The Journal of Cell Biology
The Journal of Cell Biology, Volume 162, Number 5, September 1, 2003 773–779
The Rockefeller University Press, 0021-9525/2003/09/773/7 $8.00
Differential requirements for AP-2
in clathrin-mediated endocytosis
Sean D. Conner and Sandra L. Schmid
The Scripps Research Institute, La Jolla, CA 92037
P-2 complexes are key components in clathrin-
mediated endocytosis (CME). They trigger clathrin
assembly, interact directly with cargo molecules,
and recruit a number of endocytic accessory factors. Adaptor-
associated kinase (AAK1), an AP-2 binding partner, modulates
AP-2 function by phosphorylating its
examined the effects of adenoviral-mediated overexpression
of WT AAK1, kinase-dead, and truncation mutants in HeLa
cells, and show that AAK1 also regulates AP-2 function in
vivo. WT AAK1 overexpression selectively blocks transferrin
(Tfn) receptor and LRP endocytosis. Inhibition was kinase
2 subunit. Here, we
independent, but required the full-length AAK1 as truncation
mutants were not inhibitory. Although changes in
phorylation were not detected, AAK1 overexpression signifi-
cantly decreased the phosphorylation of large adaptin sub-
units and the normally punctate AP-2 distribution was
dispersed, suggesting that AAK1 overexpression inhibited Tfn
endocytosis by functionally sequestering AP-2. Surprisingly,
clathrin distribution and EGF uptake were unaffected by
AAK1 overexpression. Thus, AP-2 may not be stoichio-
metrically required for coat assembly, and may have a more
cargo-selective function in CME than previously thought.
Clathrin-mediated endocytosis (CME) is important for a
variety of biological processes ranging from nutrient uptake
to synaptic vesicle recycling. Although more than 30 accessory
proteins are believed to control this internalization pathway
(Slepnev and De Camilli, 2000), clathrin and the adaptor
protein complex (AP-2), constitute the major coat constituents
(Brodsky et al., 2001). It is thought that the assembly of
clathrin into progressively curved lattices provides the driving
force behind the generation of coated pits and coated vesicles.
The targeting and assembly of clathrin, as well as its coupling
to cargo destined for internalization, is facilitated by the action
of AP-2. AP-2 is a multifunctional heterotetramer, consisting
of two large subunits (
and a small subunit (
2). The large subunits function in
plasma membrane targeting (Robinson, 1993) and act as a
platform to recruit other functionally relevant accessory
proteins like amphiphysin, Eps15, epsin, etc. (Traub et al.,
2 subunit interacts with the cytoplasmic do-
main of membrane-bound receptors containing tyrosine-
based internalization motifs (Kirchhausen, 1999), whereas
2 subunit appears to stabilize the AP-2 complex
(Collins et al., 2002).
2), a medium subunit (
Phosphorylation of AP-2 complexes regulates their re-
cruitment to the plasma membrane (Fingerhut et al., 2001),
their interaction with cargo molecules (Ricotta et al., 2002),
and their assembly with clathrin (Wilde and Brodsky,
1996). Several kinases copurify with clathrin-coated vesicles
(CCVs), including (1) casein kinase II, which appears to
phosphorylate clathrin light chains (Korolchuk and Banting,
2002); (2) an unknown kinase(s) that phosphorylates the
2 adaptins and may regulate their plasma membrane
recruitment (Fingerhut et al., 2001); and (3) two related ki-
nases, GAK/auxilin 2 (Umeda et al., 2000) and the newly
discovered adaptor-associated kinase (AAK1) (Conner and
Schmid, 2002), that phosphorylate
2 by AAK1 increases AP-2 affinity for tyrosine-
based internalization motifs roughly 25-fold (Ricotta et al.,
2002). Additionally, AAK1 inhibits AP-2–stimulated trans-
Tfn) internalization in perforated cell assays that re-
constitute early steps in CCV formation (Conner and
Schmid, 2002). Here, we establish that full-length AAK1
interacts with and perturbs AP-2 function in vivo
ingly, the disruption of AP-2 function by AAK1 overexpres-
sion reveals a more cargo-specific role for AP-2 in clathrin-
dependent receptor-mediated endocytosis.
2. In vitro phosphory-
The online version of this article includes supplemental material.
Address correspondence to Sandra Schmid, The Scripps Research Institute,
10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: (858) 784-2311.
Fax: (858) 784-9126. email: firstname.lastname@example.org
Key words: endocytosis; AP-2; clathrin; AAK1; kinase
Abbreviations used in this paper: AAK1, adaptor-associated kinase; CCV,
clathrin-coated vesicle; CME, clathrin-mediated endocytosis; siRNA,
small interfering RNA; Tfn, transferrin; TfnR, Tfn receptor; tTA, tetra-
The Journal of Cell Biology
774 The Journal of Cell Biology
Volume 162, Number 5, 2003
Results and discussion
To test the in vivo role of AAK1 in regulating AP-2 func-
tion, we generated recombinant tetracycline-regulatable ade-
noviruses encoding AAK1 constructs postulated to compete
for endogenous AAK1 function (Fig. 1 A). Identical con-
structs for baculovirus protein expression were also gener-
ated for biochemical analysis to allow for correlation of in
vivo and in vitro observations. Endogenous kinase(s), in-
cluding AAK1, that are known to cofractionate with AP-2
(Conner and Schmid, 2002; Korolchuk and Banting, 2002)
were first inactivated by pretreatment with FSBA, an ATP
analogue and irreversible kinase inhibitor (Fig. 1 B; Olu-
sanya et al., 2001). Addition of WT AAK1 to FSBA-inacti-
vated AP-2 resulted in efficient
incubated in the presence of [
and Schmid, 2002). As expected, point mutations of con-
served residues within the kinase domain, either K74A or
D176A, predicted to disrupt nucleotide binding and cataly-
sis, respectively, severely inhibited AAK1 activity for either
2 or autophosphorylation. A truncated
AAK1 construct (
AID) that lacks the
ing domain (AID) was efficiently autophosphorylated, thus
AID AAK1 is a fully active kinase. However, 10-fold more
AID was required to phosphorylate
P]ATP (Fig. 1 B; Conner
2 phosphorylation when
2 (Fig. 1 B). Thus,
although the majority of AP-2 interaction is supported by
the AID, other lower affinity AP-2 binding domains must
exist. Indeed, GST fusion protein pull-downs demonstrate
that most AP-2 binding is supported by the AID fragment
(Fig. 1 C); however, AP-2 binding by both the
QPA fragments was detected when
fragment–GST fusion protein was used (Fig. 1 C). We con-
clude that efficient recruitment of AAK1 to AP-2 and its
2 requires the AID and that each of the
individual domains and truncated mutants retain their ex-
We next infected tetracycline transactivator (tTA) HeLa
cells with adenoviruses encoding various AAK1 constructs
and assayed for their ability to internalize biotinylated Tfn.
Cells overexpressing WT AAK1 showed a significant, con-
centration-dependent inhibition in Tfn endocytosis com-
pared with controls (Fig. 2, A and C), a result consistent
with in vitro observations (Conner and Schmid, 2002). Al-
though somewhat less potent, inhibition was also observed
in cells overexpressing either of the kinase-inactive AAK1
mutants. This is also consistent with the weaker, but observ-
able inhibition of FSBA-treated AAK1 on endocytosis in
perforated cells (Conner and Schmid, 2002). Interestingly,
neither overexpression of the kinase-active
QPA or AID fragments of AAK1, which we expected to ef-
fectively compete for AAK1-interacting partners and thus
perturb AAK1 function, significantly affected Tfn endocyto-
sis (Fig. 2 B). From these observations, we conclude that the
internalization defects seen with full-length AAK1 con-
structs do not simply reflect rampant kinase activity, a se-
questration of AAK1-interacting partners, or interference
with other AP-2 partners that interact through the ear do-
The generality of the observed block in endocytosis re-
sulting from full-length AAK1 overexpression was con-
firmed by examining another constitutively internalized
receptor, LRP (low-density lipoprotein receptor-related
protein) and the uptake of its ligand, RAP (receptor-associ-
ated protein), in immunofluorescence assays. Cells infected
with adenovirus encoding WT AAK1 showed a significant
decrease in RAP internalization and a corresponding in-
crease in surface-associated RAP relative to controls
(Fig. S1, available at http://www.jcb.org/cgi/content/full/
jcb.200304069/DC1). Together, these data suggest that
AAK1 interacts in vivo with AP-2 to regulate its function
in the constitutive internalization of ligand complexes. In-
deed, exogenous AAK1 coimmunoprecipitated with AP-2
complexes from cells overexpressing WT AAK1 (unpub-
lished data). Inhibition by AAK1 occurred independently
of its kinase activity, but required extended interactions be-
tween full-length AAK1 and AP-2 complexes, because nei-
ther the isolated COOH-terminal AID that binds the
-ear nor an active kinase/QPA construct inhibited endocy-
tosis. These data establish that AAK1 can bind to and dis-
rupt the function of AP-2 complexes in vivo.
In vitro kinase assays did not reveal any major AAK1 tar-
gets other than
2 in either cytosolic or membrane fractions
(Conner and Schmid, 2002). Moreover,
is known to be required for endocytosis in vivo (Olusanya et
al., 2001). Thus, we expected that overexpression of full-
10-fold more AAK1
AID, nor the
phosphorylation. (A) Diagram illustrating the AAK1 constructs
used in this study. Baculovirus constructs were GST-tagged at the
COOH terminus, whereas adenovirus constructs were HA-tagged
at the NH2 terminus (see supplemental methods, available at
http://www.jcb.org/cgi/content/full/jcb.200304069/DC1). (B) FSBA-
inactivated APs (5.2 ?g) were incubated with AAK1–GST fusion
proteins, as indicated, in the presence of [?32P]ATP and phosphory-
lated protein detected by SDS-PAGE and autoradiography. Assays
were performed with 0.25 ?M of WT, K74A, or D176A AAK1 and
0.65 ?M (2.5?) or 2.5 ?M (10?) of the ?AID mutant. (C) The indi-
cated AAK1–GST fusion proteins were immobilized on glutathione–
agarose beads at either ?0.25 mg/ml (1?) or ?2.5 mg/ml (10?)
and incubated with isolated APs. Bound AP-2 was detected by
Structural requirements for AAK1-mediated ?2
The Journal of Cell Biology
Cargo selective role for AP-2 |
Conner and Schmid 775
length AAK1 constructs inhibited AP-2 function by shifting
the balance of
2 into a phosphorylated (WT AAK1) or de-
phosphorylated (K74A or D176A AAK1) state. However,
immunoprecipitation of AP-2 complexes from whole cell ly-
sates, following in vivo labeling, did not show any significant
2 phosphorylation in cells overexpressing ei-
ther WT or kinase-dead AAK1 (Fig. 3 A). Although we can-
not rule out the existence of a specifically localized subpopu-
lation of phosphorylated
2, these data suggest that
phosphorylation activity of AAK1 in vivo is tightly regulated.
Unexpectedly, a significant decrease in phosphorylation of
the large AP-2 subunits was observed. Thus, rather than al-
2 phosphorylation state, the observed receptor
internalization block appears to result from the kinase activ-
ity–independent binding of full-length AAK1 to AP-2,
which exerts a more global effect on AP-2 function.
To explore the mechanism of AAK1-mediated inhibition,
we examined its effects on AP-2 localization. Overexpression
of WT AAK1 caused a dramatic displacement of AP-2 from
the normally punctate structures at the plasma membrane
seen in control cells (Fig. 3 B). A similar, but less dramatic
phenotype was observed in cells overexpressing either kinase-
inactive AAK1 mutant (unpublished data). By comparison,
overexpression of dominant–negative dynamin mutants that
inhibit endocytosis resulted in the increased clustering of AP-
2–containing coated pits on the plasma membrane. Consis-
tent with their lack of effect on Tfn and RAP internalization,
AID, QPA, or AID fragments showed any sig-
nificant AP-2 localization defect at the plasma membrane
(Fig. 3 C; and data not depicted). To eliminate the possibil-
ity that full-length AAK1 overexpression masks the AP.6
-adaptin, we performed ELISA assays and found
that excess AAK1 did not inhibit AP.6 binding to immobi-
lized AP-2 (unpublished data). Finally, we also probed cells
with antibodies against the
2 subunit of AP-2 and identical
results were obtained (unpublished data).
Postulating that AAK1 overexpression sequesters AP-2
in the cytosol, we determined the distribution of AP-2 in
cytosol and membrane fractions. Surprisingly, WT AAK1
overexpression did not appear to alter the AP-2 distribu-
tion between soluble and particulate pools compared with
control cells, or that of cells overexpressing K74A, D176A,
AID constructs (Fig. 3 D; and data not de-
picted). Although these fractionation experiments cannot
eliminate the possibility that cytosolic AP-2 forms sedi-
mentable structures following AAK1 overexpression, or
that AAK1 overexpressions causes the mislocalization of
AP-2 to other disperse membranes within the cytosol,
these observations suggest that AAK1 overexpression in-
hibits endocytosis of the Tfn receptor (TfnR) and LRP by
functionally sequestering AP-2 complexes and preventing
their clustering on the plasma membrane.
To extend our analysis of AAK1 function in CME, we
used small interfering RNAs (siRNAs) to knock down
AAK1 expression in cells. Transfection of two different si-
RNAs that specifically target AAK1 reduced AAK1 expression
80% in either A549 or HeLa cells. However, in neither
case did we observe an alteration in Tfn internalization, AP-2
2 phosphorylation (Fig. S2, available at
cells were infected with either control adenovirus encoding the tTA
transcription activator or tetracycline-regulatable adenoviruses en-
coding either full-length WT AAK1 or kinase-inactive mutants (K74A
or D176A) (A), or AAK1 fragments (B), as indicated in the legend, and
tested for Tfn endocytosis as described in the Materials and methods.
Protein expression levels were tested by immunoblot analysis (insets),
using pAbs against ?AID AAK1 and/or the AID AAK1 fragment. Each
time point represents the average of three independent experiments ?
the standard deviation. (C) The observed inhibition is concentration
dependent. Cells were infected with WT AAK1 adenovirus in the
presence of increasing concentrations of tetracycline to titrate AAK1
expression, and Tfn endocytosis was assessed as described here.
Full-length AAK1 inhibits Tfn uptake in vivo. tTA-HeLa
The Journal of Cell Biology
776 The Journal of Cell Biology
Volume 162, Number 5, 2003
There are several possible conclusions that can be drawn
based on the inability to detect an effect of AAK1 depletion
2 phosphorylation. One possibility is that AAK1 is not
2 kinase. However, given that AAK1
copurifies with CCVs and colocalizes with endocytic clath-
rin-coated pits in both neuronal and nonneuronal cells
(Conner and Schmid, 2002), and that the AAK1 phosphor-
ylation site on
2 is required for endocytosis in vivo
(Umeda et al., 2000; Ricotta et al., 2002), we think this is
unlikely. Instead, our results may reflect functional redun-
dancy with other kinases of the Ark1/Prk1 family. This
prospect would be consistent with the functional redun-
dancy observed in yeast between the Ark1p and Prk1p pro-
teins that regulate actin dynamics and endocytosis—a yeast
phenotype is only observed following disruption of both
the Ark1 and Prk1 genes (Cope et al., 1999). Indeed, GAK,
another Ark1/Prk1 family member is known to be associ-
ated with CCVs and to phosphorylate
chuk and Banting, 2002; Umeda et al., 2000). Other
AAK1-related kinases also exist in the mammalian genome
(Conner and Schmid, 2002). Moreover, it is also possible
that the levels of AAK1 that remain following siRNA treat-
ment are sufficient to support normal levels of
2 in vitro (Korol-
based internalization motifs, whereas other AP-2 subunits are
believed to function in endocytosis by directing clathrin as-
sembly into curved lattices and by recruiting other essential
cofactors to the coated pit (Kirchhausen, 1999). Therefore,
we expected that clathrin-coated pit assembly would also be
disrupted in cells overexpressing inhibitory AAK1 constructs
that functionally sequester AP-2. Surprisingly, clathrin re-
cruitment into coated pits was not altered in WT AAK1–
overexpressing cells relative to controls (Fig. 4 A). Previous
studies have established that EGF and TfnRs are internalized
in the same coated pits (Lamaze et al., 1993). We therefore
asked if AAK1 overexpression had any effect on EGF uptake.
Surprisingly, neither WT nor K74A AAK1 overexpression
had any effect on the internalization of EGF compared with
controls (Fig. 4 , B and C). High concentrations of EGF are
known to saturate the clathrin-mediated pathway for EGFR
endocytosis (Jiang and Sorkin, 2003); therefore, care was
taken to use low concentrations (2 ng/ml) of
EGF for these assays. As an additional control for CME, cells
infected with recombinant K44A dynamin-1 adenovirus
showed the expected EGF internalization defect (Damke et
al., 1994). We cannot rule out that the small amounts of AP-2
remaining at the cell surface are selectively associated with
2 subunit of AP-2 specifically recognizes tyrosine-
function. (A) Adenovirally-infected tTA
HeLa cells overexpressing WT AAK1,
K74A AAK1, or the tTA were labeled in
vivo with 32P-orthophosphate. AP-2 was
then immunoprecipitated with the mAb
AP.6 and analyzed by SDS-PAGE (top).
Immunoblot (bottom) with antibodies
specific for ?2 (provided by J. Bonifacino,
National Institutes of Health, Bethesda,
MD) indicates equal loading. (B) Quanti-
tation of phosphorylated large adaptin
subunits from whole cell lysates relative
to the tTA control. Data shown are
representative of three independent
experiments. (C) tTA HeLa cells, cultured
in the absence of G418, were infected
with the indicated AAK1 or dynamin
adenovirus constructs. All cells are
virally infected, but only those cells
that retain tTA express the adenovirus-
encoded constructs. Infected cells were
fixed with ice-cold acetone and methanol
extracted before further processing for
immunolocalization of AAK1 using pAbs
against either the ?AID or AID fragment
and the mAb AP.6 that recognizes the
?-adaptin subunit of AP-2. Samples
were visualized by epifluorescence
microscopy using a Zeiss Axiophot with
an attached Zeiss Axiocam. (D) The
distribution of AP-2 in particulate (P)
and soluble (S) fractions, obtained as
previously described (Damke et al.,
1994), was tested in adenovirus-infected
cells by immunoblot analysis, using the
mAb 100/2 (Sigma-Aldrich).
AAK1 globally disrupts AP-2
The Journal of Cell Biology
Cargo selective role for AP-2 |
Conner and Schmid 777
coated pits engaged in EGF uptake. However, our results are
completely consistent with recent findings reporting that
siRNA-mediated AP-2–depleted cells are capable of forming
clathrin-coated pits that are competent for the internalization
of the EGFR and an LDLR chimera, but defective in TfnR
endocytosis (Motley et al., 2003). Thus, we conclude that the
functional sequestration of AP-2 by AAK1 overexpression
demonstrates an unexpected cargo-selective requirement for
this coat constituent in CME.
A currently accepted paradigm for AP-2 function is that,
in addition to cargo recognition, it is essential for the re-
cruitment and assembly of clathrin. However, our results
and recent results of others (Motley et al., 2003) suggest
that the proper localization of AP-2 is not a prerequisite for
the formation of functionally active clathrin-coated pits at
the plasma membrane. Although unexpected, this obser-
vation is also consistent with the finding that overexpres-
sion of mutant epsin, incapable of binding PtdIns(4,5)P
pits are functional for EGF uptake.
(A) WT AAK1-infected tTA HeLa cells
(cultured as described in the legend to
Fig. 3 C) show no defect in the recruitment
of clathrin to the plasma membrane or
its ability to generate coated pits, as
observed by immunolocalization using
the mAb X22. (B) EGF internalization
was tested in tTA HeLa cells infected
with adenoviruses expressing either tTA,
WT, or K74A AAK1, or K44A dynamin-1
adenoviruses, as indicated in the legend.
Protein expression levels were tested by
immunoblot analysis (inset) using pAbs
against either the ?AID AAK1 fragment
(lanes 1–3) or against dynamin (lanes 1
and 4). (C) tTA HeLa cells, cultured on
coverslips and infected with the indicated
adenoviruses, were tested for their ability
to internalize rhodamine-conjugated Tfn
and Alexa-488–conjugated EGF, simul-
taneously (Molecular Probes). Cells were
incubated in the presence of 2 ng/ml
EGF and 4 ?g/ml Tfn for 15 min at 37?C.
Cells were then transferred to ice, washed
to remove unbound ligand, fixed with
acetone, and visualized by epifluores-
cence as described in the legend to Fig. 3.
A significant decrease in Tfn accumulation
in the endosome is observed in WT
AAK1–infected cells relative to AID
AAK1– or control-infected cells (arrows),
whereas EGF uptake is unaffected.
The Journal of Cell Biology
778 The Journal of Cell Biology
Volume 162, Number 5, 2003
causes AP-2 to aggregate in the cytoplasm, having no ap-
parent effect on the punctate distribution of clathrin on
the plasma membrane (Ford et al., 2002). These findings
are also consistent with observations in yeast where clath-
rin functionality is unaffected by the absence of all known
heterotrimeric adaptor proteins (Huang et al., 1999).
Thus, it is probable that AP-2 is not stoichiometrically re-
quired for the assembly of clathrin-coated pits at the
plasma membrane and its major function is in cargo re-
cruitment. We hypothesize that cells use a wider variety of
specialized adaptors for clathrin assembly and cargo recog-
nition than previously thought. What proteins recruit and
promote clathrin assembly? It is likely that other adaptors,
such as AP180/CALM (Morgan et al., 1999; Tebar et al.,
-arrestin (Goodman et al., 1996; Lin et al., 1997),
Dab2 (Mishra et al., 2002), or HIP1R (Metzler et al.,
2001; Mishra et al., 2001) can function independently of
AP-2 to selectively package cargo molecules and trigger
Materials and methods
Protein isolation and antibody production
AAK1 constructs fused to GST were purified from baculovirus-infected Tn5
cells as previously described (Conner and Schmid, 2002). APs, isolated as
previously described (Smythe et al., 1992), were inactivated for endoge-
nous kinase activity by pretreatment with 4 mM FSBA (fluorosulphonyl-
benzoyladenosine; Sigma-Aldrich) for 2 h on ice. Unbound FSBA was re-
moved from protein preparations by two sequential gel filtrations using
G25 mini-spin columns (Amersham Biosciences).
Polyclonal antibodies against the COOH-terminal AID fragment of
AAK1 expressed in
E. coli and the NH
in baculovirus-infected Tn5 cells were generated in this laboratory as pre-
viously described (Conner and Schmid, 2002).
AID fragment expressed
Except where noted, tTA HeLa cells were cultured and infected in the pres-
ence of G418 to maintain expression of the tTA that is required for protein
overexpression. tTA HeLa cells were infected with recombinant adenovi-
ruses as previously described (Altschuler et al., 1998). Viral loads showing
nearly 100% infection and uniform protein overexpression were used, as
determined by immunolocalization. Internalization of biotinylated Tfn was
assayed as previously described (Carter et al., 1993), assessing internaliza-
tion by inaccessibility to avidin. Identical results were obtained by assess-
ing resistance to MesNa (unpubished data). For EGF internalization, virus-
infected cells were serum starved for 1.5–2 h in binding buffer (DMEM,
1% BSA) before the internalization assay. Cells were then detached from
dishes in PBS/5 mM EDTA, rinsed with ice cold binding buffer, resus-
pended in binding buffer containing 2 ng/ml
ice for 1 h. Cells were then washed with binding buffer and aliquoted. One
aliquot was kept on ice to measure total ligand binding, and the rest were
transferred to 37
C for the indicated times, returned to ice to stop endocy-
tosis, and then acid washed (0.5 M NaCl, 0.2 M acetic acid, pH 2.8) for 5
min on ice to remove surface bound ligand. Cells were pelleted, the super-
natant containing released ligand was aspirated, and the samples were
measured for internalization with a gamma counter.
I-EGF, and incubated on
Kinase assays and in vitro protein interaction tests were performed essen-
tially as described (Conner and Schmid, 2002). In vivo labeling and immu-
noprecipitation of AP-2 was performed as described using the mAb AP.6
(Wilde and Brodsky, 1996).
Online supplemental material
Fig. S1 shows immunofluorescence assays for RAP-GST endocytosis in
control and WT AAK1–overexpressing cells. Fig. S2 shows the time course
of siRNA-mediated AAK1 reduction by immunoblot analysis as well as sin-
gle round Tfn internalization assays after siRNA treatment with control and
AAK1-specific oligonucleotides. Supplemental materials and methods in-
clude information regarding AAK1 site-directed mutagenesis, the genera-
tion of AAK1 adenovirus and baculovirus constructs, RAP internalization,
and siRNA treatments. All supplemental material is available at http://
We thank Davin Henderson for help in the generation of adenovirus and
baculovirus constructs, Tricia Glen and Alisa Jones for facilitating protein
production, Miwako Ishido for help with GST-RAP isolation, and Marc Sy-
mons for siRNA advice.
S.L. Schmid and S.D. Conner were supported by National Institutes of
Health grants (R37-MH61345 and GM20632-01, respectively). This is The
Scripps Research Institute manuscript number 15361-CB.
Submitted: 14 April 2003
Accepted: 1 August 2003
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