Traffic 2011; 12: 1793–1804 © 2011 John Wiley & Sons A/S

doi:10.1111/j.1600-0854.2011.01282.x

Identification of a Novel Mono-Leucine BasolateralSorting Motif Within the Cytoplasmic Domainof Amphiregulin

Jonathan D. Gephart1, Bhuminder Singh2,

James N. Higginbotham2, Jeffrey L. Franklin1,6,

Alfonso Gonzalez3,4, Heike Folsch5 and

Robert J. Coffey1,2,6

1Department of Cell and Developmental Biology,Vanderbilt University, Nashville, TN 37232, USA2Department of Medicine, Vanderbilt University,Nashville, TN 37232, USA3Departamento de Inmunología Clínica y Reumatología,Facultad de Medicina, Pontificia Universidad Catolica deChile, 6510260 Santiago, Chile4Centro de Envejecimiento y Regeneracion, Facultad deCiencias Biologicas, Pontificia Universidad Catolica deChile, 6510260 Santiago, Chile5Department of Cell and Molecular Biology,Northwestern University, Chicago, IL 60611, USA6Department of Veterans Affairs Medical Center,Nashville, TN 37232, USA*Corresponding author: Robert J. Coffey,robert.coffey@vanderbilt.edu

Epithelial cells establish apical and basolateral (BL) mem-

branes with distinct protein and lipid compositions. To

achieve this spatial asymmetry, the cell utilizes a variety

of mechanisms for differential sorting, delivery and reten-

tion of cell surface proteins. The EGF receptor (EGFR) and

its ligand, amphiregulin (AREG), are transmembrane pro-

teins delivered to the BL membrane in polarized epithelial

cells. Herein, we show that the cytoplasmic domain

of AREG (ACD) contains dominant BL sorting informa-

tion; replacement of the cytoplasmic domain of apically

targeted nerve growth factor receptor with the ACD redi-

rects the chimera to the BL surface. Using sequential

truncations and site-directed mutagenesis of the ACD,

we identify a novel BL sorting motif consisting of a single

leucine C-terminal to an acidic cluster (EEXXXL). In adap-

tor protein (AP)-1B-deficient cells, newly synthesized

AREG is initially delivered to the BL surface as in AP-

1B-expressing cells. However, in these AP-1B-deficient

cells, recycling of AREG back to the BL surface is com-

promised, leading to its appearance at the apical surface.

These results show that recycling, but not delivery, of

AREG to the BL surface is AP-1B dependent.

Key words: μ1B, amphiregulin, AP-1B, basolateral, EGF

receptor, LLC-PK1, MDCK

Received 2 March 2011, revised and accepted for

publication 12 September 2011, uncorrected manuscript

published online 14 September 2011, published online 13

October 2011

Sheets of polarized epithelial cells line all body cavities,forming a physical barrier between external or lumi-nal environments and the internal milieu. Creation andmaintenance of this barrier require the cells to establishbiochemically and functionally diverse membrane surfaceswith distinct protein and lipid compositions (1). The api-cal membrane, which is exposed to external or luminalenvironments, acts as a protective barrier that also allowsfor the regulated uptake of nutrients and ions. The lateralmembrane is exposed to neighboring cells and functionsin cell–cell contact and communication, while the basalmembrane contacts the extracellular matrix and functionsin cell–matrix contact. Together, these domains comprisethe basolateral (BL) membrane, which regulates ion gra-dients, signal transduction and cell adhesion (2). Becauseeach surface serves a unique function critical for overallbarrier integrity and cell function, the cell has evolveda complex network of signals, proteins and compart-ments to establish and maintain the distinct membranesurfaces (3,4). Understanding the mechanisms by whichepithelial cells establish and maintain polarity is crucialto understanding normal organ function and how loss ofpolarity can lead to disease (5,6).

One family of proteins distributed to the cell surface ina polarized manner is the EGF receptor (EGFR) and itscognate ligands (7). EGFR is a transmembrane tyrosinekinase receptor involved in proliferation, migration andcell survival, and is critical for epithelial wound repairand homeostasis (8–10). Seven mammalian EGFR lig-ands have been identified [EGF, amphiregulin (AREG),transforming growth factor alpha (TGFα), heparin-bindingEGF-like growth factor (HBEGF), betacellulin, epiregulin(EREG) and epigen (EPGN)], all of which are producedas type 1 transmembrane proteins that are deliveredto the cell surface where they are cleaved by metal-loproteases to release mature, soluble ligands (7). Lig-ands bind and activate the EGFR with differing affinitiesand intensities in a paracrine, autocrine or juxtacrinemanner (7,11).

In polarized epithelial cells, EGFR, TGFα and AREG areselectively delivered to the BL surface (12–14). EGF isdelivered to both the apical and BL membranes, butis selectively cleaved by BL metalloproteases, leadingto steady-state apical accumulation of membrane-boundEGF (15). The cytoplasmic domain of EGF contains a PXXPBL sorting motif, mutation of which results in loss of EGFat the BL surface, but not the apical surface, suggest-ing that EGF contains separate apical and BL sortingmotifs (16).

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Our laboratory previously reported that TGFα is preferen-tially delivered to the BL surface through a mechanismthat depends on the myristoylated cargo recognition andtargeting (CaRT) protein Naked2 (NKD2) (13,17). TGFα-containing vesicles are coated by NKD2 after emergencefrom the trans-Golgi network (TGN) and are delivered tothe BL corner of polarized Madin–Darby canine kidney(MDCK) cells where they fuse with the plasma membranein an NKD2 myristoylation-dependent manner (17). Theshort hairpin RNA (shRNA) knockdown of NKD2 reducescell surface expression of TGFα and leads to accumulationof TGFα-containing vesicles in the cytoplasm (18). TGFα isthe only EGFR ligand known to require NKD2 for polarizedBL trafficking.

AREG is delivered to the BL surface of polarized epithelialcells (14), although by a different mechanism than EGFand TGFα. Previous studies by our laboratory showed thatremoval of the AREG cytoplasmic domain (ACD) resultsin the appearance of apical and BL membrane-associatedAREG, suggesting that this protein lacks a separate apicalsorting determinant in the extracellular and transmem-brane domains (19).

This study demonstrates that the ACD contains dominantBL sorting information, sufficient to redirect the apicallytargeted 75-kDa human nerve growth factor receptor(NGFR) to the BL surface. AREG BL trafficking is directedby a novel mono-leucine-based motif (EEXXXL) that differsfrom similar motifs found in CD147 and stem cell factor

(SCF) (20,21). Initial delivery of AREG to the BL surface isadaptor protein (AP)-1B independent. However, AP-1B isrequired for recycling back to the BL surface.

Results

The ACD contains a dominant BL sorting motif

Previous work from our laboratory showed that AREGcontains BL sorting information within the last 27 aminoacids of its 31-amino acid cytoplasmic domain, as deletionof these amino acids results in its non-polar cell surfacedistribution in MDCK cells (19). In contrast, NGFR, whichis normally localized to the apical surface of polarizedMDCK cells, contains an extracellular apical sorting deter-minant consisting of an O-glycosylated region (22,23). Todetermine whether the ACD is sufficient for BL delivery, achimeric protein consisting of the extracellular and trans-membrane domains of NGFR and the ACD (NGFR-ACD)was generated and stably expressed in MDCK cells. Con-focal immunofluorescence microscopy using an antibodyspecific to the ectodomain of NGFR showed NGFR onthe apical surface, while NGFR-ACD was mainly presenton the BL surface of polarized MDCK cells (Figure 1A).Domain-selective cell surface biotinylation showed that75% of NGFR was on the apical surface, whereas 78%of the NGFR-ACD chimera was on BL (Figure 1B and C).These data indicate that the ACD contains a BL sortingmotif that overrides the NGFR apical sorting determinant,and therefore is a dominant BL sorting motif. Interestingly,

Figure 1: The ACD redirects NGFR from the apical (Ap) to the BL surface of polarized MDCK cells. MDCK cells stablyexpressing either full-length NGFR or the NGFR extracellular and transmembrane domains fused to the ACD (NGFR-ACD) werepolarized on Transwell filters (TEER > 200 �/cm2) and then analyzed for steady-state cell surface distribution of NGFR using an NGFRectodomain-specific antibody (ME20.4). A) Polarized cells were stained for NGFR (green) under non-permeabilized conditions followedby permeabilization with 0.1% Triton and F-actin (red) staining. Analysis was performed by confocal microscopy. Bar, 10 μm. B) Polarizedcells were selectively biotinylated on the Ap or BL surface, immunoprecipitated with ME20.4, separated by SDS–PAGE, transferred tonitrocellulose and then probed with streptavidin-horseradish-peroxidase or (C) with streptavidin-IRDye680. Results were scanned on anOdyssey scanner and quantitated using ODYSSEY software (n = 3). Columns marked with an * are significantly different.

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CD147, which contains a canonical mono-leucine BLsorting motif (EDDXXXXXL), is unable to redirect NGFR tothe BL surface (20,24).

Determination of the amino acids

within the cytoplasmic domain necessary for the BL

localization of AREG

Having established the presence of a dominant BL sortingmotif within the ACD, we set out to determine thespecific amino acid sequence that contains the necessaryAREG BL sorting information. Sequential cytoplasmicdomain truncation mutants of AREG were constructedwith enhanced green fluorescent protein (EGFP) fused to

their C-termini (Figure 2A). MDCK cells stably expressingthese chimeras were selected by flow cytometric sortingfor high surface expression of AREG. The apical/BLdistribution of the truncation mutants was analyzed byconfocal immunofluorescence microscopy (Figure 2B) anddomain-selective cell surface biotinylation (Figure 2C). Full-length AREG and AREG with 6 amino acids removed fromthe cytoplasmic domain (25 amino acids) were 95 and93% localized to the BL surface, respectively. However,truncating the cytoplasmic domain by 17 amino acids(14 amino acids) resulted in only 57% of total surfaceAREG localized to the BL surface, a statistically significantdifference compared to wild-type AREG and 25-amino

Figure 2: ACD truncations reveal that residues 236–246 contain BL sorting information. A) Amino acid composition of ACD andpoints of truncation. MDCK cells stably expressing ACD truncations that were C-terminally EGFP tagged were generated and used todetermine the region of the tail that contains BL sorting information. Cells were polarized on Transwell filters and (B) surface stained forAREG (red) followed by permeabilization and F-actin (blue) staining. Analysis was performed by confocal microscopy. The AREG-EGFPchannel is shown to demonstrate the EGFP tag does not disrupt cell surface delivery of AREG. Bars, 10 μm. C) Polarized cells wereselectively biotinylated on the apical or BL surface, immunoprecipitated with anti-AREG mAb, separated by SDS–PAGE, transferred tonitrocellulose and then probed with streptavidin-IRDye680. Results were scanned on an Odyssey scanner and quantitated using ODYSSEY

software (n ≥ 3). Columns marked with an * are significantly different from wild type.

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acid mutant AREG. Further truncation of the ACD (4amino acids) did not impart significant additional apicallocalization. Combined, these results indicate that BLsorting information resides within amino acids 236 (14amino acids) through 246 (25 amino acids) of the ACD.

Identification of a mono-leucine-based BL sorting

motif

Analysis of amino acids 236 through 246 (EERKKLRQENG)revealed no obvious canonical BL sorting motifs. Compar-ison of this region across species shows it to be 73%identical and 91% similar, making it the most conservedregion of the ACD (Figure 3). This region contains an acidiccluster, a basic cluster and a mono-leucine. Mono-leucineBL sorting motifs consisting of an acidic cluster N-terminalof a mono-leucine (EDDXXXXXL) have been reported fortwo other BL proteins, SCF and CD147 (20,21). Althoughthis exact motif is not present in the ACD, we furtheranalyzed the possibility that AREG may contain a variationof the consensus mono-leucine BL sorting motif. Usingpolymerase chain reaction (PCR)-based site-directed muta-genesis, individual amino acids were substituted withalanine (A) residues (Figure 4A). When the single leucine(L) residue was changed to alanine (L241A [LA]), AREGpolar BL distribution was lost (Figure 4B) with 45% of totalsurface AREG distributed to the apical surface at steadystate (Figure 4C). When the two conserved acidic glu-tamic acid (E) residues were changed to alanine residues(EE236, 237AA [EEAA]), 33% of AREG was detectedon the apical surface at steady state (Figure 4C). Thesechanges in AREG apical distribution are both significantlydifferent from the 7% of steady-state wild-type AREGdetected on the apical surface. Combination of these twomutations (EEAA/LA) resulted in 59% of total surfaceAREG localized at the apical membrane at steady state

Figure 3: Schematic diagram of full-length AREG and amino

acid composition of the ACD. AREG is synthesized as a 252-amino acid type 1 transmembrane glycoprotein. Beginning witha signal sequence (1–19), AREG contains an N-glycosylated pro-region (20–100) that is required for proper folding and secretion.Following the pro-region is mature AREG (101–184) consistingof a heparin-binding domain (HBD) and an EGF domain. TheEGF domain contains the conserved spacing of six cysteineresidues that is preserved in all EGFR ligands. The 31-amino acidcytoplasmic domain (222–252) is aligned with other species withthe conserved residues highlighted in red.

Figure 4: Amino acid substitutions within residues 236–246

identify a mono-leucine-based sorting signal. A) Aminoacid composition of ACD between residues 236 and 246.Specific amino acid mutations are indicated in red. MDCKcells stably expressing the ACD mutants were generatedand used to determine the BL sorting motif. Cells werepolarized on Transwell filters and (B) surface stained for AREG(green) followed by permeabilization and F-actin (red) staining.Analysis was performed by confocal microscopy. Bars, 10 μm. C)Polarized cells were selectively biotinylated on the apical or BLsurface, immunoprecipitated with anti-AREG mAb, separated bySDS–PAGE, transferred to nitrocellulose and then probed withstreptavidin-IRDye680. Results were scanned on an Odysseyscanner and quantitated using ODYSSEY software (n ≥ 3). Columnsmarked with an * are significantly different from wild type.Columns marked with a † are significantly different from eachother.

(Figure 4C). To exclude the possibility that the charge ofthe region plays a role in BL sorting (25), we mutated thetwo positive lysine (K) residues (KK239 and 240AA [KKAA])to alanine residues. Similar to wild-type AREG, this mutantprotein exhibited predominant BL localization with 98%present on the BL surface (Figure 4C). These results indi-cate that both the acidic cluster and the mono-leucine inthe ACD, which are both highly conserved across species(Figure 3), are critical for BL sorting of AREG.

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Loss of AP-1B affects the polarized distribution

of AREG at steady state

Heterotetrameric AP complexes (AP-1A, AP-1B, AP-2,AP-3A, AP-3B and AP-4) selectively incorporate cargo pro-teins and facilitate vesicle formation (26). Of the knownAP complexes, only AP-1B and AP-4 have been impli-cated in BL sorting (27). AP-1B expression is epithelialcell-specific and only differs from the ubiquitous AP-1A bythe medium (μ1B) subunit (28). AP-1B is localized to recy-cling endosomes and plays a role in biosynthetic deliveryand recycling of BL proteins (29–32). Polarizing epithelialLLC-PK1 cells do not express endogenous AP-1B and,consequently, missort proteins dependent on AP-1B forBL delivery (33). To determine the role of AP-1B in AREGBL delivery, clonal lines of LLC-PK1 cells expressing eitherpCB6::μ1A (LLC-PK1::μ1A) or pCB6::μ1B (LLC-PK1::μ1B)were transiently transfected with wild-type AREG andanalyzed by confocal immunofluorescence microscopy(Figure 5A). In the LLC-PK1::μ1A cells, which lack AP-1B,AREG was detected on the apical and BL surface, suggest-ing a role for AP-1B in maintaining the polar BL distributionof AREG (Figure 5A). As expected, parental LLC-PK1 cellshad a similar apical and BL distribution of AREG (data notshown). In the LLC-PK1::μ1B cells, AREG was restrictedto the BL surface and was undetectable on the apicalsurface (Figure 5A). These results strongly support the

involvement of AP-1B in AREG BL localization. BecauseAREG was detected on both surfaces in LLC-PK1::μ1Acells at steady state, we further characterized the AREGdistribution using domain-selective cell surface biotinyla-tion. For these experiments, we used a clonal line ofMDCK cells expressing shRNA against μ1B (31) becauseprevious work has shown that LLC-PK1 cells are notideal for biotinylation studies (33). The clonal MDCK μ1Bknockdown cell line was transfected with AREG, the cellsselected with G418 and enriched by flow cytometric sort-ing to produce a pool of μ1B-knockdown MDCK cellsexpressing AREG. Knockdown of μ1B in the clonal line hasbeen previously characterized (31); this was confirmedin these AREG-expressing cells by quantitative reversetranscriptase-PCR (RT-PCR), which showed a 93% reduc-tion in μ1B message compared to parental MDCK cells(Figure 5C). AREG immunoreactivity was detected on theapical surface of polarized μ1B-knockdown MDCK cells(Figure 5A). To quantitate the cell surface distribution ofAREG in the μ1B-knockdown MDCK cells at steady state,cells were polarized on Transwell filters and selectivelybiotinylated on either the apical or BL surface. The μ1Bknockdown MDCK cells expressed 30% of the total cellsurface AREG on the apical surface at steady state, atwofold increase over parental MDCK cells expressingendogenous wild-type μ1B (Figure 5B). This result, along

Figure 5: Loss of AP-1B results in apical distribution of AREG. The μ1B-deficient LLC-PK1 cells stably expressing either the μ1B(LLC-PK1::μ1B) or μ1A (LLC-PK1::μ1A) subunit and MDCK cells stably expressing shRNA against the μ1B subunit of AP-1B (μ1B KD)were used to demonstrate the role of AP-1B in the cell surface distribution of AREG. The cells were transiently transfected with AREGand then polarized on Transwell filters. A) The polarized monolayer was surface stained for AREG (green) followed by permeabilizationand F-actin (red) staining. Analysis was performed by confocal microscopy. Bars, 10 μm. B) AREG-expressing parental and μ1B KDMDCK cells polarized on Transwell filters were selectively biotinylated on the apical or BL surface, immunoprecipitated with AREGmAb, separated by SDS–PAGE, transferred to nitrocellulose and then probed with streptavidin-IRDye680. Results were scanned on anOdyssey scanner and quantitated using ODYSSEY software (n ≥ 3). Columns marked with an * are significantly different. C) Knockdownof μ1B in μ1B KD cells was confirmed by quantitative RT-PCR using two different primer sets. RQ, relative quantity.

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with the presence of AREG on the apical surface ofLLC-PK1::μ1A cells, demonstrates that AP-1B is requiredfor steady-state BL localization of AREG.

Biosynthetic delivery of AREG to the BL surface

is dependent on the mono-leucine sorting motif

and independent of AP-1B

Figure 4 shows that under steady-state conditions, muta-tion of the mono-leucine motif within the ACD (EEAA/LA)directs approximately 60% of AREG to the apical surfaceand 40% to the BL surface (Figure 4). To determine if deliv-ery of newly synthesized AREG to the BL surface is depen-dent on the mono-leucine motif, we performed metaboliclabeling pulse-chase experiments, followed by domain-selective cell surface biotinylation. MDCK cells expressingwild-type AREG or EEAA/LA mutant AREG were polarizedon Transwell filters. The cells were metabolically labeledwith Trans35S label for 20 min, chased for 0 or 30 min andfollowed by domain-selective cell surface biotinylation.The results show that wild-type AREG is detected only onthe BL surface at 30 min (Figure 6A), supporting a previ-ously published study from our laboratory (19). In markedcontrast, the EEAA/LA mutant AREG is detected on theapical surface at 30 min, demonstrating the requirementof this mono-leucine motif in AREG for biosynthetic BL

Figure 6: BL delivery of newly synthesized AREG is depen-

dent on the mono-leucine sorting signal and independent

of AP-1B. A) MDCK cells expressing wild-type (WT) AREG orEEAA/LA mutant AREG and (B) μ1B-knockdown MDCK cells(μ1B KD) expressing WT AREG were polarized on Transwellfilters. The cells were labeled with Trans35S label for 20 minfollowed by 0- or 30-min chase. After the chase, the cells wereselectively biotinylated on the apical or BL surface and AREG wasimmunoprecipitated with anti-AREG mAb. AREG immunoprecip-itates were eluted and affinity purified with streptavidin-agaroseto capture cell surface AREG, separated by SDS–PAGE and ana-lyzed by autoradiography. BL* indicates pretreatment with thebroad-spectrum metalloprotease inhibitor batimastat (10 μM). C)Lysates from MDCK cells used above expressing endogenousμ1B (parental) and μ1B KD cells were separated by SDS–PAGE,transferred to nitrocellulose and then blotted for canine μ1B,stripped and re-blotted for alpha-tubulin as a loading control.

delivery (Figure 6A). Treatment with the broad-spectrummetalloprotease inhibitor batimastat resulted in detectionof BL EEAA/LA mutant AREG, suggesting that newly syn-thesized mutant AREG is also delivered to the BL surface,but appears to be rapidly cleaved under these experimen-tal conditions (Figure 6A). These results are consistentwith the conclusion that direct BL biosynthetic delivery ofAREG is dependent on the mono-leucine motif within thecytoplasmic domain.

In addition, we sought to test whether AP-1B is involvedin biosynthetic delivery of AREG to the BL surface. In theabsence of μ1B expression, AREG is not only presenton the BL surface, but is also detected on the api-cal membrane (Figure 5), suggesting the maintenanceof AREG on the BL surface is AP-1B dependent. Toexclude the possibility that AP-1B is involved in biosyn-thetic delivery of AREG to the BL surface, we performeda similar metabolic labeling pulse chase followed bydomain-selective cell surface biotinylation experimentsin μ1B-knockdown MDCK cells. These results show thatnewly synthesized AREG is present at the BL, but notapical, surface at 30 min (Figure 6B), similar to μ1B-expressing MDCK cells. We confirmed the reduction ofμ1B protein level by western blotting of μ1B-expressing(parental) and μ1B-knockdown (μ1B KD) MDCK cellsused in the metabolic pulse-chase experiment (Figure 6C).Taken together, these data demonstrate that direct BLbiosynthetic delivery of AREG is dependent on the AREGmono-leucine motif and independent of AP-1B.

Loss of AP-1B results in inappropriate recycling of

post-endocytic AREG to the apical surface in fully

polarized LLC-PK 1 cells

The presence of steady-state AREG on the apical sur-face of polarized epithelial cells lacking AP-1B, along withthe observation that AREG BL biosynthetic delivery isAP-1B-independent, led us to ask what role AP-1B plays inthe predominant BL distribution of AREG. Previous stud-ies have shown that AP-1B is localized to the recyclingendosomes and participates in both biosynthetic deliv-ery and recycling of proteins to the BL surface (29–32).Because the majority of steady-state cell surface AREGwas detected on the BL surface of polarized MDCK cellslacking AP-1B (Figure 5B) and the AREG biosynthetic routeis AP-1B independent (Figure 6B), we hypothesized thatrecycling of post-endocytic AREG back to the BL surfacewas AP-1B dependent. To test this hypothesis, we usedLLC-PK1::μ1A and LLC-PK1::μ1B cells transfected withAREG and asked if an AREG-specific antibody applied onlyto the BL surface could be detected on the apical sur-face of cells in the presence or absence of AP-1B. TheLLC-PK1::μ1A and LLC-PK1::μ1B cells were polarized onTranswell filters, and the integrity of the monolayer wasdetermined by both transepithelial electrical resistance(TEER) (>400 �/cm2) and the ability to inhibit diffusionof 3000-MW dextran Texas Red across the monolayer(see Materials and Methods). Once the monolayer wasintact, the cells were incubated for 30 min at 37◦C with

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Figure 7: BL-labeled AREG appears at the apical (Ap) surface of μ1B-deficient LLC-PK1 cells. AREG primary antibody was addedonly to the BL compartment of polarized μ1B-deficient LLC-PK1 cells stably expressing μ1A (LLC-PK1::μ1A) or μ1B (LLC-PK1::μ1B)and incubated at 37◦C for 30 min. Cells were washed and then selectively incubated with Alexa488-conjugated secondary antibody inthe BL compartment and Cy5-conjugated secondary antibody in the Ap compartment for 10 min at room temperature. A) BL primaryantibody-bound AREG was detected at the Ap surface in 80% of LLC-PK1::μ1A cells. Only AREG-positive cells were scored that haddetectable BL Alexa488 secondary antibody (n = 100). B) A majority of AREG expressing LLC-PK1::μ1B cells did not have Ap staining(only 35% showing apical Cy5 staining) (n = 93). The individual channels of the x –z axis are displayed. Bar, 10 μm.

an AREG ectodomain-specific antibody in the BL compart-ment only. This provided the antibody access to AREGonly on the BL surface and ample time for antibody-boundAREG to be endocytosed and then transit through therecycling endosomes back to the cell surface. The cellswere then washed and incubated for 10 min at room tem-perature with two secondary antibodies, anti-mouse-Cy5on the apical surface only and anti-mouse-Alexa488 onthe BL surface only. Primary antibody-bound AREG wasdetected on the apical surface of 80% of LLC-PK1::μ1Acells with positive BL AREG staining (Figure 7A), sug-gesting that these cells, which lack AP-1B, inappropriatelyrecycle AREG from the BL membrane to the apical surface.However, in LLC-PK1::μ1B cells, primary antibody-boundAREG was detected on the apical surface in only 35% ofBL AREG-positive cells (Figure 7B), suggesting the impor-tance of AP-1B in recycling of AREG back to the BLsurface. These data support our hypothesis that AREG isendocytosed from the BL surface and mis-recycled to theapical surface in AP-1B-deficient cells.

Discussion

Previous work from our laboratory demonstrated theimportance of the cytoplasmic domain for polardistribution of AREG (19). However, the requirements forpolarized BL AREG delivery were not described. Thisstudy extends those observations to show that the ACDcontains a dominant BL sorting motif consisting of a mono-leucine C-terminal to an acidic cluster, and show that theadaptor protein, AP-1B, is necessary to maintain the polarBL distribution of AREG.

The 31-amino acid ACD is 35% identical across species(Figure 3). We showed that the cytoplasmic domain issufficient for BL distribution (Figure 1), and using cytoplas-mic domain truncation mutants, we identified amino acids236–246 in the ACD as necessary for the polarized BL dis-tribution of AREG (Figure 2). These amino acids constitute

the most conserved region of the cytoplasmic domain thatis 73% identical and 91% similar across species (Figure 3).In addition, extending the deletion to 4 amino acids didnot significantly disrupt the polarized distribution of AREGfurther than the 14 amino acids (Figure 2C). Of note, thisdeleted region (amino acids 226–235) is only 22% identicalacross species. However, we cannot exclude the possibil-ity that additional information in the juxtamembrane regioncontributes to the steady-state BL localization of AREG.

We previously reported that the juxtamembrane region ofTGFα was sufficient to maintain its BL distribution (34).There are similarities between the amino acid composi-tion of the AREG (QLRRQYVRK) and TGFα (HCCQVRKH)juxtamembrane regions. However, in contrast to TGFα,the presence of this region alone in AREG does not main-tain polar BL distribution (Figure 2) (34). Also, unlike TGFα,AREG BL delivery is not dependent on NKD2, which,in part, binds the juxtamembrane region of TGFα (17).The AREG juxtamembrane region does contain tyro-sine residues at positions 227 and 231. Because tyro-sine residues are established constituents of BL sortingmotifs (35,36), we mutated these tyrosine residues indi-vidually and in combination to alanine residues, but nochange in the AREG polarity was observed (Table 1 anddata not shown). In addition, we confirmed that removal ofall but four amino acids from the cytoplasmic domain stillresulted in 35% of total surface AREG being distributed tothe BL surface (Figure 2C) (19). We believe that this indi-cates the absence of an apical sorting determinant withinthe ectodomain of AREG.

Canonical BL sorting motifs (YXX�, FXNPXY, [DE]XXXL[LI])(35,37) are not present within amino acids 236–246(EERKKLRQENG) of AREG. However, a less well-char-acterized BL sorting motif consisting of a mono-leucineis present. The consensus motif for a mono-leucine BLsorting signal consists of a single leucine five residuesC-terminal to an acidic cluster (EEDXXXXXL) and ispresent in the only other two proteins identified to

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Table 1: ACD and its mutants

Domain mutation Amino acid composition Localization

Wild-type AREG QLRRQYVRKYEGEAEERKKLRQENGNVHAIA BL25-aa AREG QLRRQYVRKYEGEAEERKKLRQENG BL14-aa AREG QLRRQYVRKYEGEA Ap/BL4-aa AREG QLRR Ap/BLL241A QLRRQYVRKYEGEAEERKKARQENGNVHAIA Ap/BLEE236/237AA QLRRQYVRKYEGEAAARKKLRQENGNVHAIA Ap/BLEEL236/237/241A QLRRQYVRKYEGEAAARKKARQENGNVHAIA Ap/BLKK239/240AA QLRRQYVRKYEGEAEERAALRQENGNVHAIA BLY231A QLRRQYVRKAEGEAEERKKLRQENGNVHAIA BLY227A QLRRQAVRKYEGEAEERKKLRQENGNVHAIA BLY227/231A QLRRQAVRKAEGEAEERKKLRQENGNVHAIA BL

The 31-aa ACD (222–252) is aligned with the mutant AREG protein sequences. Alanine substitutions are highlighted in red. Wild-typeand mutant AREG protein surface localization is noted and is based on the analysis performed in Figures 2 and 3, and data not shown.BL localization is indicated as BL and non-polar delivery to both surfaces is indicated as Ap/BL.

contain mono-leucine BL sorting motifs, CD147 andSCF (20,21,38). Although AREG does not contain thisexact consensus motif, it does contain a mono-leucineC-terminal to an acidic cluster (EEXXXL). The AREG BLsorting motif differs from CD147 not only in the con-sensus motif, but also in its dominance over the NGFRapical sorting determinant (Figure 1). CD147 and SCFcan both redirect the apical reporter Tac to the BLsurface, but CD147 is incapable of redirecting NGFRto the BL surface (20,21,24). CD147 also has a muchmore pronounced apical distribution compared to AREGafter mutation of the mono-leucine, with 80% of L252ACD147 distributed apically compared to 45% of L241AAREG (Figure 4C) (20). This may indicate that additionalBL sorting information is present in the ACD, althoughremoval of the ACD results in only 65% apical distribution(Figure 2C). Also, removal of the ACD does not result in astatistically significant difference in apical distribution com-pared to the 59% apical distribution of the mono-leucinemotif mutant (EEAA/LA) (Figure 4C). Another commonal-ity between SCF and CD147 is their formation of homo-and heterodimers, respectively. SCF homodimerizationis necessary for efficient cell surface expression whileCD147 heterodimerization with the proton-coupled mono-carboxylate transporter 1 (MCT1) directs the localizationof MCT1 (24,39). It is unknown if AREG dimerizes.

The AREG mono-leucine BL sorting motif is criticalfor the polarized BL biosynthetic delivery of AREG asdemonstrated by metabolic labeling pulse-chase domain-selective cell surface biotinylation (Figure 6A). Mutation ofthe mono-leucine motif (EEAA/LA) results in delivery ofnewly synthesized EEAA/LA AREG to both apical and BLsurfaces with accumulation on the apical surface. BecauseAREG is more rapidly cleaved from the BL surface than theapical surface (19), pretreatment with the broad-spectrummetalloprotease inhibitor batimastat is necessary for thedetection of newly synthesized EEAA/LA AREG deliveredto the BL surface. Batimastat is not necessary for thedetection of BL wild-type AREG because all of the wild-type AREG is delivered to the BL surface, whereas only

a portion of the EEAA/LA is delivered to the BL surface.However, over time enough EEAA/LA AREG does accu-mulate on the BL surface to result in approximately 40%of total surface EEAA/LA AREG to be on the BL surfaceat steady state (Figure 4C). The dramatic difference insurface distribution of newly synthesized EEAA/LA AREGcompared to wild-type AREG highlights the importance ofthe mono-leucine sorting motif in AREG BL delivery.

The adaptor protein AP-1B is not necessary for the deliveryof newly synthesized wild-type AREG to the BL sur-face (Figure 6B). After a 30-min chase, wild-type AREGis detectable only on the BL surface of μ1B-knockdownMDCK cells. Pretreatment with batimastat is not neces-sary to detect BL AREG in μ1B-knockdown cells, suggest-ing all of the AREG is delivered to the BL surface, similar toμ1B-expressing MDCK cells. Therefore, polar steady-stateBL distribution of AREG is AP-1B-dependent, in that newlysynthesized AREG is delivered to the BL surface in AP-1B-deficient cells and, subsequently, mis-recycled to theapical membrane after internalization (Figures 5–7). Thissuggests a role for AP-1B in the recycling of AREG and notin the biosynthetic delivery in fully polarized MDCK cells,as previous studies have demonstrated for low-densitylipoprotein receptor (LDLR), transferrin receptor (TfR) andCoxsackie-adenovirus receptor (31,32,38,40). These previ-ous studies demonstrated an AP-1B-independent biosyn-thetic route for TfR in a mature, fully polarized monolayer,but an AP-1B-dependent trans-endosomal biosyntheticroute in an immature, recently confluent MDCK mono-layer (31). It should be noted that all our experimentswere performed on mature fully polarized monolayers.

To investigate the possibility that AP-1B affects AREGpolar BL distribution due to regulation of AREG recycling,we performed an antibody binding and transcytosis assayin LLC-PK1::μ1A and LLC-PK1::μ1B cells transfected withAREG. Because the integrity of the monolayer is impor-tant in this type of assay and LLC-PK1 cells are known toform incomplete monolayers, we used stringent criteriato validate the monolayer was fully intact, performing the

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assay only on monolayers that prevented the diffusion ofa molecule 50 times smaller than mouse IgG (3000-MWdextran Texas Red) across the monolayer. Our results sup-port the hypothesis that post-endocytic AREG is missortedto the apical surface in AP-1B-deficient cells (Figure 7), ashas been shown for the LDLR and Coxsackie-adenovirusreceptor (29,40). Future work will determine how AP-1Binteracts with AREG.

Mutant EEAA/LA AREG appears to have aberrant biosyn-thetic delivery to the apical membrane, suggesting thisregion is involved in biosynthetic BL delivery. However,AP-1B does not seem to be involved in AREG biosyntheticdelivery because newly synthesized wild-type AREG ispresent on the BL surface and undetectable on the apicalsurface in MDCK cells with μ1B knocked down. Theseresults are consistent with the idea that an adaptor pro-tein other than AP-1B recognizes the mono-leucine motifand is required for BL delivery of AREG. Currently, μ1Bis thought to mainly interact with tyrosine-based sortingmotifs (27,36,41). However, mutation of the two tyrosineresidues in the ACD does not disrupt AREG BL distributionin MDCK cells (Table 1). AREG may interact with AP-1Bthrough a linker/adaptor protein, as has been recentlyreported for LDLR and the autosomal recessive hyperc-holesterolemia (ARH) protein (42). Extensive efforts by ourlaboratory have yet to identify an interacting protein for theACD. In the case of LDLR, ARH interacts with the proxi-mal FXNPXY motif in the LDLR cytoplasmic domain (43).One possible explanation as to why mutation of the AREGtyrosine residues does not produce the same phenotypeas knockdown of μ1B is that the tyrosine residues are alsoinvolved in the endocytosis of AREG. Mutation of theseresidues could prevent post-endocytic AREG from reach-ing recycling endosomes where mis-sorting to the apicalmembrane can occur (Figure 7). However, the AREG tyro-sine motifs do not resemble the well-characterized NPXYinternalization motif or the YXX� motif demonstrated tointeract with the medium subunit, μ2, of AP-2 (44–47).AREG does contain a YXXXXEE motif, similar to the prox-imal BL sorting motif in LDLR, but mutation of the acidicresidues in the LDLR motif did not affect endocytosis (45).Mutation of the tyrosine in the LDLR proximal motif resultsin apical distribution of truncated LDLR (48), while theY231A mutation in AREG did not affect BL delivery. Also, ifthis YXXXXEE motif in AREG acts as a BL signal, we wouldexpect the L241A mutant to maintain polar BL distribution,as is the case if only one BL motif in LDLR is mutated (48).Future work to determine the AREG motif recognized byAP-1B, the AREG endocytosis motif and identification ofinteracting proteins for the ACD will be addressed.

The goal of this study was to identify the componentsnecessary for the polarized BL distribution of AREG. Wehave demonstrated the presence of a dominant BL sort-ing motif in the ACD that consists of a mono-leucineand an acidic cluster (EEXXXL) and differs from previouslydescribed mono-leucine motifs. We have also revealed arole for AP-1B in the maintenance of AREG polarity.

Materials and Methods

Reagents and antibodiesCell culture media was purchased from Media Tech Inc. and fetalbovine serum from Hyclone Laboratories. All chemicals were purchasedfrom Sigma unless otherwise stated. Sulfo-NHS-LC-Biotin and micro-BSA protein assay kits were purchased from Pierce Biotechnology. Allelectrophoresis reagents were purchased from Bio-Rad Laboratories.Rainbow markers were purchased from Bio-Rad or Fermentas LifeSciences. Enhanced chemiluminescence reagents were purchased fromPerkin Elmer. Nitrocellulose was purchased from Whatman. DNA mini-prep and cleanup kits were purchased from Qiagen Sciences. ProteinG agarose beads were purchased from Invitrogen. All conjugatedsecondary antibodies were purchased from Jackson ImmunoResearchLaboratories Inc. Labeled phalloidin was purchased from Invitrogen.Mouse monoclonal antibody 6R1C2.4 against human AREG wasas previously described (19,49,50). Anti-NGFR p75 (ME20.4) mousemonoclonal antibody was purchased from Santa Cruz Biotechnology Inc.Streptavidin-IR-Dye680 was purchased from LI-COR Biosciences. The μ1B-specific antibodies that do not recognize μ1A were produced in rabbitsimmunized with a synthetic peptide (CK-EKEEVEGRPPIGV) correspondingto the C-terminal 366–271 residues of human μ1B conjugated withmollusk Concholepas concholepas hemocyanin (Blue Carrier; BiosondaBiotechnology) (32). The antibody was affinity purified using the samepeptide linked to Affi15 resin (Bio-Rad). Bound antibodies were eluted with0.1 M of glycine at pH 2.5 and promptly neutralized with 1 M Tris at pH 9.

Cells and cell cultureMDCK II cells were obtained from Enrique Rodriguez-Boulan (Cornell Uni-versity Medical College). MDCK μ1B KD cells were obtained from EnriqueRodriguez-Boulan and were previously described (31). LLC-PK1::μ1A andLLC-PK1::μ1B cells were previously described (33). Cells were cultured aspreviously described (13). Cells were grown on 12 or 24 mm Transwells(0.4 μm pores; Corning Inc.) as previously described (19).

AREG constructsHuman AREG cDNA encoding wild-type pro-AREG was obtained fromDr Greg Plowman (Sugen) (51) and Dr Gary Shipley (Oregon HealthSciences University) (52) and expressed in a pCB6 vector (50). All untaggedconstructs were expressed in a pCB6 vector (53). All EGFP-taggedconstructs were expressed in the Clontech vector pEGFP-N1 (GenBankAccession number U55762). Human NGFR cDNA was obtained from DrAndre Le Bivic (54). A chimera of the extracellular and transmembranedomains of NGFR with the ACD (NGFR-ACD) was constructed by creatinga BsmI site at the transmembrane–cytoplasmic domain junction of NGFR.The cytoplasmic domain of NGFR was removed with a BsmI/XbaI digest.Using PCR, ACD fragment was ligated to the NGFR to create an NGFR-ACDchimera. ACD truncations and amino acid mutations were obtained by PCRQuikChange® site-directed mutagenesis of wild-type pro-AREG in a pCB6vector as per the manufacture’s instructions (Stratagene catalog number200518). All DNA constructs were confirmed by sequencing prior to use.

Domain-selective cell surface biotinylationCells were plated on 12-mm Transwell inserts at a cell density of 1 × 105

cells/Transwell. Four days after plating, the TEER for each Transwell wasconfirmed to be >200 �/cm2. Cells were then treated with 5 mM sodiumbutyrate overnight. On day five, the cells were washed three times withcold PBS containing 0.1 mM CaCl2 and 1.0 mM MgCl2 (1× PBS-CM) onice. All subsequent steps were done on ice or at 4◦. Either the apical orBL cell surface was biotinylated with 0.5 mg/mL biotin in 1× PBS-CM.Cells were incubated with biotin for 20 min and then used biotin wasremoved and replaced with fresh biotin for an additional 20 min. The biotinwas quenched with five washes of 1× PBS-CM, 0.2% BSA, 100 mM

glycine followed by two washes with 1× PBS-CM. Filters were cut fromthe inserts and placed in 250 μL of 1% Nonidet P-40 (NP-40) lysis buffer

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(50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% NP40 and 2 mM ethylenedi-aminetetraacetic acid) plus protease inhibitor cocktail (Sigma P2714) androtated for 30 min. Cell lysates were transferred to new eppendorf tubesand centrifuged for 15 min at 13 000 r.p.m. (18 000 × g). The supernatantswere then transferred to new tubes and rotated for 1 h with 10 μL of50% slurry recombinant Protein G agarose beads to preclear the sam-ples. The protein concentration of each sample was determined using abicinchoninic acid (BCA) protein assay. Equal protein concentration of eachsample was transferred to a new tube with 1 μg of mouse anti-AREG anti-body (6R1C2.4) and rotated overnight. Twenty microliters of recombinantProtein G agarose bead slurry was added to each sample and rotated for3 h. Beads were gently pelleted and washed three times with 1 mL of lysisbuffer. The final pellet was resuspended in 20 μL of 1× bromophenol bluesample buffer (2× sample buffer: 125 mM Tris–HCl pH 6.8, 2% glycerol,4% SDS (w/v) and 0.05% bromophenol blue) and heated at 75◦ for 10 min.Proteins were separated by 12.5% SDS–PAGE and electrophoreticallytransferred to nitrocellulose membranes. All subsequent steps were per-formed with filtered PBS. Membranes were blocked overnight with 1× PBSand 3% BSA. Membranes were probed for 30 min at room temperaturewith streptavidin-IR-Dye680 diluted 1:25 000 in 1× PBS, 3% BSA, 0.1%Tween20 and 0.01% SDS. Membranes were then washed three timeswith 1× PBS, 0.1% Tween and two times with 1× PBS before imagedon an Odyssey® infrared imaging system (LI-COR Biosciences). ODYSSEY®software (version 3.0, LI-COR Biosciences) was used to determine the inte-grated intensity of each band. The integrated intensities for all the bands inboth the apical and BL lanes were added together to obtain a ‘total cell sur-face’ value [(apical integrated intensity) + (BL integrated intensity) = total].The integrated intensities of the bands in either the apical or BL lanes weredivided by the total cell surface value to give the percentage of total value[(apical integrated intensity/total) × 100 = percentage of total on apicalsurface].

Metabolic labeling and pulse-chase analysis followed

by domain-selective cell surface biotinylationTrans35S label (Met-Cys) was purchased from ICN Biomedicals. Five hun-dred thousand MDCK cells were seeded on 24-mm Transwell filters andgrown for 5 days until the TEER reached 200 �/cm2 at which time cellswere treated with 5 mM sodium butyrate overnight. The next day, filterswere starved for 30 min with DMEM (-cys, -met) at 37◦C. One hundredmicroliter puddles of DMEM (-cys, -met) containing Trans35S label (2mCi/mL) were spotted on to Parafilm and filters were placed on top ofthese puddles and incubated for 20 min at 37◦C. The label was thenwashed with DMEM (10% bovine growth serum, BGS) containing 10times excess L-cysteine and L-methionine at 18◦C, and then chased withDMEM/BGS containing 10 times excess L-cysteine and L-methionine forvarying periods of time at 37◦C. After the chase, filters were placed in1× PBS-CM, 0.2% BSA at 4◦C and processed for domain-selective cellsurface biotinylation as described above. After the biotinylation, cells werelysed in 1% NP-40 lysis buffer and the lysates were precleared with 40 μL50% protein G agarose bead slurry (2 h, 4◦C). Twenty microliters of anti-AREG antibody pre-coated beads (1 μg antibody/μL beads) were added andincubated overnight at 4◦C. Beads were washed three times with lysisbuffer followed by elution in 40 μL of buffer containing 5% SDS by boilingfor 5 min. Supernatant was removed and diluted with additional 460 μLof lysis buffer. Twenty microliters of 50% streptavidin-agarose bead slurrywas added to each sample and incubated for 4 h at 4◦C. Beads were againwashed three times with lysis buffer. Samples were finally boiled for 5 minin 20 μL of 2× loading buffer containing 65 mM DTT, resolved on 10%SDS–PAGE, dried and exposed to autoradiographic film to detect labeledcell surface AREG (n = 3).

Immunofluorescence and confocal microscopyLLC-PK1 cells were processed and stained for microscopic analysis aspreviously described (33). Cells were plated on 12-mm Transwell inserts ata cell density of 1 × 105 cells/Transwell. Cells were grown on Transwellsfor 4 days and reached appropriate TEER (MDCK >200 �/cm2, LLC-PK1>400 �/cm2) before staining. MDCK cells were washed three times with

ice-cold PBS-CM and then stained for 1 h on ice with primary antibodydiluted in 1× PBS-CM. Cells were washed six times with ice-cold 1×PBS-CM prior to fixation with 4% paraformaldehyde/PBS-CM for 15 minon ice and then quenched with 50 mM ammonium chloride in 1× PBS-CM for 10 min. Cells were washed and blocked in 1× PBS-CM, 1%BSA and 0.2% fish skin gelatin (blocking buffer) three times over 1 h.Secondary antibodies were diluted 1:200 in blocking buffer and incubatedon cells for 1 h. Cells were washed three times followed by a 10-minwash with blocking buffer plus 0.1% Triton-X-100. Cells were incubatedwith labeled phalloidin diluted 1:200 in blocking buffer for 30 min priorto three washes with blocking buffer and mounting in ProLong® Gold(Invitrogen P36934). Immunofluorescence imaging was acquired on aZeiss LSM 510 confocal microscope (Zeiss Microscope Imaging Inc.) usinga 40× objective with a 2× zoom at 1024 × 1024 resolution. Contents of theimage window were exported as Tiff files using LSM IMAGE BROWSER software(version 4.2.0.121, Carl Zeiss GmbH Jena 1997-2006; Zeiss MicroscopeImaging, Inc.). Tiff files were processed and cropped using ADOBE

PHOTOSHOP software (version 12.0, Adobe Systems Incorporated). Levelsfor each channel were independently modified and include all availabledata.

Transcytosis assayLLC-PK1 cells were plated on 12 mm Transwell inserts at a cell densityof 1 × 105 cells/Transwell. Cells were grown on Transwells for 6 days andreached appropriate TEER (>400 �/cm2). Integrity of the monolayer wasconfirmed using 3000-MW dextran Texas Red (Invitrogen D3328) dilutedto 63 μg/mL in phenol red-free complete media. Labeled dextran wasadded to the apical compartment of the Transwell only and incubated at37◦C for 30 min. One hundred microliters of media was removed from theBL compartment and replaced with fresh media every 5 min. The diffusionof the dextran across the monolayer was measured using a Synergy 4BioTek plate reader and GEN5 OLE AUTOMATION software (version 1.06.10)(BioTek Instruments Inc.). Once the monolayer was determined to beintact, mouse anti-AREG antibody (6R1C2.4) diluted to 4 μg/mL in serum-free media was added to the BL compartment only for 30 min at 37◦C.Cells were washed three times at room temperature with 1× PBS-CMand 1% BSA to remove unbound antibody. In 1× PBS-CM, 1% BSA andAlexa-488-conjugated anti-mouse secondary antibody (1:200) were addedto the BL compartment only, and Cy5-conjugated anti-mouse secondaryantibody (1:200) was added to the apical compartment only. Cells wereincubated with secondary antibodies for 10 min at room temperature,washed three times with 1× PBS-CM and 1% BSA and then fixed with 4%paraformaldehyde/PBS-CM for 15 min at room temperature. Cells werewashed three times with 1× PBS-CM, 1% BSA and then one 15-min washwith 1× PBS-CM, 1% BSA and 0.1% Triton-X-100. F-actin was stainedwith phalloidin-rhodamine (Invitrogen Corp) diluted 1:200 in 1× PBS-CMand 1% BSA for 30 min followed by three washes with 1× PBS-CM, 1%BSA and then mounted with ProLong Gold. Images were acquired asdescribed above.

Quantitative RT-PCRTwo sets of primers were used to determine the mRNA level of canineμ1B in μ1B-knockdown MDCK cells. Primer set 1 (RealTimePrimers.com)(annealing and extension: 61◦C, 45 seconds) contained the forwardprimer 5′-ACA AGA CGG TGG AGG TTT TC-3′ and reverse primer 5′-CCT GCT GCG TGA TGT ACT CT-3′. Primer set 2 (Sigma) (annealing andextension: 65◦C, 45 seconds) was self-designed and contained the forwardprimer 5′-CCT GAT CAG CCG CAA CTA CAA GG-3′ and reverse primer5′-GTA CTC AGA GAA AAC CTC CAC CG-3′. Primers for canine beta-actin (RealTimePrimers.com) (annealing and extension: 61◦C, 45 seconds)contained the forward primer 5′-CCC AGA TCA TGT TCG AGA CT-3′ andreverse primer 5′-CAT GAG GTA GTC GGT CAG GT-3′. Platinum® SYBR®

Green qPCR supermix-UDG (Invitrogen catalog number 11733-038) wasused as per the manufacture’s instructions. Samples were run and analyzedon a StepOnePlus real-time PCR system with STEPONE software version2.1 (Applied Biosystems).

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Statistical analysisAnalysis of variance was used to compare apical distribution of AREG inpolarized MDCK cells. Tukey’s honestly significant difference was used forpair-wise comparisons. Statistical significance was declared for p < 0.05.

Acknowledgments

This work was funded by NCI grants CA046413 and CA095103 to R.J. C. H. F. was supported by GM070736. J. D. G. was supported by theBiochemical and Chemical Training for Cancer Research NCI training grants2 T32 CA009582. Experiments and data analysis were performed in partthrough the use of the VUMC Cell Imaging Shared Resource (supportedby NIH grants CA68485, DK20593, DK58404, HD15052, DK59637 andEY08126). Generation of anti-μ1B antibodies was funded by a CONICYTgrant number PFB-12/2007 to A. G.

Special thanks to Enrique Rodriguez-Boulan for providing the MDCK μ1B-knockdown cells, Gregory D. Ayers in the VICC Division of CancerBiostatistics for statistical analysis and Michelle Demory Beckler forreviewing the manuscript.

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