
evaluation of the manuscript. Supported by funding
from the Sandler Program in Basic Sciences (M.B.).
Supporting Online Material
Materials and Methods
References and Notes
16 August 2002; accepted 15 October 2002
Signaling of Rat Frizzled-2 Through
Phosphodiesterase and Cyclic GMP
Adriana Ahumada,1Diane C. Slusarski,2Xunxian Liu,1
Randall T. Moon,3Craig C. Malbon,1Hsien-yu Wang4*
The Frizzled-2 receptor (Rfz2) from rat binds Wnt proteins and can signal
by activating calcium release from intracellular stores. We show that wild-
type Rfz2 and a chimeric receptor consisting of the extracellular and trans-
membrane portions of the ?2-adrenergic receptor with cytoplasmic domains
of Rfz2 also signaled through modulation of cyclic guanosine 3?,5?-mono-
phosphate (cGMP). Activation of either receptor led to a decline in the
intracellular concentration of cGMP, a process that was inhibited in cells
treated with pertussis toxin, reduced by suppression of the expression of the
heterotrimeric GTP–binding protein (G protein) transducin, and suppressed
through inhibition of cGMP-specific phosphodiesterase (PDE) activity. More-
over, PDE inhibitors blocked Rfz2-induced calcium transients in zebrafish
embryos. Thus, Frizzled-2 appears to couple to PDEs and calcium transients
through G proteins.
The Wnt proteins are secreted signaling pro-
teins that play diverse roles in cell polarity,
cell proliferation, and specification of cell
fate (1–3). Wnt proteins signal through friz-
zled (Fz) gene products (4, 5), members of
the superfamily of G-protein–coupled recep-
tors (GPCRs) (6–8). Wnt-Fz family mem-
bers can be grouped into functionally distinct
classes. Activation of the Wnt–?-catenin
pathway increases nuclear accumulation of
?-catenin (1, 2, 9), thus activating transcrip-
tion (10–14). The Rfz2 receptor, by itself,
transduces binding of Wnt-5A to increases in
intracellular calcium release (15) and activa-
tion of calcium-calmodulin–dependent pro-
tein kinase II (16, 17) and protein kinase C
(18) without appreciably activating the ca-
nonical Wnt–?-catenin pathway. Because pu-
rified, active Wnt proteins are not available
for analysis of Rfz2 receptor function, we
engineered a chimeric receptor to substitute
the three cytoplasmic loops and the COOH-
terminal tail of the Rfz2 receptor for the
corresponding regions of the hamster ?2-
adrenergic receptor (?2AR) (19). The Rfz2
chimeric receptor, whose cytoplasmic do-
mains display no similarity to that of ?2AR
(fig. S1A) (20), is functional insofar as it
couples to calcium mobilization (19) and to
rapid activation of calcium-calmodulin–de-
pendent kinase II (16), as does the wild-type
For functional analysis of Rfz2 signal-
ing, we expressed the Rfz2 chimera in
mouse totipotent F9 teratocarcinoma cells
and in Chinese hamster ovary (CHO) cells
that lack endogenous ?2AR. We identified
stable transfectants expressing the Rfz2
chimera in CHO clones by reverse tran-
scription (RT) and polymerase chain reac-
tion (PCR) amplification (fig. S1B) (19,
20), immunoblotting with antibodies to
?2AR (fig. S1C) (19, 20), and specific
binding of the ?2AR antagonist [125I]iodo-
1Department of Molecular Pharmacology, Diabetes
and Metabolic Diseases Research Center, University
Medical Center, SUNY–Stony Brook, Stony Brook, NY
11794–8651, USA.2Department of Biological Scienc-
es, University of Iowa, Iowa City, IA 52242, USA.
3Howard Hughes Medical Institute, Department of
Pharmacology and Center for Developmental Biology,
University of Washington School of Medicine, Seattle,
WA 98195, USA.
Biophysics, Diabetes and Metabolic Diseases Research
Center, University Medical Center, SUNY–Stony
Brook, Stony Brook, NY 11794–8661, USA.
4Department of Physiology and
*To whom correspondence should be addressed. E-
R E P O R T S
6 DECEMBER 2002VOL 298SCIENCE www.sciencemag.org
on May 18, 2007