JOURNAL OF VIROLOGY, Oct. 2007, p. 10437–10450
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 19
A Tyrosine-Based Signal Plays a Critical Role in the Targeting and
Function of Adenovirus RID? Protein?
Nicholas L. Cianciola,1Denise Crooks,1,2† Ankur H. Shah,1,2and Cathleen Carlin1,2,3*
Department of Physiology and Biophysics,1Molecular Virology Training Program,2and
Case Western Reserve University Cancer Center,3School of Medicine,
Case Western Reserve University, Cleveland, Ohio 44106-4970
Received 23 February 2007/Accepted 9 July 2007
Early region 3 genes of human adenoviruses contribute to the virus life cycle by altering the trafficking of
cellular proteins involved in adaptive immunity and inflammatory responses. The ability of early region 3 genes
to target specific molecules suggests that they could be used to curtail pathological processes associated with
these molecules and treat human disease. However, this approach requires genetic dissection of the multiple
functions attributed to early region 3 genes. The purpose of this study was to determine the role of targeting
on the ability of the early region 3-encoded protein RID? to downregulate the EGF receptor. A fusion protein
between the RID? cytoplasmic tail and glutathione S-transferase was used to isolate clathrin-associated
adaptor 1 and adaptor 2 protein complexes from mammalian cells. Deletion and site-directed mutagenesis
studies showed that residues 71-AYLRH of RID? are necessary for in vitro binding to both adaptor complexes
and that Tyr72 has an important role in these interactions. In addition, RID? containing a Y72A point
mutation accumulates in the trans-Golgi network and fails to downregulate the EGF receptor when it is
introduced into mammalian cells as a transgene. Altogether, our data suggest a model where RID? is trafficked
directly from the trans-Golgi network to an endosomal compartment, where it intercepts EGF receptors
undergoing constitutive recycling to the plasma membrane and redirects them to lysosomes.
Adenoviruses (Ads) are nonenveloped DNA viruses that
replicate and assemble in the host cell nucleus (13). Ads are
responsible for approximately 5% of acute upper respiratory
tract (3) and 15% of lower respiratory tract (1) infections in
infants and children. Similar to other DNA viruses, Ads are
also capable of establishing persistent infections, because they
have evolved numerous strategies to evade host antiviral sur-
veillance mechanisms. Some of these same mechanisms also
facilitate survival of the virus during an acute infection. Thus,
a thorough understanding of viral genes that control host im-
mune and inflammatory responses is fundamental to our ability
to prevent and treat virus-induced disease at multiple levels.
These mechanisms have also influenced strategies for design-
ing Ad-based vectors for gene therapy (14).
The early region 3 (E3) transcription genes of human Ads
encode several proteins that exploit the intracellular trafficking
machinery to modify host immune or inflammatory responses
(11, 29). Thus, E3 proteins have served as novel probes of
membrane transport mechanisms in addition to providing in-
sights to Ad pathophysiology. The most abundant E3 protein,
E3/19K, suppresses the host adaptive immunity response by
retaining class I MHC molecules in the endoplasmic reticulum.
Other E3 proteins affect the functions of molecules involved in
proliferation and apoptosis, intracellular cell signaling events
linked to NF-?B, and secretion of proinflammatory chemo-
kines. Two E3 proteins, RID? (also called E3-10.4 and E3-
13.7) and RID? (also called E3-14.5), that act to remove sev-
eral receptors from the surface of host cells, including death
receptors FAS and TRAIL, tumor necrosis factor receptor 1
(TNFR1), and EGF receptor (EGFR), have been identified.
These two E3 proteins have independent functions and are
also capable of forming a molecular complex that has been
named the receptor internalization and degradation (RID)
complex. However, mutations that affect the ability of the RID
complex to downregulate one receptor molecule have no effect
on expression with a second class of receptors, suggesting that
regulation of different targets by the RID complex involves
multiple mechanisms (9).
RID? is a type II membrane protein expressed by all human
Ad serotypes (27). Its basic structural organization consists of
a short N-terminal cytosolic domain (?4 residues), two trans-
membrane domains connected by an exocytic loop domain
(?17 residues), and a C-terminal cytosolic tail domain (?28
residues). We have shown previously that RID? is sufficient to
downregulate the EGFR (25) and that it localizes to the en-
docytic pathway, where it diverts EGFRs in a constitutive re-
cycling loop to lysosomes (10). However, other laboratories
have reported that RID? functions only in the context of the
RID complex that localizes to the plasma membrane, where it
regulates EGFR internalization (43). Although the precise
role of EGFR downregulation during an Ad infection is not
completely understood, the EGFR has been linked to TNF-?-
dependent NF-?B activity (22) and to the release of proinflam-
matory chemokines (38) in other biological settings.
The RID? cytosolic tail has several consensus sorting signals
that likely engage the host cell machinery necessary for its
* Corresponding author. Mailing address: Department of Physiol-
ogy and Biophysics, Case Western Reserve University School of Med-
icine, 10900 Euclid Avenue, Cleveland, OH 44106-4970. Phone: (216)
368-8939. Fax: (216) 368-3952. E-mail: firstname.lastname@example.org.
† Present address: Technology Transfer Branch, National Cancer
Institute, Executive Plaza South, Suite 450, 6120 Executive Boulevard,
Bethesda, MD 20892.
?Published ahead of print on 18 July 2007.
localization and function. The goal of this study was to identify
these sorting signals and to disable them by site-directed mu-
tagenesis. The study focused on potential interactions with
clathrin adaptor protein (AP) complexes involved in mem-
brane protein sorting at the level of the trans-Golgi network
(TGN) (AP class 1 [AP-1]) and the plasma membrane (AP-2)
(42). Our results indicate that RID? residues 71-AYLRH bind
to both classes of APs. In addition, we show that a Y72A point
mutation traps RID? in the TGN but not the plasma mem-
brane, suggesting that the Ad protein encounters AP-1 first in
the biosynthetic pathway. Our data also support a model where
AP-2 serves as a quality control function to capture RID? mol-
ecules that leak to the plasma membrane and redirect them back
to endosomes, similar to many other membrane cargoes with
overlapping AP binding sites (2). These studies were carried out
with RID? transgenes, confirming the original Ad mapping stud-
ies that demonstrated the ability of this protein to modulate
EGFR trafficking independent of other E3 proteins (7).
MATERIALS AND METHODS
Cell lines, antibodies, Ad stocks, and transfections. Human hepatocellular
carcinoma-derived N-PLC-PRF/5 cells (6) and fetal lung-derived WI-38 cells
(20) were maintained in minimal essential medium (MEM). Human epithelial-
derived A431 carcinoma cells (12) and human embryonic kidney 293 (HEK-293)
cells (16) were maintained in Dulbecco’s modified MEM. Chinese hamster ovary
(CHO) cells (40) were maintained in MEM alpha modification. Human lung
carcinoma-derived A549 cells (12) were maintained in Ham’s F12 medium. All
media were supplemented with 10% fetal bovine serum and 2 mM glutamine.
Cells were grown at 37°C with 5% CO2and were subcultured with 0.25%
trypsin-0.1% EDTA in phosphate-buffered saline (PBS).
The following antibodies were used: ?-adaptin, ?-adaptin, FLAG M2, and
FLAG-BioM2 monoclonal antibodies from Sigma (St. Louis, MO); transferrin
receptor (TfR) monoclonal antibody from Zymed (San Francisco, CA); the
human-specific EGFR EGF-R1 monoclonal antibody (46); RID? rabbit poly-
clonal antibody produced with a synthetic peptide corresponding to residues 76
to 91 (27); furin convertase rabbit polyclonal antibody from ABR (Golden, CO);
glutathione S-transferase (GST) goat polyclonal antibody from Amersham-Phar-
macia (Piscataway, NJ); and Golgi gp125 antibody obtained from the Develop-
mental Studies Hybridoma Bank developed under the auspices of the National
Institute of Child Health and Human Development and maintained by the
Department of Biological Sciences, The University of Iowa, Iowa City, IA.
Secondary antibodies were purchased from Amersham-Pharmacia (horseradish
peroxidase [HRP]) or Jackson ImmunoResearch (fluorescent and HRP; West
Ad stocks were grown in HEK-293 cells, and titers were determined by plaque
assay, using standard techniques. The Ad2-Ad5 recombinant Ad mutant that
overproduces the RID? protein has been described elsewhere (7). Cells were
acutely infected with approximately 200 PFU per cell, according to established
CHO cells were transiently transfected using TransIt-CHO (Mirus, Madison,
WI) as directed by the manufacturer, and HEK-293 cells were transfected using
FuGENE 6 (Roche, Basel, Switzerland) following the manufacturer’s instruc-
Cloning and mutagenesis. Wild-type and modified RID? carboxyl-terminal se-
quences were subcloned into pGEX-3X (Amersham-Pharmacia) as follows. RID? res-
idues Asp64 to Leu91 (see Fig. 1A) were subcloned in the M13mp18 single-stranded
DNA phage vector (New England BioLabs, Ipswich, MA). Y72A and Y79A point
mutations were introduced using the M13mp18 RID? template, a Sculptor in vitro
mutagenesis system (Amersham-Pharmacia), and the following mutagenic primers to
introduce tyrosine-to-alanine substitutions (in boldface type): 5?-TGTGCGCATTGCG
GCCCTCAGGCACCAT-3? for Y72A, and 5?-GCACCATCCGCAAGCCAGAGAC
a forward primer (5?-CAUCAUCAUCAUGATATCGACTGGGTTTGTGTGCGCAT
TGC-3?) designed to anneal to the codon for Asp64 (in boldface type) and a reverse
TTTTCG-3?) to anneal to M13mp18 vector sequences 3? to the polylinker region.
Both primers incorporate EcoRV restriction sites (underlined) and also have
noncomplementary dU repeats on the 5? ends (in italics). The dU repeats in the
PCR products were depurinated with uracyl DNA glycosidase to expose single-
stranded 3? ends and enable base-pairing to complementary ends in the linear
shuttle vector pAMP (Invitrogen, Gaithersburg, MD). EcoRV fragments were
subcloned in frame at the SmaI site in pGEX-3X, which reconstituted the RID?
Ile63 codon. The GST fusion plasmid encoding RID? carboxyl-terminal se-
quences truncated to residues Tyr70 or Tyr79 were prepared using PCR and
full-length RID? cloned in the pcDNA1/Amp eukaryotic expression vector
(Clontech, Mountain View, CA) as a template. Sequences were amplified with a
forward primer 5?-CTAACTAGAGAACCCACTGC-3? designed to anneal to
vector sequences 5? to the RID? coding region, and reverse mutagenic primer
5?-AGTGGGATCCTAGTATTGCGGGTGGTGCCTGA-3? to incorporate an
A71Stop codon or 5?-CCTAGGATCCTAAATGCGCACACAAACCCA-3? to
incorporate an R80Stop codon, respectively (both stop codons are in boldface
type). The reverse mutagenic primers also incorporated BamHI sites (under-
lined) compatible with the pcDNAI/Amp polylinker. The cytoplasmic tail se-
quences were amplified using forward (5?-GTTTCCCGGGATTGACTGGGTT
TGTG-3?) and reverse (5?-GAGTCCCGGGTTGAGAGTCAGCAGTAGC-3?)
primers complementary to Ile63 and sequences in pcDNAI/Amp 3? to the RID?
coding region, respectively. Both primers incorporate SmaI sites (underlined) to
facilitate in-frame cloning at the SmaI site in the pGEX-3X polylinker region.
The GST fusion plasmid encoding RID? carboxyl-terminal sequences truncated
to residue His75 was created using an overlap PCR extension technique, the
RID?/pcDNA1/Amp template, and the following four primers. Primers 1 and 4
are forward and reverse flanking primers that anneal to unique restriction en-
zymes in the polylinker region at the 5? and 3? ends of the RID? coding region
that were already described, and primers 2 and 3 are forward (5?-ACCTCAGG
CATAACCGCAATACA-3?) and reverse (5?-CTGTATTGCGGTTAGTGCCT
GAG-3?) mutagenic primers that replace His76 with a stop codon (in boldface
type). The first round of PCRs were carried out with primers 1 and 3 or primers
2 and 4, creating two overlapping PCR products. Both fragments were gel
purified and used as templates for a second PCR with the flanking primers to
generate a 627-bp product with the H76Stop mutation. This fragment was in-
serted into pcDNA1/Amp using restriction enzymes incorporated in the ends of
the product compatible with restriction sites in the polylinker region. Carboxyl-
terminal sequences encoding the H76Stop were amplified and subcloned in
pGEX-3X exactly as described for the A71Stop and R80Stop constructs.
The cDNAs encoding full-length wild-type RID? and RID? with a single Y72A
or Y79A amino acid substitution for expression in eukaryotic cells were constructed
using forward (5?-ATCGTAAAGATCTTGATTCCTCGAGTTCTTATATTATT
G-3?) and reverse (5?-CTAAGATCTCCTTAAAGAATTCTGAGAAGATCAGC
TATAGTCCTG-3?) primers to amplify the RID? open reading frame and incor-
BglII and ligated to an amino-terminal FLAG epitope in the polylinker region of the
pExchange2 plasmid (Stratagene, La Jolla, CA) digested with the same restriction
enzyme. The Y72A and Y79A point mutations were incorporated into pExchange2/
RID? using a QuikChange XL site-directed mutagenesis kit (Stratagene), and PCR
primers 5?-GTGTGCGCATTGCGGCCCTCAGGCACCATC-3? (forward) and 5?-
GATGGTGCCTGAGGGCCGCAATGCGCACAC-3? (reverse) (Y72A substitu-
tion in boldface type), or 5?-AGGCACCATCCGCAAGCCAGAGACAGGACTA
TAG-3? (forward) and 5?-CTATAGTCCTGTCTCTGGCTTGCGGATGGTGCC
T-3? (reverse) (Y79A substitution in boldface type), according to the manufacturer’s
PCR primers were designed using the DNASTAR software package
(DNASTAR, Inc., Madison, WI). PCR amplifications were carried out using a
RoboCycler 40 temperature cycler (Stratagene, La Jolla, CA). All PCR products
and religated recombinant products were sequenced by automated DNA se-
quencing (Cleveland Clinic Foundation Genomics Core, Cleveland, OH).
Fusion proteins. GST fusion proteins were purified from BL21 cells that were
freshly transformed with pGEX-3X plasmids. Bacteria were cultured at 37°C to
an optical density at 600 nm of approximately 0.6, induced with 0.1 mM isopro-
pyl-?-D-thiogalactopyranoside (IPTG) for 16 h at room temperature, and col-
lected by low-speed centrifugation. Cells were subjected to one freeze-thaw cycle,
resuspended in a solution of 50 mM Tris (pH 7.7), 0.1 M NaCl, 0.2 mM EDTA,
and protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride and 1 ?M leu-
peptin), and then digested with lysozyme (0.1 mg/ml) for 1 h at room tempera-
ture. MgSO4was added to a final concentration of 3 mM, and bacterial lysates
were digested for an additional 1 h at room temperature with 0.02 mg (each) of
DNase and RNase. Lysates were adjusted to pH 7.4 and incubated with 1.5%
L-lauryl sarcosine to solubilize inclusion bodies for 15 min on ice, followed by
centrifugation at 12,000 ? g for 10 min at 4°C. Supernatants were adjusted to 3%
Triton X-100 and incubated with glutathione-Sepharose beads (Amersham-
Pharmacia) for 20 min at 4°C with rotation. Beads with attached fusion proteins
10438 CIANCIOLA ET AL.J. VIROL.
were washed three times with a solution of 50 mM Tris (pH 7.4), 10 mM MgCl2,
and 1% Triton X-100 supplemented with 0.4 M NaCl (high-salt wash) and twice
with the same solution supplemented with 0.15 M NaCl (low-salt wash). Beads
with attached fusion proteins were incubated with crude subcellular fractions
enriched for clathrin adaptors, by using the method described in the next para-
graph, for 20 min at 4°C, and the beads were washed three times with the
high-salt solution and then twice with the low-salt solution. Fusion protein
complexes were solubilized with Laemmli buffer and resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for immunoblotting us-
ing standard methods.
Cell fractionation methods. Crude subcellular fractionation was carried out
essentially as described in reference 41. Briefly, cells were rinsed twice and then
scraped in PBS supplemented with 2 mM EDTA, 5 mM EGTA, and protease
inhibitors. Cells were resuspended in 0.1 M morpholineethansulfonic acid (MES;
pH 6.5), 0.2 M EDTA, 0.5 mM MgCl2, 0.02% NaN3, 10 mg/ml bovine serum
albumin, and protease inhibitors and then lysed with 1% NP-40 for 5 min at room
temperature. Postnuclear supernatants were centrifuged at 60,000 ? g for 30 min
at 4°C in an Optima TL Ultracentrifuge (Beckman Instruments, Inc., Palo Alto,
CA) to collect the supernatant corresponding to crude cytosol. The resulting
membrane pellet was resuspended in the NP-40 lysis buffer and incubated with
0.5 M Na2CO3for 5 min on ice to release peripheral membrane proteins. The
mixture was then centrifuged at 50,000 ? g for 20 min at 4°C, and the peripheral
membrane protein-enriched supernatant was incubated with 0.5 M KH2PO4for
1 h on ice. Integral membrane proteins were solubilized by incubating membrane
pellets with radioimmunoprecipitation assay (RIPA) detergent (1% NP-40, 0.5%
sodium deoxycholate, and 0.1% SDS) in 50 mM Tris, pH 8.0, supplemented with
150 mM NaCl, 2 mM EDTA, 5 mM EGTA, and protease inhibitors, for 30 min
Cell homogenates were also fractionated on Percoll (Amersham-Pharmacia)
gradients essentially as described in reference 18. Briefly, cells were rinsed twice
with PBS supplemented with 2 mM EDTA and 5 mM EGTA and then scraped
in ice-cold homogenization buffer (HB) consisting of 10 mM HEPES (pH 7.5),
0.25 M sucrose, 1 mM EDTA, and protease inhibitors. Cells were collected by
centrifugation, resuspended in HB, and homogenized with 22 strokes of a
Dounce homogenizer. The homogenate was diluted with an equal volume of
fresh HB and centrifuged at 400 ? g for 10 min at 4°C to pellet unbroken cells
and nuclei. Postnuclear supernatants were adjusted to a final concentration of
27% Percoll in 0.25 M sucrose using a 90% Percoll stock solution and then
layered over a 1-ml sucrose cushion consisting of 10? HB. Gradients were
centrifuged for 90 min at 25,000 ? g in an SS34 fixed-angle rotor (Sorvall
Instruments, Newtown, CT) without braking. A total of nine 1.2-ml fractions
were collected manually, starting from the top of the gradient. Fractions were
analyzed by comparing equal aliquots of total cellular protein determined by
Bradford assay (Bio-Rad Laboratories, Hercules, CA) or by immunoprecipita-
tion after membranes were solubilized with the RIPA lysis buffer and centrifuged
at 100,000 ? g for 30 min at 4°C in a TL 100.3 fixed-angle rotor (Beckman
Instruments) to precipitate Percoll. Alternatively, fractions were assayed for
?-hexosaminidase activity by incubating 20 ?g of protein in a solution of 0.1 M
MES (pH 6.5), 1 mM p-nitrophenyl-?-D-glucosaminide, and 0.2% Triton X-100
for 90 min at 37°C. The reaction was quenched with 0.5 M glycine, pH 10, and
absorbance was read at 405 nm using a model 3550 automatic microplate reader
(Bio-Rad). Gradients were also collected from cells that had been incubated with
125I-EGF (50 ng/ml, 150 ?Ci/?g; PerkinElmer-NEN) or125I-transferrin (200
ng/ml, 0.5 ?Ci/?g; PerkinElmer-NEN) to label plasma membrane receptors.
Fractions from these gradients were analyzed by gamma counting (Cobra Auto-
Gamma counter; Packard Instruments, Meriden, CT).
Immunoprecipitations and immunoblotting. Immunoprecipitations were car-
ried out using antibodies adsorbed to protein A-Sepharose CL-4B beads
(Sigma), or streptavidin agarose (Pierce, Rockford, IL). Immune complexes were
centrifuged at 14,000 ? g for 15 min at 4°C, washed five times with lysis buffer,
solubilized with Laemmli buffer, and resolved by SDS-PAGE using standard
methods. Immunodepleted supernatants were centrifuged a second time to en-
sure removal of immune complexes before they were added to GST fusion
protein pull-down assays. Gels with radioactive proteins were treated with
En3Hance (PerkinElmer-NEN, Wilmington, DE) for fluorography. Immuno-
blotting was carried out using proteins transferred to nitrocellulose membranes
by standard methods. Nitrocellulose filters were incubated with primary antibod-
ies and the appropriate HRP-conjugated secondary antibodies (Amersham Life
Sciences, Inc., Arlington, IL; Jackson ImmunoResearch Laboratories, Inc.) for
detection by enhanced chemiluminescence (Amersham Life Sciences). For quan-
tification, immunoblots were incubated with125I-labeled goat anti-mouse sec-
ondary antibody (1 ? 106cpm/ml, 8.07 ?Ci/?g; PerkinElmer-NEN) for 1 h at
room temperature. Blots were air-dried after extensive washing, and radiola-
beled proteins were quantified by phosphor storage autoradiography. Digitized
images were analyzed using the ImageQuant software package (Molecular Dy-
namics, Sunnyvale, CA), which averages five measurements of light emission for
each pixel location, to give a pixel value that is proportional to the amount of
Confocal microscopy. HEK-293 cells or CHO cells transiently transfected with
RID? expression plasmids were prepared for staining essentially as described in
reference 8. Briefly, cells were permeabilized with 0.5% ?-escin in a solution of
80 mM piperazine-N,N?-bis(2-ethanesulfonic acid) (PIPES), pH 6.8, supple-
mented with 5 mM EGTA and 1 mM MgCl2for 5 min and then fixed with 3%
paraformaldehyde-PBS for 15 min. Cells were stained with primary antibody or
secondary antibodies and 4?,6?-diamidino-2-phenylindole (DAPI) for 45 min at
room temperature. Antibodies were diluted in a solution containing 0.5% ?-
escin and 3% radioimmunoassay-grade bovine serum albumin and were blocked
with a solution containing 1% normal serum from the host animal used to
generate the secondary antibody. Cells were examined with a Leica TCS SP2 with
AOBS confocal microscope (Leica, Mannheim, Germany) using the 405-nm
wavelength line of a UV laser and the 488/568-nm lines of an argon-krypton
laser. Image resolution using a Leica 100?, 1.4-numerical-aperture oil immer-
sion lens and Leica LCS software was 512 by 512 pixels. Some cells were
pretreated with nocodazole (100 ?M; Sigma) or vehicle (dimethyl sulfoxide) for
30 min prior to staining. Phase contrast images were collected, and the outline of
the cells was drawn using Metamorph (Molecular Devices, Sunnyvale, CA) and
overlaid onto the respective confocal image.
Surface reduction of extracellular disulfide bonds. Cells were pulse-labeled
for 1 h with L-[35S]cysteine (50 mCi/ml, 1,075 Ci/mmol; PerkinElmer-NEN) in
cysteine-free MEM supplemented with 10% dialyzed fetal bovine serum and
0.2% bovine serum albumin and then incubated in chase medium (complete
Dulbecco’s MEM supplemented with 500 ?M nonradioactive cysteine) for 3 h to
allow proteins to achieve steady-state localization. Radiolabeled cells were in-
cubated twice (25 min/incubation) with an ice-cold solution of 80 mM L-cysteine,
75 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.5 N NaOH, and 1% fetal bovine
serum to reduce external disulfide bonds of surface proteins (36). Cells were
rinsed twice with PBS containing iodoacetamide (1 mg/ml) and then incubated
with the RIPA lysis buffer supplemented with iodoacetamide (1 mg/ml) and
protease inhibitors for analysis by immunoprecipitation. Immunoprecipitates
were solubilized in Laemmli buffer supplemented with 1 mg/ml iodoacetamide
and separated by SDS-PAGE under reducing (0.1 M dithiothreitol [DTT]) or
nonreducing (no DTT) conditions.
EGFR protein stability assay. CHO cells, which lack endogenous EGFRs,
were cotransfected with plasmids encoding human EGFR (23) and FLAG-
tagged wild-type or Y72A or Y79A RID? plasmids or empty vector. Cells were
radiolabeled with [35S]cysteine for 30 min starting at 48 h posttransfection as
described previously. Cells were harvested either immediately or following a 5-h
chase in medium supplemented with a 10-fold excess of nonradioactive cysteine.
Cells were lysed with RIPA buffer and analyzed by immunoprecipitation, SDS-
PAGE, and fluorography.
The C-terminal cytoplasmic tail of RID? interacts with AP
complexes. We have shown previously that RID? localizes to
the endocytic pathway, where it redirects constitutively recy-
cling EGFRs to lysosomes in Ad-infected cells (10, 26). The
RID? cytoplasmic tail contains a number of putative sorting
motifs that may regulate these sorting properties (2), including
a dileucine motif at residues 87-LL and two possible tyrosine-
based sorting motifs containing Tyr72 and Tyr79, respectively
(Fig. 1A). To determine whether the RID? cytoplasmic tail
interacts with clathrin AP complexes involved in membrane
protein sorting, we utilized a fusion protein consisting of the
30-amino-acid cytoplasmic domain of RID? fused to GST
(characterized in Fig. 1B) to pull down complexes from mam-
malian cell extracts. The mammalian cell fractionation scheme
involved purification of a peripheral membrane protein frac-
tion to enrich for functional AP complexes according to the
method described in reference 41. Figure 1C validates this
purification scheme, showing that ?-adaptin and ?-adaptin
VOL. 81, 2007RID? TARGETING AND FUNCTION 10439
(subunits of AP-2 and AP-1, respectively) are both enriched in
the peripheral membrane fraction, but not in integral mem-
brane fractions that include both of the known molecular
weight forms (e.g., 13.7-kDa and 11.3-kDa) of the RID? pro-
tein. A small amount of each of these adaptin molecules was
also present in crude cytosol, as expected for AP complexes
that cycle on and off membranes. Thus, the crude cytosol and
peripheral membrane protein fractions were mixed with GST
or the C-terminal fusion protein, precipitated with glutathione
agarose, washed, and subjected to SDS-PAGE and immuno-
blot analysis with monoclonal antibodies to ?-adaptin and
?-adaptin. As shown in Fig. 1D, ?-adaptin and ?-adaptin from
a human hepatocellular cell line were both coprecipitated with
the C-terminal fusion protein in contrast to GST alone. Similar
results were obtained using peripheral membrane protein frac-
tions from a number of other cell lines, including epithelial
carcinoma-derived A431 cells and normal human diploid
WI-38 fibroblasts (Fig. 1E) and human lung carcinoma-derived
A549 cells and HEK cells (data not shown).
Analysis of RID? cytoplasmic tail mutants. Our next goal
was to identify the sequence(s) within the cytoplasmic tail of
RID? that interacts with each of the AP complexes identified
FIG. 1. Binding of RID? cytosolic tail to AP complexes. (A) Schematic showing membrane topology of RID? and the complete amino acid
sequence (in single-letter code) of the carboxyl terminus (Ile63-Leu91) that was linked to GST for expression in Escherichia coli. Tyr72 and Tyr79
or residues that were changed to stop codons (Fig. 2) are highlighted in gray or black, respectively. Nuclear magnetic resonance studies have shown
that residues 87-LLRIL comprise an amphipathic helix stabilized by interactions with membrane mimetic micelles (45). Two additional residues
are presented in the exocytic loop connecting the two membrane spanning helices: Ala23, comprising the amino terminus of the 11.3-kDa cleaved
form of the protein produced cotranslationally by signal peptidase, and Cys31, which forms intermolecular disulfide bonds (27). (B) GST or the
C-terminal (C-Term) fusion protein was purified from E. coli by glutathione affinity chromatography and resolved by SDS-PAGE for detection by
Coomassie staining or by immunoblotting (IB) with GST or RID?-specific antibodies. (C) Ad-infected cells were fractionated into crude cytosol
(Cyt) and peripheral (Peri) and integral membrane (IM) proteins, as described in Materials and Methods. Equal aliquots of total cell equivalents
were resolved by SDS-PAGE and immunoblotted with antibodies to RID?, ?-adaptin, or ?-adaptin. (D) GST or C-terminal fusion proteins were
incubated with crude cytosol or peripheral membrane proteins isolated from N-PLC-PRF/5 cells, and bound proteins were immunoblotted with
adaptin-specific antibodies. (E) Fusion proteins were incubated with peripheral membrane proteins from A431 or WI-38 cells and analyzed as
described for panel D. Molecular weight standards: ?-galactosidase, 116,300; phosphorylase B, 97,400; carbonic anhydrase, 30,000; lysozyme,
14,400; aprotinin, 6,000.
10440CIANCIOLA ET AL.J. VIROL.
in the first set of experiments. As already noted, RID? con-
tains putative dileucine and tyrosine motifs that could be in-
volved in adaptor complex formation. Thus, we constructed a
series of fusion proteins with cytoplasmic tail truncations that
are shown in Fig. 2A and B to assess a role for each of these
putative signals. The fusion protein with an R80Stop substitu-
tion has the carboxyl-terminal dileucine motif deleted, the
H76Stop has the distal tyrosine residue Tyr79 deleted, and the
A71Stop has all putative sorting motifs deleted. In vitro pull-
down assays with these fusion proteins revealed that RID?
residues 71-AYLRH are responsible primarily for binding both
types of AP complexes (Fig. 2D and E). These data also show
that the carboxyl-terminal dileucine motif does not bind APs
directly, contradicting published reports from another labora-
tory (21). Consistent with the results shown here, we have also
observed that the conserved 679-LLRIL sequence localized to
the EGFR juxtamembrane region that regulates ligand-in-
duced postendocytic sorting to lysosomes (32, 33) does not
bind APs in vitro (data not shown).
The AP binding site identified in the in vitro mapping stud-
ies has one tyrosine at residue Tyr72. To assess its role in AP
binding, fusion proteins were constructed with Y72A as well as
Y79A point mutations incorporated into the full-length cyto-
plasmic tail (Fig. 2C). These fusion proteins were incubated
with peripheral membrane protein fractions, and adaptin bind-
ing was quantified by immunoblotting with125I-labeled second-
ary antibodies and phosphor storage autoradiography after the
bound proteins were resolved by SDS-PAGE and transferred
to nitrocellulose filters. Results from one set of experiments
are summarized in Table 1, and similar results were obtained
in three separate determinations. We found that the Y72A
substitution was associated with reduced binding to both AP-1
and AP-2, although its effect on AP-2 binding was consistently
greater than that on AP-1 binding. Interestingly, the Y79A
substitution adjacent to the primary AP binding site identified
in Fig. 2 was associated with increased binding to AP-2 but
reduced binding to AP-1, suggesting that residues outside of
the binding motif may be important for AP interactions.
AP-1 and AP-2 bind to the RID? cytoplasmic tail with dis-
tinct properties. Results presented to this point suggest that
FIG. 2. Mapping the sites of interaction of RID? with AP complexes using truncation mutants. (A) Schematic showing sequences of
cytoplasmically truncated GST fusion proteins used in the mapping studies. Critical residues in putative sorting signals are highlighted by a black
background. (B and C) Proteins were purified from E. coli by glutathione affinity chromatography and resolved by SDS-PAGE for detection by
Coomassie staining. Proteins in panel B were used in panels D to E of this figure, and those in panel C were used to obtain the data in Table 1.
(D and E) Fusion proteins shown in panel B were incubated with peripheral membrane proteins isolated from N-PLC-PRF/5 cells, and bound
proteins were immunoblotted (IB) with adaptin-specific antibodies. C-Term, C terminal.
VOL. 81, 2007RID? TARGETING AND FUNCTION 10441
AP-1 and AP-2 bind overlapping but distinct regions in the
RID? cytoplasmic tail. To further test this hypothesis, studies
were carried out to determine whether there is competition
between AP-1 and AP-2 for binding to the C-terminal fusion
protein. These studies were possible because the ?-adaptin
antibody immunoprecipitates intact AP-1 complexes com-
prised of all four of the protein subunits (Fig. 3A). Thus,
peripheral membrane protein fractions were immunodepleted
for AP-1 prior to incubation with GST or the C-terminal fusion
protein. AP-2 binding was essentially identical when the C-
terminal fusion protein was incubated with cell fractions that
were precleared with an isotype-matched nonspecific immuno-
globulin G versus the ?-adaptin specific antibody; in compar-
ison, AP-1 binding was substantially reduced in the immunode-
pleted samples in the same experiment (Fig. 3B). Although the
reverse experiment was not possible since immunoprecipita-
tions with the ?-adaptin antibody must be carried out under
denaturing conditions, these results strongly suggest that AP-1
and AP-2 binding to the RID? cytoplasmic tail is noncompet-
Figure 3D provides further evidence that AP-1 and AP-2
bind to RID? by distinct mechanisms. This experiment was
carried out by incubating cell fractions with increasing amounts
of the C-terminal fusion protein, followed by quantitative im-
munoblotting for ?-adaptin and ?-adaptin binding exactly as
described for Table 1. Data are represented as the percent of
total adaptin protein detected in combined crude cytosol and
peripheral membrane fractions that were also quantified by
phosphor storage autoradiography. This experiment demon-
strates that ?-adaptin binding is saturated rapidly compared to
?-adaptin. To evaluate the relative importance of ionic inter-
actions in the binding of AP complexes to the cytosolic tail of
RID?, the in vitro binding assay was carried out in the pres-
ence of increasing concentrations of salt or by changing the pH
of the binding solution. Ionic interactions are highly sensitive
to changes in salt or pH, while hydrophobic interactions are
not. Binding of ?-adaptin and ?-adaptin to RID? cytosolic tail
sequences was not appreciably affected by changes in salt con-
125I-labeled secondary antibodies and
centration (Fig. 3E) or in pH (data not shown), suggesting that
these protein-protein interactions are enabled primarily by hy-
drophobic amino acids with nonpolar side-chains. Thus, Ala71
and/or Leu73 in the 71-AYLRH motif may be important for
AP binding; in addition, hydrophobic residues in the surround-
ing tail sequences may not be necessary but nevertheless con-
tribute to the overall strength of AP binding.
The Y72A point mutation alters subcellular localization of
RID? in mammalian cells. The Y72A and Y79A substitutions
were engineered into the full-length viral protein to study their
expression and localization in mammalian cells. Wild-type and
mutant proteins were transiently expressed in HEK-293 cells,
and subcellular localization was determined by collecting con-
focal images of cells stained for the viral protein and then
costained for furin or the TfR, which are well-characterized
markers of the biosynthetic and endocytic compartments, re-
spectively. The wild-type protein was largely excluded from the
biosynthetic compartment defined by furin that is enriched in
the TGN (Fig. 4A) and was found to colocalize with the TfR
(Fig. 4B), a marker for endocytic vesicles as previously shown
(10). The Y72A mutant, however, colocalized almost exclu-
sively with the TGN marker furin (Fig. 4A) and did not colo-
calize with the TfR (Fig. 4B), consistent with the idea that the
Y72A mutation blocks exit from the biosynthetic compart-
ment. Similar to the wild type, the Y79A mutant did not co-
localize with furin (Fig. 4A), but it partially colocalized with
the TfR (Fig. 4B). This result indicates that while the Y79A
mutation does affect AP-1 binding in vitro (Table 1), the pro-
tein is able to efficiently exit the biosynthetic compartment.
The subcellular distribution of wild-type and mutant pro-
teins was confirmed by cell fractionation of membrane com-
partments on Percoll gradients. Fractions were initially char-
acterized by quantifying radioactivity in fractions from cells
that were incubated with125I-EGF or125I-transferrin to label
plasma membrane receptors, by assaying fractions for ?-hex-
osaminidase activity enriched in lysosomes (Fig. 5A), and by
immunoblotting with antibodies to well-characterized markers
of biosynthetic (gp125) and endocytic (TfR) compartments
(Fig. 5B). Based on these analyses, we concluded that biosyn-
thetic compartments are concentrated in fractions 2 through 5,
the plasma membrane is enriched in fractions 1 and 2, and
TfR-positive endosomes are concentrated in fractions 2 and 3.
Both molecular weight forms of the wild-type protein exhibited
a subcellular distribution that is most similar to TfR in light
fractions 2 and 3, and distribution of the mutant protein was
most similar to the Golgi glycoprotein gp125 (Fig. 5C). Thus,
these data are consistent with the images obtained by confocal
microscopy showing that the wild-type protein localizes to an
endocytic compartment, compared to the mutant protein,
which localizes to the biosynthetic pathway.
In contrast to results shown here and published elsewhere
(10, 24, 26, 28), other laboratories have reported that RID? is
localized to the plasma membrane (21, 43). Thus, the obser-
vation that the wild-type protein is enriched in fraction 2 could
also be interpreted as plasma membrane localization, based on
the distribution of radioactivity in gradients obtained from
cells labeled with125I-labeled ligands (Fig. 5A). In addition,
disabling a critical AP-2 binding site could result in plasma
membrane accumulation of the Y72A mutant protein. Addi-
tional experiments were therefore carried out to exclude the
TABLE 1. Quantification of adaptin binding to fusion proteins
Amt bound% Difference
657 ? 33
140 ? 8
1,009 ? 97
618 ? 54
222 ? 19
373 ? 23
aPeripheral membrane proteins were incubated with 100 ?g of fusion protein
attached to glutathione beads. The fusion proteins tested had either wild-type
RID? Ile63-Leu91 sequences or Ile63-Leu91 sequences with Y72A or Y79A
bBound proteins were immunoblotted with adaptin-specific antibodies fol-
lowed by a125I-labeled secondary antibody, and blots were exposed to a phos-
cVolume integration of pixel values in a defined area with background sub-
tracted. Pixel values are arbitrary units that are proportional to the amount of
radiation stored and re-emitted from the phosphor screen. Values in the table
are the means from three independent determinations for each band ? standard
errors of the means and have been multiplied by 10?3. Similar results were
obtained in two independent determinations.
dPercent difference relative to the amount of protein bound to C-terminal
fusion protein, which is set to 100%.
10442 CIANCIOLA ET AL.J. VIROL.
possibility that RID? is targeted to the plasma membrane.
RID? exists in two molecular weight forms: one corresponding
to the full-length molecule, which has two membrane-spanning
?-helices and cytosolic amino and carboxy termini, and a lower-
molecular-weight species lacking the amino-terminal ?-helix,
which is cleaved by signal peptidase in the endoplasmic retic-
ulum (Fig. 1A) (27). Sequences connecting the ?-helices
should be exposed at the cell exterior in either form of the
FIG. 3. Lack of competition between AP-1 and AP-2 for RID? C-terminal binding. (A) Peripheral membrane proteins from cells that were
metabolically labeled with L-[35S]cysteine for 16 h were incubated with the ?-adaptin monoclonal antibody, which immunoprecipitates (IP) intact AP-1
complexes comprised of ?, ?, and ? subunits in addition to ?-adaptin. (B and C) Peripheral membrane fractions were preincubated with nonspecific
mouse immunoglobulin G (IgG) or with the ?-adaptin monoclonal antibody, and the immunodepleted samples were then incubated with GST or
C-terminal (C-term) fusion proteins. Aliquots of the immunodepleted samples (B) and proteins bound to glutathione beads (C) were analyzed by
immunoblotting (IB) with adaptin-specific antibodies. (D) Equal aliquots of peripheral membrane proteins were added to increasing concentrations of
the C-terminal fusion protein indicated on the x axis. Bound proteins were transferred to nitrocellulose filters, which were incubated with125I-labeled
The amount of bound protein was calculated as a percentage of total adaptin proteins in the peripheral membrane fractions, based on immunoblots of
five times with wash buffers supplemented with various concentrations of NaCl, as indicated in the figure.
VOL. 81, 2007RID? TARGETING AND FUNCTION 10443
molecule located at the plasma membrane. However, this re-
gion cannot be labeled by conventional surface biotinylation,
since it lacks residues with free amino groups. We therefore
took advantage of the fact that both RID? molecular weight
species form disulfide-linked dimers at Cys31 (highlighted in
Fig. 1A) (27). Hence, disulfide bonds on proteins located at the
cell surface should be reduced if cells are exposed to an exter-
nal reducing agent, such as cysteine, while intracellular pro-
teins are protected (4, 36). Transfected cells were subjected to
pulse-chase metabolic labeling and then incubated with cys-
teine-containing medium and lysed in the presence of iodo-
acetamide to prevent de novo disulfide bond formation. When
RID? immune complexes were resolved under nonreducing
conditions (i.e., in the absence of DTT), radiolabeled proteins
FIG. 4. Colocalization of wild-type (WT), Y72A, and Y79A RID? with membrane-compartment markers. HEK-293 cells transiently expressing
wild-type, Y72A, or Y79A mutant proteins were costained with antibodies to the FLAG-tagged viral protein (red channel) and to the TGN marker
furin (A) or the endocytic marker TfR (B) (green channel) and DAPI (blue channel) for analysis by confocal microscopy. Red and green channels
were merged, and “yellow” indicates the overlap of red and green fluorescence. Higher-magnification images of outlined areas are shown to the
right of each set of panels. Size markers, 10 ?m.
10444CIANCIOLA ET AL.J. VIROL.
FIG. 5. Subcellular distribution of wild-type and Y72A RID? proteins. (A) CHO cells were fractionated on Percoll gradients, and individual
fractions were assayed for ?-hexosaminidase activity (open circles). Cells were also fractionated after they had been incubated with125I-transferrin
for 30 min on ice (open squares) or after they were transfected with a human EGFR expression plasmid and incubated with125I-EGF for 30 min
on ice 48 h later (open triangles). Enzyme activity or radioactivity was plotted as a percentage of total activity or radioactivity detected across the
entire gradient. (B) Equal aliquots of total cellular protein from individual cell fractions were immunoblotted (IB) with antibodies to well-
characterized markers of the Golgi body (gp125) and of the plasma membrane and early endosomes (TfR). (C) CHO cells were transfected with
wild-type (WT) or Y72A RID? plasmids, and immune complexes from individual Percoll gradient fractions were analyzed by immunoblotting with
a RID?-specific antibody. (D and E) CHO cells transfected as described for panel C and nontransfected CHO cells were pulse-labeled and then
switched to chase medium to allow proteins to reach their steady-state localization. Proteins were immunoprecipitated and analyzed by SDS-PAGE
under standard reducing conditions (?DTT) or were immunoprecipitated from cells subjected to surface reducing (?) or sham (?) conditions
followed by SDS-PAGE under nonreducing conditions (?DTT).
VOL. 81, 2007 RID? TARGETING AND FUNCTION 10445
migrated as high-molecular-weight dimers, regardless of
whether or not cells were exposed to extracellular cysteine
(Fig. 5D). Introduction of the Y72A mutation had no effect on
the resistance of high-molecular-weight dimers to surface re-
duction. This result was in contrast to that for disulfide-linked
TfRs, which were partially reduced by external cysteine (Fig.
5E), consistent with approximately 20% to 45% of this mole-
cule exhibiting plasma membrane localization, with the re-
mainder in endocytic compartments (34, 47). These data con-
firm that the majority of wild-type and mutant proteins are
localized to intracellular membrane compartments at steady
Endosomal compartments are dispersed throughout the cy-
toplasm following a brief exposure to the microtubule-depoly-
merizing agent nocodazole (30). We have shown previously
that wild-type RID? displays the same behavior, consistent
with the viral protein’s localization to endocytic compartments
(A. H. Shah, N. L. Cianciola and C. Carlin, submitted for
publication). We therefore reasoned that a similar treatment
should have little or no effect on localization of the RID?
protein with the Y72A substitution if the mutant protein ac-
cumulates in a biosynthetic compartment, as is indicated by the
results shown in Fig. 4 and 5. Results in Fig. 6 indicate that
nocodazole has a very modest effect on the distribution of the
Y72A mutant protein compared to vesicles with wild-type
RID? that were redistributed from the perinuclear region to
the peripheral cytosol. Altogether, these data suggest that the
mutant protein is retained in the biosynthetic pathway and
mostly excluded from a nocodazole-sensitive endocytic path-
Y72A and Y79A point mutations prevent EGFR downregu-
lation by RID?. Although many functions have now been
associated with this Ad protein, RID? was first identified be-
cause of its ability to specifically reduce EGFR plasma mem-
brane expression (7). Thus, studies were carried out to deter-
mine the effect of the Y72A and Y79A mutations in RID? on
EGFR metabolic stability in CHO cells expressing both pro-
teins. Cells were cotransfected with viral protein and human
EGFR plasmids and harvested for total cellular protein or
pulse-labeled with [35S]cysteine for 30 min to measure protein
stability. When viral protein immune complexes were exam-
ined by immunoblotting, we observed that wild-type and mu-
tant viral proteins were expressed at similar levels at 48 h
posttransfection (Fig. 7A). The EGFR acquires seven to nine
N-linked high-mannose oligosaccharides cotranslationally that
are processed to complex carbohydrates during Golgi body
maturation, resulting in retarded migration on SDS-polyacryl-
amide gels (5). A molecular weight species corresponding to
the high-mannose oligosaccharide EGFR precursor was de-
tected in cells cotransfected with an empty vector control or
FIG. 6. Effect of nocodazole on RID? protein subcellular localization. CHO cells transiently expressing wild-type or Y72A mutant proteins
were pretreated with dimethyl sulfoxide vehicle (Veh) or nocodazole (Noc; 100 ?M) for 30 min prior to staining with antibody to the FLAG-tagged
viral protein (red channel) and with DAPI (blue channel). Cell outlines (in green) were made with the MetaMorph program using phase contrast
images as a guide. Size markers, 10 ?m.
10446 CIANCIOLA ET AL.J. VIROL.
plasmids encoding wild-type or mutant viral protein in cells
that were harvested immediately after a 30-min pulse-label
(Fig. 7B), showing that the EGFR is synthesized to similar
extents in all four populations of transfected cells. The molec-
ular weight species corresponding to the mature EGFR pro-
tein with complex carbohydrates was detected in cells trans-
fected with the empty vector control or with plasmids encoding
the Y72A or Y79A mutant proteins after a 5-h chase with
nonradioactive amino acid precursors. This was in contrast to
cells expressing wild-type RID?, where mature EGFR protein
was not detected, consistent with the known ability of RID? to
target EGFR to lysosomes for degradation in the absence of
other Ad proteins (25). Thus, the Y72A mutation appears to
block the ability of the viral protein to facilitate EGFR degra-
dation. RID? with the Y79A mutation also does not down-
regulate the receptor, even though these mutants appear to
localize to endosomes (Fig. 4B). The loss-of-function pheno-
type associated with the Y79A mutant can likely be attributed
to the fact that this region mediates protein-protein interac-
tions with ORP1L (Shah et al., submitted), a Rab7 effector that
links late endocytic vesicles to minus end-directed microtubule
motor complexes (31).
The E3 region is not required for viral replication; never-
theless, it plays a critical role in Ad pathogenesis (15). The
importance of this region is underscored by the fact that the
first generation of Ad gene therapy vectors which contained
large E3 deletions were ultimately deemed unsafe (14). E3
genes encode integral membrane proteins that regulate a va-
riety of host cell functions involved in innate immunity and
inflammatory responses. The ability of these proteins to modify
host cell function is due in part to cytosolic tail sequences that
interact with sorting machinery and target membrane proteins
to specific intracellular compartments. The E3 protein RID?
was originally identified because of its ability to downregulate
the EGFR (7) and other related receptor tyrosine kinases (35).
In this study, we have demonstrated that RID? residues 71-
AYLRH comprise a binding site for AP complexes and that
Tyr72 is required for RID? localization to endosomes and its
ability to downregulate the EGFR. These findings support
previous studies concluding that RID? acts by targeting
EGFRs undergoing constitutive recycling to the plasma mem-
brane (10, 26, 28). The fact that 71-AYLRH is precisely con-
served in all Ad serotypes that have been sequenced except for
Ad12 suggests its fundamental importance in a majority of
Ad-induced diseases (7).
Although the mutation of Tyr72 to an alanine residue leads
to a clear reduction in AP binding in vitro (Table 1), 71-
AYLRH does not conform to classical tyrosine-dependent
YXXØ motifs that have a preference for hydrophobic residues
with bulky side chains at the Ø position (39). Instead, it has a
histidine residue that is mildly basic and hydrophilic. However,
many other factors contribute to sorting signal recognition,
including the position of the signal relative to the membrane
and to the carboxyl terminus and the presence of amino acid
residues in areas adjacent to the signal. We have reported
previously that 71-AYLRH exists in an amphipathic helix that
is stabilized by interactions with a membrane-mimetic phos-
pholipid micelle surface based on data obtained using nuclear
magnetic resonance spectroscopic methods (45). This close
degree of membrane association could have an important role
in regulating the availability of the signal for interaction with
APs. Thus, 71-AYLRH may be masked when the cytoplasmic
tail is intimately associated with membrane, while cellular
events that result in its translocation into the cytosol could
make it available for binding APs. Some examples that could
bring about such a change include modulation by another
membrane protein, changes in the endosomal pH or ionic
environment as RID? traverses different cellular compart-
ments, posttranslational modification, or a dramatic shift in the
tilt or membrane placement of the adjacent transmembrane
helix. For example, we have recently demonstrated that RID?
function is highly dependent on reversible palmitoylation at a
residue in the cytosolic tail (N. L. Cianciola and C. Carlin,
unpublished data), suggesting that the association of the RID?
cytosolic tail with membranes is tightly regulated in cells (17).
The possibility that 71-AYLRH availability is regulated by a
transmembrane mechanism is particularly intriguing since the
RID? loop domain connecting its two membrane spanning
domains resides in compartmental lumens. Thus, the trans-
membrane domain could act as a conduit to fine-tune 71-
AYLRH recognition at specific subcellular organelles.
Even though 71-AYLRH recognizes two different classes of
APs, we have demonstrated that AP-1 and AP-2 do not com-
pete for binding to RID? in vitro (Fig. 3C) and that mutation
FIG. 7. Effect of Y72A and Y79A point mutations on RID? ex-
pression and function in mammalian cells. CHO cells were cotrans-
fected with plasmid expressing the human EGFR along with a plasmid
expressing wild-type RID? (WT), plasmids expressing RID? with a
Y72A or Y79A substitution, or an empty vector (Sham). (A) Equal
aliquots of total cell protein were resolved by SDS-PAGE, transferred
to nitrocellulose, and immunoblotted with a RID?-specific antibody.
(B) The cells were radiolabeled with [35S]cysteine for 30 min, starting
at 48 h posttransfection, and harvested either immediately or following
a 5-h chase in medium with an excess of nonradioactive cysteine.
EGFR immune complexes were resolved by SDS-PAGE and detected
by autoradiography. IP, immunoprecipitation.
VOL. 81, 2007RID? TARGETING AND FUNCTION10447
of adjacent residue Tyr79 leads to increased binding of AP-2
but diminished binding to AP-1 (Table 1). Thus, it seems likely
that AP-1 and AP-2 recognize distinct but overlapping sets of
tyrosine-based sorting signals in RID?. The Y72A mutation
traps RID? in the TGN (Fig. 4B) but not the plasma mem-
brane (Fig. 5D) suggesting the Ad protein encounters AP-1
first in the biosynthetic pathway. AP-2 is known to interact with
a majority of tyrosine-based signals identified for other mole-
cules, in agreement with studies showing that most naturally
occurring signals mediate internalization (37). The broad spec-
ificity of AP-2 recognition implies that it serves a quality con-
trol function to retarget membrane proteins to their correct
intracellular location that escape to the plasma membrane (2).
This would suggest that the plasma membrane may not be an
obligatory membrane transport destination for RID? and that
a majority of RID? is delivered directly to endosomes (see
summary model in Fig. 8). Accordingly, even though RID? can
be detected on the plasma membrane, this localization corre-
lates with high levels of protein expression and constitutes a
relatively minor fraction of the total protein in the cell (10).
This is entirely consistent with the quality control role for AP-2
that has been proposed for membrane proteins that leak to the
In addition to the Tyr72-based sorting motif, RID? also has
a potential dileucine-based motif located at residues 87-LL
(Fig. 1A). Although another laboratory has published a study
saying that these residues constitute an AP-2 binding site (21),
those results were not substantiated in the present study. We
did observe that AP binding was remarkably insensitive to pH
or salt (Fig. 3D), supporting a role for hydrophobic interac-
tions either within the signal itself or in adjacent regions. It is
possible that the dialanine substitution at 87-LL analyzed in
reference 21 lowers the overall strength of AP binding. Inter-
estingly, 87-LL is part of a larger motif that is precisely con-
served in the EGFR (26), suggesting its involvement in cargo
selection and/or targeting EGFRs to lysosomes. This conjec-
ture is supported by evidence that this sequence in the EGFR
is necessary for ligand-induced trafficking to lysosomes (32, 33)
and also RID?-mediated diversion of recycling EGFRs to
lysosomes (44). Thus, although 87-LL may not be directly
involved in AP recognition, it undoubtedly has an important
role in RID? function at least as it relates to EGFR down-
regulation. The 87-LL motif shared with the EGFR is found in
group C Ad2 and Ad5; however, this region is not precisely
conserved in other serotypes (7). Thus, different Ads may vary
in their ability to specifically target the EGFR.
RID? has been associated with other activities besides
EGFR downregulation. For example, the RID complex (com-
prised of RID? and RID?) downregulates death receptors,
including TNFR1 and FAS (9). However, mutagenesis studies
support a model where the RID complex acts on TNFR1 at the
plasma membrane, in contrast to FAS, where the functional
interaction occurs intracellularly. These seemingly paradoxical
results are best understood by considering the many steps
involved in receptor downregulation. These include cargo se-
lection, sorting to specific endocytic compartments involved in
transport to lysosomes, and coupling to microtubules necessary
for transporting MVB intermediates to the perinuclear region
(19). We have already discussed the idea that the molecular
basis for RID?-mediated EGFR cargo selection likely involves
FIG. 8. Summary model. Data presented in this study suggest that newly synthesized RID? is delivered directly from the TGN to endosomes
(open arrows) where it encounters EGFRs undergoing constitutive recycling to the plasma membrane (dashed arrows). Since RID? proteins with
a defective AP binding site accumulate in the Golgi body and TGN, we conclude that newly synthesized RID? proteins encounter AP-1 necessary
for incorporation into clathrin-coated vesicles (CCV), which are necessary for exiting the biosynthetic compartment. Data also show that RID?
is delivered directly to endosomes and not the plasma membrane, suggesting that the ability of the viral protein to bind AP-2 serves a quality control
function in which proteins that “leak” to the plasma membrane are quickly endocytosed and retargeted to their appropriate intracellular locations.
Although the EGFR and RID? are both transported to late endosomes (LE) via multivesicular body (MVB) transport intermediates (solid
arrows), only EGFR is degraded. EE and RE, early and recycling endosomes, respectively; MT, microtubule.
10448CIANCIOLA ET AL. J. VIROL.
the dileucine motif that is conserved in EGFR and RID?
encoded by Ad2 and Ad5. In addition, we have recently dis-
covered that RID? interacts with Rab7 effectors, including
RILP and ORP1L, which are necessary for microtubule-de-
pendent transport (Shah et al., submitted). Thus, it is likely
that RID? regulates EGFR downregulation at multiple levels.
Other cargoes could require accessory molecules to deliver
specific receptors to endosomes once the maturation process is
under way. Thus, RID? may promote TNFR1 uptake to en-
dosomes, whereupon they are then sorted to lysosomes by a
mechanism involving RID?-dependent endosomal maturation.
This conjecture is consistent with the observation that RID?
binds AP-2 and that AP-2 is required for RID-mediated down-
regulation of TNFR1 but not FAS (9). The ability to “mix and
match” different aspects of RID function may have evolved to
allow Ads to fine-tune RID activity in different cell types or
during acute versus persistent infections.
These studies were done with the support of the Pediatric Imaging
Center of University Hospitals of Cleveland, with the help of Sarah
Richer Dawson. We also thank Kim Preston for technical assistance
and Song J. Kil for critically reviewing the manuscript.
This work was supported by Public Health Service grant RO1
GM64243. N.L.C. was supported in part by NIH HL-007653, and
A.H.S. was supported in part by a Cell and Molecular Biology Training
Grant awarded through the National Institute of General Medical
1. Avila, M. M., G. Carballal, H. Rovaletti, B. Ebekian, M. Cusminsky, and M.
Weissenbacher. 1989. Viral etiology in acute lower respiratory infections in
children from a closed community. Am. Rev. Respir. Dis. 140:634–637.
2. Bonifacino, J., and L. Traub. 2003. Signals for sorting of transmembrane
proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72:395–447.
3. Brandt, C. D., H. W. Kim, A. J. Vargosko, B. C. Jeffries, J. O. Arrobio, B.
Rindge, R. H. Parrott, and R. M. Chanock. 1969. Infections in 18,000 infants
and children in a controlled study of respiratory tract disease. I. Adenovirus
pathogenicity in relation to serologic type and illness syndrome. Am. J.
4. Bretscher, A. 1989. Rapid phosphorylation and reorganization of ezrin and
spectrin accompany morphological changes in A431 cells induced by EGF.
J. Cell Biol. 108:921–930.
5. Carlin, C. R., and B. B. Knowles. 1986. Biosynthesis and glycosylation of the
epidermal growth factor receptor in human tumor-derived cell lines A431
and Hep 3B. Mol. Cell. Biol. 6:257–264.
6. Carlin, C. R., D. Simon, J. Mattison, and B. B. Knowles. 1988. Expression
and biosynthetic variation of the epidermal growth factor receptor in human
hepatoma-derived cell lines. Mol. Cell. Biol. 8:25–34.
7. Carlin, C. R., A. E. Tollefson, H. A. Brady, B. L. Hoffman, and W. Wold.
1989. Epidermal growth factor receptor is down-regulated by a 10,400 MW
protein encoded by the E3 region of adenovirus. Cell 57:135–144.
8. Chavrier, P., R. G. Parton, H. P. Hauri, K. Simons, and M. Zerial. 1990.
Localization of low molecular weight GTP binding proteins to exocytic and
endocytic compartments. Cell 62:317–329.
9. Chin, Y. R., and M. S. Horwitz. 2005. Mechanism for removal of tumor
necrosis factor receptor 1 from the cell surface by the adenovirus RID?/?
complex. J. Virol. 79:13606–13617.
10. Crooks, D., S. J. Kil, J. M. McCaffery, and C. Carlin. 2000. E3-13.7 integral
membrane proteins encoded by human adenoviruses alter epidermal growth
factor receptor trafficking by interacting directly with receptors in early
endosomes. Mol. Biol. Cell 11:3559–3572.
11. Fessler, S., F. Delgado-Lopez, and M. Horwitz. 2004. Mechanisms of E3
modulation of immune and inflammatory responses. Curr. Top. Microbiol.
12. Giard, D. J., S. A. Aaronson, G. J. Todaro, P. Arnstein, J. H. Kersey, H.
Dosik, and W. P. Parks. 1973. In vitro cultivation of human tumors. J. Natl.
Cancer Inst. 51:1417–1423.
13. Ginsberg, H. 1999. The life and times of adenoviruses. Adv. Virus Res.
14. Ginsberg, H. 1996. The ups and downs of adenovirus vectors. Bull. N.Y.
Acad. Med. 73:53–58.
15. Ginsberg, H. S., and G. A. Prince. 1994. The molecular basis of adenovirus
pathogenesis. Infect. Agents Dis. 3:1–8.
16. Graham, F. L., J. Smiley, W. C. Russell, and R. Nairn. 1977. Characteristics
of a human cell line transformed by DNA from human adenovirus type 5.
J. Gen. Virol. 36:59–74.
17. Greaves, J., and L. H. Chamberlain. 2007. Palmitoylation-dependent protein
sorting. J. Cell Biol. 176:249–254.
18. Green, S. A., K. P. Zimmer, G. Griffiths, and I. Mellman. 1987. Kinetics of
intracellular transport and sorting of lysosomal membrane and plasma mem-
brane proteins. J. Cell Biol. 105:1227–1240.
19. Gu, F., and J. Gruenberg. 1999. Biogenesis of transport intermediates in the
endocytic pathway. FEBS Lett. 452:61–66.
20. Hayflick, L. 1965. The limited in vitro lifetime of human diploid cell strains.
Exp. Cell Res. 37:614–636.
21. Hilgendorf, A., J. Lindberg, Z. Ruzsics, S. Honing, A. Elsing, M. Lofqvist, H.
Engelmann, and H.-G. Burgert. 2003. Two distinct transport motifs in the
adenovirus E3 proteins act in concert to down-modulate apoptosis receptors
and the epidermal growth factor receptor. J. Biol. Chem. 278:51872–51884.
22. Hirota, K., M. Murata, T. Itoh, J. Yodoi, and K. Fukuda. 2001. Redox-
sensitive transactivation of epidermal growth factor receptor by tumor
necrosis factor confers the NF-kappa B activation. J. Biol. Chem. 276:25953–
23. Hobert, M. E., S. Kil, and C. R. Carlin. 1997. The cytoplasmic juxtamem-
brane domain of the epidermal growth factor receptor contains a novel
autonomous basolateral sorting signal. J. Biol. Chem. 272:32901–32909.
24. Hoffman, B. L., K. Takishima, M. R. Rosner, and C. Carlin. 1993. Adeno-
virus and protein kinase C have distinct molecular requirements for regu-
lating epidermal growth factor receptor trafficking. J. Cell. Physiol. 157:
25. Hoffman, B. L., A. Ullrich, W. Wold, and C. Carlin. 1990. Retrovirus-medi-
ated transfer of an adenovirus gene encoding an integral membrane protein
is sufficient to down regulate the receptor for epidermal growth factor. Mol.
Cell. Biol. 10:5521–5524.
26. Hoffman, P., and C. Carlin. 1994. Adenovirus E3 protein causes constitu-
tively internalized EGF receptors to accumulate in a prelysosomal compart-
ment, resulting in enhanced degradation. Mol. Cell. Biol. 14:3695–3706.
27. Hoffman, P., M. B. Yaffe, B. L. Hoffman, S. Yei, W. S. M. Wold, and C.
Carlin. 1992. Characterization of the adenovirus E3 protein that down-
regulates the epidermal growth factor receptor. J. Biol. Chem. 267:13480–
28. Hoffman, P. H., P. Rajakumar, B. Hoffman, R. Heuertz, W. S. M. Wold, and
C. R. Carlin. 1992. Evidence for intracellular down-regulation of the
epidermal growth factor receptor during adenovirus infection by an EGF-
independent mechanism. J. Virol. 66:197–203.
29. Horwitz, M. S. 2004. Function of adenovirus E3 proteins and their interac-
tions with immunoregulatory cell proteins. J. Gene Med. 6(Suppl. 1):S172–
30. Husain, M., and B. Moss. 2003. Intracellular trafficking of a palmitoylated
membrane-associated protein component of enveloped vaccinia virus. J. Vi-
31. Johansson, M., N. Rocha, W. Zwart, I. Jordens, L. Janssen, C. Kuiji, V. M.
Olkkonen, and J. Neefjes. 2007. Activation of endosomal dynein motors by
stepwise assembly of Rab7-RILP-p150Glued, ORP1L, and the receptor ?lll
spectrin. J. Cell Biol. 176:459–471.
32. Kil, S., and C. Carlin. 2000. EGF receptor residues Leu679, Leu680mediate
selective sorting of ligand-receptor complexes in early endocytic compart-
ments. J. Cell. Physiol. 185:47–60.
33. Kil, S. J., M. E. Hobert, and C. Carlin. 1999. A leucine-based determinant
in the EGF receptor juxtamembrane domain is required for the efficient
transport of ligand-receptor complexes to lysosomes. J. Biol. Chem. 274:
34. Klausner, R. D., J. Van Renswoude, G. Ashwell, C. Kempf, A. N. Schecter, A.
Dean, and K. R. Bridges. 1983. Receptor-mediated endocytosis of transferrin
in K562 cells. J. Biol. Chem. 258:4715–4724.
35. Kuivinen, E., B. L. Hoffman, P. A. Hoffman, and C. R. Carlin. 1993. Struc-
turally related class I and class II receptor protein tyrosine kinases are
down-regulated by the same E3 protein coded by human group C adenovi-
ruses. J. Cell Biol. 120:1271–1279.
36. Low, S. H., B. L. Tang, S. H. Wong, and W. Hong. 1992. Selective inhibition
of protein targeting to the apical domain of MDCK cells by brefeldin A.
J. Cell Biol. 118:51–62.
37. Marks, M. S., H. Ohno, T. Kirchhausen, and J. S. Bonifacino. 1997. Protein
sorting by tyrosine-based signals: adapting to the Ys and wherefores. Trends
Cell Biol. 7:124–128.
38. Monick, M. M., K. Cameron, J. Staber, L. S. Powers, T. O. Yarovinsky, J. G.
Koland, and G. W. Hunninghake. 2005. Activation of the epidermal growth
factor receptor by respiratory syncytial virus results in increased inflamma-
tion and delayed apoptosis. J. Biol. Chem. 280:2147–2158.
39. Ohno, H., R. C. Aguilar, D. Yeh, D. Taura, T. Saito, and J. S. Bonifacino. 1998.
The medium subunits of adaptor complexes recognize distinct but overlapping
sets of tyrosine-based sorting signals. J. Biol. Chem. 273:25915–25921.
40. Puck, T. T., S. J. Cieciura, and A. Robinson. 1958. Genetics of somatic
mammalian cells. III. Long-term cultivation of euploid cells from human and
animal subjects. J. Exp. Med. 108:945–956.
VOL. 81, 2007RID? TARGETING AND FUNCTION10449
41. Robinson, M. 1993. Assembly and targeting of adaptin chimeras in trans- Download full-text
fected cells. J. Cell Biol. 123:67–77.
42. Robinson, M. S., and J. S. Bonifacino. 2001. Adaptor-related proteins. Curr.
Opin. Cell Biol. 13:444–453.
43. Stewart, A., A. Tollefson, P. Krajcsi, S. Yei, and W. Wold. 1995. The adeno-
virus E3 10.4K and 14.5K proteins, which function to prevent cytolysis by
tumor necrosis factor and to down-regulate the epidermal growth factor
receptor, are localized in the plasma membrane. J. Virol. 69:172–181.
44. Tsacoumangos, A., S. J. Kil, L. Ma, F. D. Sonnichsen, and C. Carlin. 2005. A
novel dileucine lysosomal-sorting-signal mediates intracellular EGF-receptor
retention independently of protein ubiquitylation. J. Cell Sci. 118:3959–3971.
45. Vinogradova, O., C. R. Carlin, F. D. Sonnichsen, and C. R. Sanders. 1998. A
membrane setting for the sorting motifs present in the adenovirus E3-13.7
protein which down-regulates the epidermal growth factor receptor. J. Biol.
46. Waterfield, M. D., E. L. V. Mayes, P. Stroobant, P. L. P. Bennet, S. Young,
P. N. Goodfellow, G. S. Banting, and B. Ozanne. 1982. A monoclonal anti-
body to the human epidermal growth factor receptor. J. Cell. Biochem.
47. Weiel, J. E., and T. A. Hamilton. 1984. Quiescent lymphocytes express
intracellular transferrin receptors. Biochem. Biophys. Res. Commun. 119:
10450 CIANCIOLA ET AL.J. VIROL.