Article

https://doi.org/10.1038/s41467-023-43931-7

Proximity extracellular protein-protein
interaction analysis of EGFR using AirIDconjugated fragment of antigen binding
Received: 27 January 2023

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Accepted: 24 November 2023

Kohdai Yamada1, Ryouhei Shioya1, Kohei Nishino2, Hirotake Furihata 1,
Atsushi Hijikata3, Mika K. Kaneko 4,5, Yukinari Kato 4,5, Tsuyoshi Shirai
Hidetaka Kosako 2 & Tatsuya Sawasaki 1

6,

Receptor proteins, such as epidermal growth factor receptor (EGFR), interact
with other proteins in the extracellular region of the cell membrane to drive
intracellular signalling. Therefore, analysis of extracellular protein-protein
interactions (exPPIs) is important for understanding the biological function ofreceptor proteins. Here, we present an approach using a proximity biotinylation enzyme (AirID) fusion fragment of antigen binding (FabID) to analyse theproximity exPPIs of EGFR. AirID was C-terminally fused to the Fab fragment
against EGFR (EGFR-FabID), which could then biotinylate the extracellularregion of EGFR in several cell lines. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis indicated that many known EGFR interactors wereidentiﬁed as proximity exPPIs, along with many unknown candidate interactors, using EGFR-FabID. Interestingly, these proximity exPPIs were inﬂuenced by treatment with EGF ligand and its speciﬁc kinase inhibitor, geﬁtinib.
These results indicate that FabID provides accurate proximity exPPI analysis oftarget receptor proteins on cell membranes with ligand and drug responses.

Membrane proteins account for more than 30% of the human proteome and play important roles in many cellular functions, such asenvironmental responses, signal transduction, and cell-cellinteractions1,2. Additionally, the extracellular regions of many membrane proteins interact with other proteins for these cellularfunctions3. Therefore, analysis of extracellular protein–protein interactions (exPPIs) is a key issue for understanding the biological functions of membrane proteins. General methods to identify proteinsinteracting with the target protein include the yeast two-hybrid
system4,5, mass spectrometry analysis after immunoprecipitation6,7,
and cell-free protein arrays8,9. However, these methods are not suitable
for the exPPI analysis of membrane proteins because they have been

designed speciﬁcally for the analysis of soluble proteins localised in
the cytoplasm and nucleus. In addition, because many extracellularregions make speciﬁc specialised structures on the membrane andextracellular environments, exPPI analysis requires an environment
similar to that of the cell membrane.
When considering the exPPI of membrane proteins, it is important
to note that the environment is vastly different outside and inside
the cell membrane. Furthermore, the biological responses of membrane proteins, such as ligand binding and cell-cell interactions, occur
in the extracellular region of the cell membrane. However, because theextracellular domains of many membrane proteins function as regulatory and response regions for localisation, transport, and ligand

1

Division of Cell-Free Life Science, Proteo-Science Center, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan. 2Division of Cell Signaling,
Fujii Memorial Institute of Medical Sciences, Institute of Advanced Medical Sciences, Tokushima University, Tokushima 770-8503, Japan. 3Laboratory ofComputational Genomics, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji 192-0392, Japan. 4Department of Antibody Drug
Development, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan. 5Department of Molecular Pharmacology, Tohoku University
Graduate School of Medicine, Sendai 980-8575, Japan. 6Department of Bioscience, Nagahama Institute of BioScience and Technology, 1266 Tamura,e-mail: kosako@tokushima-u.ac.jp; sawasaki@ehime-u.ac.jp
Nagahama 526-0829, Japan.

Nature Communications | (2023)14:8301

1

Articlebinding, using the native protein without a detectable peptide tag or
insertion protein is the best way to perform exPPI analysis. Under these
conditions, some studies have used a highly speciﬁc monoclonal
antibody (mAb) against the extracellular domains of the target membrane protein10,11 because unnatural modiﬁcations are not required,
and they can be used directly for cell analysis.
Proximity-labelling technology has been widely used to identify
partner proteins12–14. Since proximity labelling detects proteins that arevery close together, it possibly obtains more precise information about
interacting proteins15,16. Currently, several proximity-labelling-based
techniques, such as the enzyme-mediated proximity cell labellingsystem (EXCELL)17, pupylation-based interaction tagging (PUP-IT)18,
engineered ascorbate peroxidase (APEX)19,20, and µMap, have beendeveloped for the interactome analysis of membrane proteins10. These
technologies were conjugated to proximity-labelling probes for atarget-speciﬁc mAb. However, because these methods did not identify
the labelling sites, it is not clear whether the interaction regions
between the target membrane protein and interacting proteins were
exPPIs.
BioID (proximity-dependent biotin identiﬁcation)21–24 has beendeveloped as an enzymatic proximity-labelling technology. Recently,
we developed a new enzyme AirID for the BioID method24,25. However,currently, BioID has not been conjugated to mAb and is used as a direct
tool to analyse exPPIs through genetic insertions26. This genetic
approach requires the insertion of BioID enzymes between thedomains of membrane proteins, which may have a substantial effecton membrane protein function. Therefore, an exPPI analysis using aprotein that fuses a BioID enzyme with an antibody capable of recognising a membrane protein, may be an alternative to the current exPPI
analysis approach.
Here, we show an AirID fusion antibody technology suitable for
exPPI analysis of membrane proteins. We used a speciﬁc AirID-fusion
antibody for exPPI analysis of epidermal growth factor receptor (EGFR)
in cancer cell lines. We produced modiﬁed antibodies by fusing AirID
to the fragment of antigen binding (Fab) region (FabID) or to the fulllength of the antibody (mAbID). We achieved higher biotinylation
efﬁciency with FabID in vitro and in cells than with mAbID. LC-MS/MS
analysis of biotinylated peptides indicated that EGFR-FabID (FabID thatrecognises EGFR) biotinylated the extracellular regions of many
membrane proteins, including well-known EGFR interactors. Geﬁtinib(Iressa), a pharmaceutical compound widely used for the treatment oflung cancer, is a highly speciﬁc EGFR inhibitor for tyrosine kinase
activity in the intracellular region27. Interestingly, these proximity
exPPI interactions were inﬂuenced by the EGF ligand and geﬁtinib
treatment, suggesting that ligand binding and inhibition of receptor
tyrosine kinase (RTK) activity have the potential to change exPPIs.
These results indicate that the FabID system is a useful method for
exPPI analysis of receptor proteins.

Results
Biotinylation of antigen having an epitope AGIA tag by AGIAmAbID or AGIA-FabID
We recently created a proximity biotinylation enzyme, AirID, for the
BioID method, based on an artiﬁcial intelligence algorithm24. Because
AirID provided an analysis of highly speciﬁc PPIs in cells25, we used the
AirID enzyme for proximity biotinylation of antigens. To design an
AirID-fusion antibody, the AGIA-tag system we developed previously
was utilised because the AGIA peptide tag (EEAAGIARP) is speciﬁcallyrecognised by rabbit anti-AGIA mAb28, and we had the gene sets for theconstruction and production of its mAb. Proximity biotinylation prefers a short distance between the antibody and antigen because it
occurs within a very short range (~10 nm)13. To integrate AirID with antiAGIA mAb, we attempted to use two types of antibodies: (1) AGIAmAbID: AirID is fused to the C-terminus of the heavy chain in fulllength IgG antibody or (2) AGIA-FabID: AirID is fused to one of the

Nature Communications | (2023)14:8301

https://doi.org/10.1038/s41467-023-43931-7

fragments of antigen binding instead of the Fc region (Fig. 1a). We
considered the effect of the presence or absence of the fragment
crystallisable region (Fc region) on the biotinylation efﬁciency.
To obtain AGIA-mAbID and AGIA-FabID proteins, they wereexpressed in Expi293F cells and puriﬁed by afﬁnity chromatographyusing a nickel column (Fig. 1b) because they have a 10x-histidine tag attheir C-terminus. Biotinylation of these puriﬁed proteins was mainly
found in the heavy chain but not in each light chain (Fig. 1c). To checkthe biotinylation ability to the antigen (Fig. 1d), FLAG-GST fused to the
C-terminal region of human DRD1 (FLAG-GST-DRD1 CTD) was usedbecause the anti-AGIA mAb recognised it as the epitope28. Immunoblotting with an anti-biotin antibody showed that antigen biotinylation
by AGIA-FabID was higher than that by AGIA-mAbID (arrowhead in
Fig. 1e). AGIA-FabID also biotinylated other proteins with an AGIA tag(Fig. 1f). Furthermore, to investigate whether AGIA-FabID could biotinylate a protein interacting with an antigen, an interaction between
AGIA-tagged p53 and FLAG-GST-fusion MDM2 (FG-MDM2), previously
used to check proximity biotinylation as a PPI model24, was used
(Fig. 1g). AGIA-FabID biotinylated both AGIA-p53 and FG-MDM2(Fig. 1h) but did not biotinylate FLAG-p53 and FG-MDM2. Taken
together, these results showed that FabID could biotinylate bothantigens and their interacting proteins.
We previously showed that AirID functions in cells24,25. Since thepH conditions of the extracellular environment (pH ~ 7.429) differ fromthose of the intracellular environment, we investigated the biotinylation activity of AGIA-FabID under various pH conditions. These results
showed that AGIA-FabID has biotinylation activity between pH 7 and 8(Supplementary Fig. 1a), suggesting that FabID can be used in this pH
range, mimicking extracellular conditions.

Biotinylation of EGFR by EGFR-FabID or EGFR-mAbID
EGFR is a critical signalling mediator in many epithelial cancers30. EGFR
consists of extracellular, transmembrane, and intracellular domains,with the C-terminal domain having tyrosine kinase activity. When aligand such as EGF or TGF-α binds to EGFR, the structure of theextracellular domain of EGFR is altered, and EGFR forms a homodimer
with other EGFR or heterodimers with other ERBB family members.
This dimerisation activates the intracellular tyrosine kinase domain,which phosphorylates the tyrosine residues in the C-terminal domain
and transmits signals downstream31. In addition, EGFR interacts withmany membrane proteins32,33. However, it is often unclear whether
these interactions occur in the extracellular or intracellular regions of
EGFR. Thus, we used EGFR as a model membrane protein for proximity
exPPI analysis. To make a proximity biotinylation probe, we combined
AirID and a previously reported anti-human EGFR mouse mAb (clone
EMab-134) because it showed high afﬁnity and selectivity for the
extracellular epitope (epitope region no. 377–386) of EGFR34,35. The
EGFR-FabID and EGFR-mAbID were constructed similarly to the antiAGIA mAb shown in Fig. 1. Both EGFR-FabID and EGFR-mAbID were
puriﬁed and conﬁrmed to make disulﬁde bonds on the Fab and IgG(Fig. 2a). Full-length human EGFR was synthesised using a nonreducing wheat cell-free protein production system because its
extracellular region (25–645) has S-S bonds36,37. Biotinylation assays
showed that full-length human EGFR was biotinylated in vitro by EGFRFabID and EGFR-mAbID (Fig. 2b) but not by negative controls, AGIAFabID and AGIA-mAbID.
In a method using an AirID-fused antibody, the accessibility of the
AirID-antibody is a very important factor for increasing the detection
sensitivity. Many proximity biotinylation methods use suspension cells10
because antibodies can access the entire surface area of suspension
cells. Accordingly, Expi293F cells were used in suspension culture
conditions and transfected with a plasmid containing the human EGFR
gene (EGFR) or pcDNA3.1 empty plasmid (Mock). To validate the biotinylation ability of cells, EGFR-FabID or EGFR-mAbID was added to theserum-free medium and incubated with Expi293F cells transiently2Article(kDa)

250150-Proxymetry
biotinylation
enzyme

AirID

AirID

AirID

Anti-AGIA
antibody(kDa)
250150-250150-10075-

10075-

50-

50-

37-

37-

25-

25-

250150-

100-

100-

75-

75-

AGIA-mAbID
Heavy chain
AGIA-FabID
Heavy chain

AGIA-mAbID
50-

Fusion50-

AirID

Anti-AGIA
Fab

37-

37-

AGIA-FabID

d

e

AirID

B
Biotin

+
+

+
-

1

AirID

AGIA-FabID

+ -

+ -

+

37-

FLAG-GSTDRD1 CTD

25-

IB:Biotin

250150100755037-

AGIA-FabID
AGIA-OTULIN
FLAG-GST-DRD1
CTD

2520-

AGIA-SHB

15-

IB:AGIA10-

IB: Biotin

ID

Air

100-

Air

ID

B

-

(kDa)

37-

B

AirID

TD

50-

Protein AGIA

Protein AGIA

C

R
N
LI
-D
B
TU
H
ST
O
G
-S
G
IA
IA
A
G
G
A
A
FL

+
+

50-

AGIA-mAbID

IB:His

D

75-

AirID

B

f

(kDa)
250150100-

Protein AGIA25-

Non reduce

FLAG-GST-DRD1 CTD

AGIA-FabID +AGIA-mAbID Biotin -AGIA-FabID

Protein AGIA

Light chain

25-

IB:Biotin

Reduce75-5037-

Biotin
IB:His50-ghFLAG GST(kDa)

AG
IAAG FabI
D
I AmA
bID

Fusionc

AG
IA
AG -Fab
IAI
mA D
bID

AG
IA
AG -FabI
IAmA D
bID

b

a

AG
IAFa
bID
AG
IAmA
b ID

https://doi.org/10.1038/s41467-023-43931-7(kDa)

250150-AGIA

p53

IB:Biotin- + ++ - + - +- + FLAG-GST-MDM2

10075-

AGIA-FabID
AGIA-p53

B

AirID

IB: AGIA

AGIA-FabID

AGIA-p53 +FLAG-p53 FLAG-GST-MDM2 +FLAG-LHBs -

MDM2

25-

50-

BiotinFLAG-LHB
37150-

FLAG

100-

B
GST

MDM2
AGIA

p53

FLAG-GST-MDM2

75-

AirID

IB:FLAGFLAG-p53
50-

B

FLAG-LHBs
37-

IB:AGIA
5075-

IB:His50-

overexpressing EGFR for 2 h (Fig. 2c). Similar to the in vitro experiments
above, both EGFR-FabID and EGFR-mAbID clearly biotinylated EGFR,
whereas AGIA-FabID did not. A comparison of both probes indicatedthat the biotinylation efﬁciency of EGFR-FabID was much higher thanthat of EGFR-mAbID. The biotinylation of EGFR was also conﬁrmed
using a streptavidin-pull-down assay (STA-PDA in Fig. 2c).

Nature Communications | (2023)14:8301

To understand why EGFR was more biotinylated in cells treatedwith EGFR-FabID than with EGFR-mAbID, we performed structuralmodelling of FabID and mAbID with the extracellular region of EGFR,
as previously reported36. The modelling showed that the proximity
distance between the extracellular domain and EGFR-FabID (Fig. 2d) or
EGFR-mAbID (Fig. 2e) is 9.1 and 13.9 nm respectively, indicating that3

Articlehttps://doi.org/10.1038/s41467-023-43931-7

Fig. 1 | Biotinylation of AGIA-tag protein by AGIA-FabID and AGIA-mAbID.
a Schematic of AGIA antibody fused with the proximity biotinylation enzyme AirID.
AirID is fused to AGIA-Fab or AGIA-mAb, b SDS-PAGE and Coomassie Brilliant Blue
(CBB) staining in reducing and non-reducing state of AGIA-FabID and AGIA-mAbID
synthesised by Expi293F expression system and puriﬁed by Ni Sepharose excel.
c Detection of self-biotinylation of AGIA-FabID and AGIA-mAbID by immunoblotting using an anti-biotin antibody. Detection of AGIA-FabID and AGIA-mAbID using
an anti-His tag antibody. d Schematic of biotinylation targeting the AGIA-tagged

protein using AGIA-FabID and AGIA-mAbID. e Comparison of the activities of AGIAFabID and AGIA-mAbID targeting FLAG-GST-DRD1 CTD (FLAG-GST fused to the
C-terminus of DRD1) in vitro. f Targeting various AGIA-tagged proteins biotinylated
by AGIA-FabID. g Schematic of the biotinylation-targeting complex of p53-MDM2
using AGIA-FabID. h Conﬁrmation of biotinylation of FLAG-GST-MDM2 and AGIAp53 by immunoblotting blotting. The SHB and LHB are S-protein of hepatitis B
virus. b, c, e, f, h, The experiment was independently repeated twice, with similar
results. Source data are provided as a Source Data ﬁle.

EGFR-FabID is less distant than EGFR-mAbID and thus may have greater
biotinylation efﬁciency than EGFR-mAbID.
TurboID, another highly active biotinylation enzyme22, was also
fused with the same Fab against EGFR. The expression of TurboIDfusion Fab was lower than AirID-based EGFR-FabID in Expi293F cells(IB: His in Supplementary Fig. 1b), whereas high biotinylated proteins
were found in the culture medium expressing TurboID-based EGFRFabID (IB: Biotin). Puriﬁed TurboID-based EGFR-FabID also showed
higher biotinylation than AirID-based EGFR-FabID in the heavy chain
fragment (Supplementary Fig. 1c). Moreover, in vitro biotinylation
activity of TurboID-based EGFR-FabID was much lower than that of
AirID-based EGFR-FabID (arrowhead in Supplementary Fig. 1d).
Although TurboID-based EGFR-FabID was attempted, we could not
purify TurboID-fused cells from the culture media of Expi293F cells. As
shown in Fig. 1, AirID-based AGIA-FabID was puriﬁed and had highproximity biotinylation activity. Taken together, these results suggestthat AirID is suitable for fabricating Fab-fusion molecules.
We performed ﬂow cytometry to determine whether EGFR-FabID
recognises EGFR on the cell surface and biotinylates proteins on theplasma membrane. Expi293F cells stably expressing EGFR were biotinylated with EGFR-FabID for 2 h, stained with STA-Alexa488, and its
ﬂuorescence was measured using FACS (Supplementary Fig. 2a). The
results showed an increase in the population of cells biotinylated on
the cell surface by treatment with EGFR-FabID and biotin. This suggested that EGFR-FabID biotinylated the cell surface after binding to
EGFR. An MTS assay using A431 cells was also performed to verify thecytotoxicity of EGFR-FabID (Supplementary Fig. 2b). The results of the
MTS assay conﬁrmed that addition of EGFR-FabID or AGIA-FabID did
not affect cell survival.
Next, to conﬁrm whether biotinylation by EGFR-FabID occurs in
the extracellular region, biotinylated peptides were puriﬁed by using
Tamavidin 2-REV and analysed by LC-MS/MS according to previous
reports38,39. MS analysis indicated that EGFR-FabID biotinylated ﬁve
and seven lysine residues in the extracellular and intracellular regionsof EGFR, respectively (Fig. 2f). Furthermore, since the anti-EGFR antibody recognises an epitope around Ser380 in the extracellular region34
and the 3D structure of the extracellular region of EGFR has beenreported (PDB code: 1IVO [https://doi.org/10.2210/pdb1ivo/pdb])36, weestimated the distance between the binding site of EGFR-FabID andbiotinylation sites. These ﬁve biotinylation sites in the extracellularregion were localised within 5 nm of the epitope (Fig. 2g). These
interval values seem reasonable because the biotinylation range of the
BioID enzyme is presumably ~10 nm13, strongly suggesting that EGFRFabID binds to the epitope and subsequently biotinylates it. Taken
together, these results indicated that EGFR-FabID functions as a speciﬁc proximity biotinylation probe in the extracellular region.

(Fig. 3a). Further, when AGIA-FabID was used as a negative control, 189
peptides were signiﬁcantly biotinylated by EGFR-FabID (P < 0.05 and
EGFR-FabID/AGIA-FabID Ratios >1 in Supplementary Data 1). Since ourdetection method using Tamavidin 2-REV could identify biotinylationsites on proteins38,39, we analysed whether the biotinylation sites werelocalised in the extracellular regions using TMHMM (https://services.
healthtech.dtu.dk/service.php?/TMHMM-2.0/)40 and Uniprot (https://
uniprot.org) databases. Statistically predominant biotinylation sites of
64 peptides were found in the extracellular region (red dots in Fig. 3b),and the remaining 125 peptides were localised in intracellular or
unknown cellular regions (black dots, Supplementary Data 1).
Further analysis focused on the 22 proteins that were biotinylated
in their extracellular region because EGFR-FabID recognised theextracellular epitope. Based on the IntAct database, which includes
EGFR-interacting proteins (https://ebi.ac.uk/intact/)41, 12 proteins were
known to interact with EGFR (see black characters in Fig. 3c). Twelveout of 22 proteins (55%) showed high follow-up capability with recorded evidence, suggesting that the remaining 10 proteins were also
unknown interacting or proximal proteins (see blue characters in
Fig. 3c). To compare the proximal exPPIs with the existing PPI database, these interactions were analysed using the Drug Target Excavator(DTX) (https://harrier.nagahama-i-bio.ac.jp/dtx/). This database
depicts human protein/metabolite interaction pathways between agiven pair of proteins. For DTX analysis, a direct protein-protein
interaction pathway is presented as one edge. If a protein-protein
interaction pathway had two edges, it indicated that the protein
interaction was mediated by another protein. Proximal exPPIs detected by EGFR-FabID were found within three edges (see the DTX edge in
Fig. 3c). In addition, pathway analysis of the 22 proteins showed individual interactions with EGFR rather than multiple interactions,forming a large network (Fig. 3d). Taken together, these results indicate that EGFR-FabID provides a proximity interactome of extracellularregions between EGFR and other membrane proteins in transiently
EGFR-overexpressing suspended cells.

Proximity exPPI analysis of EGFR by EGFR-FabID on suspension
culture Expi293F cells overexpressing EGFR transiently
As shown in Fig. 2, EGFR-FabID catalysed extracellular biotinylation of
EGFR by incubation with serum-free medium suspension of Expi293F
cells transiently overexpressing EGFR. EGFR reportedly interacts withother membrane proteins32,33. LC-MS/MS analysis to determine thebiotinylation of other proteins revealed that EGFR-FabID biotinylated
639 peptides in transiently EGFR-overexpressing Expi293F cells

Nature Communications | (2023)14:8301

Biotinylation analysis of endogenous EGFR by EGFR-FabID in
human A431 cells
Next, we attempted to determine whether EGFR-FabID works on
endogenous EGFR located on the surface of adherent cells. To selectan adherent cell type, the protein level of endogenous EGFR and thereactivity of EGFR-FabID were investigated using immunoblotting
and STA-PDA. The anti-EGFR (clone EMab-134) reacted with the
EGFR protein in eight cell lines (Supplementary Fig. 2c). The
expression level of EGFR protein was the highest in human squamous carcinoma epithelial cells (A431), whereas it was not detected
in HEK293T and Expi293F cells. Comparison of EGFR expression
indicated that EGFR protein expression in A431 cells was higher thanthat in stably EGFR-overexpressing Expi293F cells and similar to thatin transiently EGFR-overexpressing Expi293F. To validate the performance of EGFR-FabID, we compared ﬁve cell lines with different
EGFR expression levels: A431 (high), Expi293F-EGFR stable (middle),
HeLaS3 (low), NCI-H1975 (low), and NCI-H226 (low) cells. Immunoblotting showed that EGFR biotinylation by EGFR-FabID was found in
all lysates (red arrowheads in Fig. 4a); this was conﬁrmed using STAPDA. In addition, immunostaining indicated that EGFR-FabID
4

Articlehttps://doi.org/10.1038/s41467-023-43931-7

(kDa)

EG
FR
EG -Fab
ID
FR
-m
Ab
ID

EG
FR
EG -Fab
ID
FR
-m
Ab
ID

a

b

(kDa)
250-

250-

c

Mock EGFREGFR-FabID + - - + EGFR-mAbID - + - - +AGIA-FabID - - + - -EGFR

EGFR-FabID
AGIA-FabID
EGFR-mAbID
AGIA-mAbID
(kDa)
250-

- + + - - - - - +

+
-

(kDa)
250150-

EGFR

150-

150-

150100-

100-

100-

75-

75-

75-

EGFR

10075-

50-

Input
37-

IB:Biotin
50250150-

503737-50-

IB:Biotin

250150-

IB:EGFR

50-

IB:Tubulin

10075-

75-

25-

Reduce

IB:EGFR

250150-

100-

25-

100-

50-

STA-PDANon reduce

37-

IB:Biotin

IB:His50-de
AirID EGFR-FabID

250150-

IB:EGFREGFR-mAbID

EGFR

EGFR

9.1 nm13.9 nm

AirID

g

f
RegionBiotinylationsite (K)

Extracellular

133
479
454
487
489

Intracellular

716
745
754
739
846
852
1188

Distancefrom Ser380 (nm)
4.5
4.5
4.5
3.1
4.2

biotinylated EGFR on the extracellular region of the cell membranein A431 cells (Fig. 4b), whereas AGIA-FabID did not. These results
suggest that EGFR-FabID biotinylates endogenous EGFR in cells at
low and high expression levels. We next performed experiments
using the well-known endocytosis inhibitor Dynasore to check that
EGFR-FabID works under conditions in which endocytosis does notoccur. After treatment of non-small-cell lung cancer-derived NCI-

Nature Communications | (2023)14:8301

K133

S380

K489

K487

K479
K454

H226 cells with Dynasore, the cells were biotinylated with EGFRFabID (Supplementary Fig. 2d). The immunoblotting of cell lysates
showed that EGFR was biotinylated even in the endocytosisinhibited cells and the band intensity was similar between treatments with and without Dynasore. This indicates that endocytosis is
not essential for biotinylation of EGFR-FabID and the majority ofbiotinylated EGFR is present at the cell surface.
5Articlehttps://doi.org/10.1038/s41467-023-43931-7

Fig. 2 | Biotinylation analysis of EGFR by EGFR- FabID on the cell membrane.
a SDS-PAGE of EGFR-FabID and EGFR-mAbID synthesised by the Expi293F expression system, puriﬁed by Ni Sepharose in reducing and non-reducing states, andstained with CBB. b Biotinylation of EGFR using EGFR-FabID and EGFR-mAbID
in vitro. EGFR was synthesised using a wheat cell-free protein synthesis systembased Disulﬁde Bond PLUS Expression Kit. c Immunoblot analysis of EGFR biotinylated by EGFR-FabID and EGFR-mAbID on the plasma membrane using EGFRoverexpressing Expi293F cells. a–c The experiment was independently repeated
twice, with similar results. d EGFR-FabID modelling diagram calculated fromstructural information and AlphaFold. Green represents EGFR, and yellow and blue
represent anti-EGFR Fab. Pink represents AirID. e EGFR-mAbID modelling diagram
calculated from structural information and AlphaFold. Green represents EGFR;
grey, and blue represent anti-EGFR mAbs. Pink represents AirID. f Biotinylation siteof EGFR detected by LC-MS/MS; the intermolecular distance between S380 of
EMab-134 epitope and biotinylated lysine residue was measured by PyMOL basedon structural information (PDB 1IVO [https://doi.org/10.2210/pdb1igt/pdb]) and(g), biotinylation sites of extracellular domain EGFR. Source data are provided as a
Source Data ﬁle.

Many researchers have used A431 cells for EGFR analysis because
the EGFR gene has been cloned from this cell line42. In addition, EGFRFabID clearly biotinylated the endogenous EGFR protein in A431 cells(Fig. 4b). Based on these results, further analysis of exPPI interactions
using EGFR-FabID on adherent cells was focused on A431 cells.
Previous structural analysis data by Ogiso et al. suggested that site
1 in domain 1 and sites 2 and 3 in domain 3 were important for EGF
recognition by EGFR36. EGFR recognition site 2 in EGFR-FabID was
located between amino acids 377–386 in domain 3. We examined theeffect of FabID fusion on EGF/EGFR binding because the EGFR-FabID
recognition site was located at site 2. We examined whether the
addition of EGFR-FabID affected the EGFR ligand response using geﬁtinib. In A431 cells, after EGF ligand treatment with or without geﬁtinib,
EGFR-FabID was supplied to the cells. Using the phospho-EGFR speciﬁc
antibody, immunoblotting indicated that the kinase activity of EGFR
dramatically increased by the treatment of EGF ligand in the presenceof EGFR-FabID (Fig. 4c) and was completely inhibited by geﬁtinib
treatment. Notably, the large β-sheets at sites 1 and 3 of EGFR were
important for the interaction between EGFR and the EGFR ligand TGFα, which had a structure similar to that of EGF37. Binding of EGFR-FabID
to site 2 did not decrease EGFR phosphorylation, suggesting that the
EGF-EGFR complex was stable during the binding of EGFR sites 1 and 3
to EGF. These reports would support the understanding of the resultsthat EGFR-FabID did not affect EGFR activity after treatment with the
EGF ligand.
Considering these responses of EGFR in A431 cells, biotinylationof EGFR by EGFR-FabID was attempted under three conditions, with orwithout EGF ligand and with geﬁtinib treatment in the presence of EGF.
Biotinylation of EGFR was analysed using LC-MS/MS, as shown in Fig. 2.
Interestingly, three biotinylated peptides on K454, K479, and K487 orK489 were found under all conditions, and the K489-biotinylated
peptide increased on treatment with EGF ligand (red dots in Fig. 4d).
Two biotinylated peptides on K479 and K487 or 489 increased ontreating EGFR with geﬁtinib and EGF ligand (Fig. 4e), indicating thatbiotinylation sites changed in the presence or absence of EGF ligand or
the presence of a kinase inhibitor. These results suggest that K489 orK479 biotinylation could be a marker of EGF binding or geﬁtinib
treatment of EGFR using EGFR-FabID, respectively.
As shown in Fig. 2, biotinylation sites of EGFR from transiently
EGFR-overexpressing Expi293F cells were analysed and compared withbiotinylation sites on endogenous EGFR in A431 cells. In the extracellular region, almost the same pattern of biotinylation sites wasobserved (Supplementary Fig. 3a). Interestingly, the intracellularregion of endogenous EGFR in A431 cells was not biotinylated by EGFRFabID. However, in transiently EGFR-overexpressing Expi293F cells,
the intracellular region was biotinylated at seven sites (black dots in
Fig. 4c and Supplementary Fig. 3b), suggesting that transiently overexpressed EGFR proteins contain a mixture of forms that differ inconformation from endogenous EGFR proteins on the plasma membrane. We performed MS analysis of Expi293F stably expressing EGFR
cells to determine whether transient overexpression of EGFR causes
biotinylation of the intracellular domain of EGFR by EGFR-FabID(Supplementary Data 2). We found no biotinylation of the intracellulardomain of EGFR in stably expressing Expi293F cells. All biotinylated

lysine residues were in the extracellular domain of EGFR (Supplementary Fig. 3a). This result suggested that the intracellular domain of
EGFR was biotinylated due to its transient overexpression. Taken
together, these results indicate that EGFR-FabID proﬁling of biotinylation sites on the extracellular region could help understand thevarious conformations of EGFR on the cell membrane.

Nature Communications | (2023)14:8301

Total proximity exPPI analysis using EGFR-FabID among threecell lines
The biotinylation sites of the EGFR protein were detectable in adherent
A431 cells (Fig. 4). Therefore, to evaluate the potential versatility of
EGFR-FabID in adherent cells and the commonality of proximity proteins of EGFR, the human lung squamous cell carcinoma cell line NCIH226 was also used for EGFR exPPI analysis as a new adherent cancer
cell line. Biotinylated proteins from A431 and NCI-H226 cells, with orwithout EGF ligand and with geﬁtinib treatment in the presence of EGF,
were analysed (Fig. 5a, Supplementary Data 3, 4). LC-MS/MS analysis
revealed 481 and 849 biotinylated peptides from A431 and NCI-H226
cells, respectively, under the three treatment conditions. The biotinylation site in the intracellular region of EGFR was also not found in
NCI-H226 cell lines expressing endogenous EGFR (Supplementary
Fig. 3c), similar to A431 cells. The location of biotinylation sites was
identiﬁed using TMHMM (http://www.cbs.dtu.dk/services/TMHMM/)
and Uniprot (https://uniprot.org) databases; 216 and 347 biotinylated
peptides from A431 and NCI-H226 cells, respectively, were annotatedas peptides on the cell surface (red characters in Fig. 5a and red dots in
Fig. 6a). The remaining peptides were localised in intracellular or
unknown cellular regions (Supplementary Data 3, Supplementary
Data 4). Finally, of these extracellular biotinylated peptides, 44 and 66
proteins, including EGFR, were found to be membrane proteinsshowing proximal exPPI with EGFR in A431 and NCI-H226 cells,
respectively (Supplementary Fig. 4a). The DTX analysis revealed 15 and16 proteins from A431 and NCI-H226 cells, respectively, interactingwith EGFR. The remaining 29 and 50 proteins from the A431 and NCIH226 cells, respectively, were unknown proximal exPPI candidates(denoted by blue numbers and protein names in Fig. 5a and Supplementary Fig. 4a).
Commonality analysis was performed based on the biotinylated
protein data from the three cell lines (EGFR-overexpressing Expi293F,
A431, and NCI-H226). Except for EGFR, ﬁve proteins: EEF1A1 (elongation factor 1-alpha 1), ENO1 (alpha-enolase), MICA (MHC class I
polypeptide-related sequence A), INSR (insulin receptor), and CD44
(cell differentiation 44), were the most common proximal exPPI proteins, of which EEF1A133 and ENO143 were known EGFR interactors (seeblack characters in Fig. 5b), and MICA, INSR, and CD44 were unknown
ones (see blue characters). Surprisingly, the translation elongation
factor EEF1A1 was biotinylated by EGFR-FabID. However, the noncytotoxicity of EGFR-FabID was conﬁrmed by MTS (Supplementary
Fig. 2b). This suggested that the biotinylated EEF1A1 detected was notthat of lysed cells. Although EEF1A1 was a translation elongation factor,
it has also been found on the plasma membrane44. Then we examinedthe co-localisation of EGFR with EEF1A1 that was biotinylated in all of
the three cell lines (Supplementary Fig. 10b). Immunostaining without
permeabilization conﬁrmed the co-localisation of EEF1A1 and EGFR on
6

Articlehttps://doi.org/10.1038/s41467-023-43931-7

a

Known
HasTM or Cell membrane or membrane

Expi293F

AirID

B

B

AirID

AirID

LC-MS/MSwith Tamavidin

639

P<0.05
Ratio>1

Identification of peptides
biotinylation sites detected

EGFRTransfection FabID

189

22

PeptidesProteins

125

New proteinsc6

Proteinname-Log10(p-value)

1210Intracellular

pcDNA3.1
-EGFR

b

64

4

2

P=0.050-50

5Log2(EGFR-FabID/AGIA-FabID)d

SLC39A10
CDH2
CD44
DSG2

MICA

P27824CANX
Q02487DSC2Q14126
DSG2
P68104EEF1A1
P00533
EGFR
P06733ENO1
P15311
EZR
P04406GAPDH
HSP90AA1 P07900
HSP90AB1 P08238
P11142HSPA8SLC25A5 P05141
P16070
CD44
P19022
CDH2
P22223
CDH3P07814
EPRS
P06213
INSR
Q29983MICA
Q9HC56PCDH9Q99623PHB2SLC39A10 Q9ULF5P21796VDAC1

DTX edge
1
2
2
1
0
2
1
1
1
1
1
1
2
2
2
2
2
2
3
2
3
2

EZR

ENO1
HSPA8

CDH3

HSP90AB1

GAPDH
EGFR

CANX
INSR

HSP90AA1
EEF1A1
SLC25A5
PHB2

Uniprot
code

VDAC1

EPRS
PCDH9

Directly connected
Connected via one nodeConnected via two nodes
Not connected

DSC2

Fig. 3 | Proximity extracellular interactome of over-expressing EGFR by EGFRFabID on cells. a Workﬂow of PPI analysis targeting EGFR overexpression. LC-MS/
MS identiﬁed 640 biotinylated peptides, of which 189 were selected as those with
AGIA-FabID ratios >1 and P < 0.05. The number of peptides was then narrowed to 64
peptides that had transmembrane (TM) regions or contained “membranes” in thegene ontology (GO) term. 64 peptides were matched into 22 proteins. Twelve of the
22 proteins were known to interact with EGFR, and 10 were novel EGFR-interacting
proteins. b Volcano blot (3 biological replicates) of peptides detected as biotinylated peptides by LC-MS/MS in (a). Peptides derived from cell membrane proteins
are indicated by red dots. c Table of extracellular proteins identiﬁed by mass
spectrometry (three biological replicates, EGFR-FabID/AGIA-FabID ratios >1 and

P < 0.05). The number of DTX edges for each protein and EGFR are shown in thetable. Proteins in black font are those already known to interact with EGFR,
whereas those in blue represent unknown EGFR-interacting proteins. d Pathway
analysis of extracellular proteins detected using mass spectrometry. The Gene
Ontology software Drug Target Excavator (DTX) (https://harrier.nagahama-i-bio.
ac.jp/dtx/) and IntAct database, including EGFR-interacting proteins (https:///ebi.ac.uk/intact/), were used to analyse protein interactions. b, c Signiﬁcantchanges in the volcano plots and heat map were calculated by Student’s twosided t test and the false discovery rate (FDR)-adjusted P-values calculated using
Benjamini–Hochberg method are shown in the Supplementary Data 1.

the cell membrane. These results suggested that EGFR-FabID biotinylates EEF1A1 on the plasma membrane. In addition, 15 common proteins were found between the A431 and NCI-H226 cell lines andconsisted of 6 known and 9 unknown proteins. Although Expi293F
cells overexpressing EGFR are ﬂoating cells and the remaining two celllines, A431 and NCI-H226, are adherent cells, common proteins,
including known interactors, were found between each cell, indicatingthat EGFR-FabID has similar functions in both adherent and nonadherent cells. The remaining 8, 19, and 41 proteins were independently observed in each cell line, suggesting that exPPIs with EGFR may

Nature Communications | (2023)14:8301

7

Articlediffer by cell type. The proximity exPPI results of the three cell lines
with EGFR-FabID revealed many known interacting proteins, suggesting that EGFR exPPI may occur in complex with known interacting
proteins.
To further compare the proximity exPPIs with the existing PPI
database, these interactions were analysed using DTX [https://harrier.
nagahama-i-bio.ac.jp/dtx/)]. Interaction network diagrams of

Nature Communications | (2023)14:8301

https://doi.org/10.1038/s41467-023-43931-7

biotinylated proteins in A431 and NCI-H226 cells showed that many
proteins interacted with EGFR within three nodes (Fig. 5c). In A431
cells, all but DSG3 interacted with EGFR within two nodes. Some biotinylated proteins also interacted with each other. In NCI-H226 cells, allproteins, except L1CAM, interacted with EGFR within two nodes. Theinteraction network diagram, including proteins mediating interactions with EGFR, also conﬁrmed that some biotinylated proteins8Articlehttps://doi.org/10.1038/s41467-023-43931-7

Fig. 4 | Biotinylation analysis of endogenous EGFR by EGFR-FabID in humanA431 cells. a Streptavidin pull-down assay of endogenous EGFR on the plasma
membrane biotinylated by EGFR-FabID in various cells. Red arrowheads indicate
the molecular weight of EGFR. b Immunostaining with EGFR-FabID in A431 cells.
Control-treated areas included those without FabID treatment and those treatedwith AGIA-FabID. Scale bar represents 50 µm. c Kinase assay using A431 cells.
Phosphorylation of EGFR and STAT3 activated by EGF stimulation was then conﬁrmed by immunoblotting. a–c The experiment was independently repeated twice,with similar results. d Volcano plot of biotinylated EGFR peptides identiﬁed by mass

spectrometry (three biological replicates). Red dots represent peptides derived
from the extracellular domain of EGFR, and black dots represent peptides derived
from the intracellular domain. Signiﬁcant changes in the volcano plots and heat
map were calculated by Student’s two-sided t test and the false discovery rate(FDR)-adjusted P-values calculated using Benjamini–Hochberg method are shownin the Supplementary Data 1, 3. e Heat map showing changes in biotinylated peptidesites upon EGF and geﬁtinib treatment of A431 cells (three biological replicates).
Schematic of the changes in the EGFR biotinylated site. Error bars represent standard deviations. Source data are provided as a Source Data ﬁle.

mediate common molecules in the three cell lines (Supplementary
Fig. 4b). Taken together, these results indicate that this approach using
EGFR-FabID could be used to analyse the proximal extracellularinteracting proteins of EGFR on the cell membranes of both adherent
and ﬂoating cells.
The EGF ligand was not detected by mass spectrometry as a biotinylated peptide in A431 and NCI-H226 cells. To understand why EGF
was not biotinylated by EGFR-FabID, a model illustration of EGFRFabID bound to the EGFR-EGF complex was generated using its crystal
structure and AlphaFold (Supplementary Fig. 5a). The proximity biotinylation enzyme biotinylated accessible lysines that were exposed on
the protein surface by releasing short-lived, hydrophilic biotinoyl-5’AMP. The amino acid sequence of the EGF ligand contained lysine
residues at positions 28 and 48. The model ﬁgure suggested that K48,
which was located inside EGFR, was not easily accessed by the proximity biotinylation enzyme. Furthermore, K28 was located at theboundary between the light chains of EGFR-FabID and EGFR, which was
also potentially difﬁcult for hydrophilic biotinoyl-5’-AMP to access. We
then examined whether EGFR-FabID could biotinylate the ligand iflysine residues are present on the surface of the ligand. To ensurelysine sites for biotinylation, we used the FLAG-GST (glutathione Stransferase) tag was used because it has totally 23 lysine residues. We
then constructed a vector of FLAG-GST-EGF where a TEV-His-AGIA-GS
linker inserted between FLAG-GST and EGF. The FLAG-GST-EGF ligands
induced the phosphorylation of EGFR (IB: P-EGFR1173 in Supplementary Fig. 5b), indicating the activity as a ligand. The cells were cultured
for two days after transfection with this vector and biotinylated by
EGFR-FabID. The culture medium was analyzed by the immunoblotting
after GST pull-down by glutathione-sepharose beads, and biotinylationof FLAG-GST-EGF ligand was detected (Supplementary Fig. 5c). This
result showed that EGFR-FabID can biotinylate the ligand if there are
sufﬁcient lysine residues on the ligand surface and suggested that nobiotinylation of EGF by EGFR-FabID was due to the lack of exposed
lysine residues on the ligand surface.

decreased by EGF treatment compared with that by DMSO treatment.
Furthermore, geﬁtinib treatment in the presence of EGF (EGF+geﬁtinib) also affected biotinylation of the proximal exPPIs with EGFR,
suggesting that treatment with EGF ligands and tyrosine kinase inhibitors changes the proximity exPPIs of EGFR. Additionally, different
cells exhibited different patterns of biotinylation induced by EGFRFabID in the presence of EGF and/or geﬁtinib.
EGFR-FabID detected exPPI changes of EGFR after EGF stimulationin the presence and absence of Geﬁtinib treatment (Fig. 6a, b). Geﬁtinib binds to the ATP binding site of EGFR and suppresses its phosphorylations in the presence of EGF45,46. Testing of geﬁtinib binding to
EGFR in the absence of EGF could lead to a better understanding of theeffects of Geﬁtinib. Therefore, to conﬁrm the biotinylation of EGFR
exPPI proteins with geﬁtinib treatment alone, we attempted MS analysis and identiﬁed proximity exPPI proteins using EGFR-FabID in
geﬁtinib treatment (Supplementary Fig. 9a, b, Supplementary Data 7
and 8). Interestingly proteins other than those biotinylated during EGFstimulation or EGF-stimulated geﬁtinib treatment were also biotinylated to EGFR-FabID in the geﬁtinib treatment alone (SupplementaryFigs. 6a, 9b). These results suggest that even in the absence of ligand,drugs that bind to the intracellular region of RTKs have a potential tomodify the interaction of the extracellular region.
To conﬁrm the presence of exPPIs with EGFR, we attempted tobiochemically detect the interaction between the extracellular regionof EGFR using the AlphaScreen method. PTK7 (tyrosine receptor
kinase 7), INSR, and ADAM17 (disintegrin and metalloproteinasedomain-containing protein 17), commonly found in proximity exPPIs
with EGFR-FabID in both A431 and NCI-H226 cells, were selected. PTK7
and INSR share downstream signalling with EGFR47–49, although they
are not known as EGFR interactors. ADAM17 is also a major protease of
the EGF family50. For the biochemical interaction of these proteins withthe extracellular domain of EGFR, the extracellular region of EGFR was
synthesised by a wheat cell-free system to form S-S complexes, and theextracellular interactions were analysed using the AlphaScreen
method. Since the alpha subunit of INSR is known as the extracellular
insulin-binding site51, it was used in the assay. The AlphaScreen method
showed that PTK7, INSR alpha, and ADAM17 signiﬁcantly bound to theextracellular domain of EGFR (Fig. 6c). Furthermore, the interaction of
PTK7 and INSR with EGFR was also conﬁrmed using the NanoBiT
method, which allows the measurement of interacting proteins in
cultured cells (Fig. 6d). However, ADAM17 did not work on the NanoBiT method. We also performed immunostaining for INSR, ADAM17,
and PTK7 to examine their co-localisation with endogenously expressed EGFR. ADAM17 and PTK7 co-localised with EGFR in A431 cells(Supplementary Fig. 10a). The results showed that endogenous EGFR
co-localised with endogenous ADAM17 and PTK7 on the plasma
membrane of A431 cells. INSR was also conﬁrmed to colocalize with
EGFR on the plasma membrane of NCI-H226 cells (Supplementary
Fig. 10b). These biochemical and cellular interaction analyses suggested that INSR, ADAM17, and PTK7 interacted with the extracellularregion of EGFR.
Next, the change in proximity exPPIs in EGF, with or without
geﬁtinib treatment, was validated by proximity ligation assay (PLA)
method in A431 cells. As shown in Fig. 6b (A431 cell panel), proximity

Dynamic responses of proximity exPPIs on treatment with EGF
ligand with or without geﬁtinib
In the EGF signalling response, dimeric EGFR phosphorylate each otherat tyrosine residues in the intracellular region to form a signal complex. Geﬁtinib is a highly speciﬁc tyrosine kinase inhibitor of EGFR27. To
understand the effect of geﬁtinib on exPPI, we compared the proximal
exPPIs in A431 and NCI-H226 cells in the presence of EGF ligand (EGF)
and EGF plus geﬁtinib (EGF+geﬁtinib) with a control without ligand(DMSO). From each treatment of the two cell lines, proximity exPPI
analysis of EGFR yielded approximately 30 and 35–50 proteins,respectively (Fig. 6a, Supplementary Fig. 6). These proximity exPPI
proteins were analysed using a heatmap and scatter plot based onprotein data from LC-MS/MS (Supplementary Data 5 and 6). In both
A431 and NCI-H226 cells, 20 proteins were common proximity exPPIs(Fig. 6b, Supplementary Fig. 7) (HLA-C was removed from the common
proteins because no values were detected in the protein MS data for
A431 cells.), and the remaining 23 and 46 proteins were speciﬁc to A431
and NCI-H226 cells, respectively (Supplementary Fig. 8a, b). Interestingly, EGFR-FabID-driven biotinylation was dramatically increased or

Nature Communications | (2023)14:8301

9

Articlehttps://doi.org/10.1038/s41467-023-43931-7

a

A431

EGFR
B

Cell surface

AirID

216

B

EGFR-FabID

AirID

BiotinKnown

P<0.05
Ratio>1

15
44
Proteins

481
AirID

B

EGF

EGF

EGF

AirID

AirID

A431 or NCI-H226

P

EGF

B

Identification of
biotinylation sites

P

P

AirID

LC-MS/MSwith Tamavidin

B

B

P

B

Intracellular or
unknown localization

NCI-H226
347

AirID

AirID

Known

Cell surface

B
EGF

EGF

P<0.05
Ratio>1

16
66

Proteins

849bProximal exPPIs

502
Intracellular or
unknown localization

CANX

A431
NCI-H226

A431
Expi293F HSP90AB1(oxEGFR) HSP90AA1

A431
NCI-H226
Expi293F(oxEGFR)

CDH3

A431

Expi293F
Transiently overexpression EGFR

8

NCI-H226
Expi293F(oxEGFR)

4

ANXA2
ICAM1
GAPDH
DSG2
EFNB1
SLC3A2
HLA-A
PTK7
DCBLD2CA2GPC1PLXNB2ITGA6
ADAM17
HLA-C

EGFR
EEF1A1
ENO1
MICA
INSR
CD44

19

6

4

DSG2
EZR
GAPDH
SLC39A10

50
New

peptidesdetected

Gefitinib

29
New

265

peptidesdetected

15
41
NCI-H226c

A431

NCI-H226
CDH5

CKMT1BADAM17

PTK7

CA2

LAMB3

ENO1
RUVBL1

LAMA3

CD44
HSP90AB1
HSPA8MAPT
SLC3A2

PPIB
NEO1

EEF1A1
TGFBI

ST14

CDH3

CFL1
EGFR

ICAM1

DCBLD2NFASCEFNA5
COL18A1
ARFGAP2
LAMA5
OLR1
SQSTM1BCAMDSC3STXBP1
MICA
AGRN
ITGB1
EPHB6
ALCAM LRP1
ADAM17
GAPDH
PTPRF
CLU

PODXL

INSR

EFNB1

VASN
GAPDH

HSP90AA1

ANXA2
CANX
HLA-A

PCDH1

F11R

GABARAP

SLC39A10

HLA-C
DCBLD2GPC1PLXNB2ITGA6CA2FGFBP1

EPCAM

DSG3

EGFR

DSG2

GPC1

CD44 INSR
BSG

ICAM1

EFNB1

KHDRBS1

ITGB4

ANXA2
HLA-A

CLINT1TENM2S100A10 PLXNB2
FAS
CD99L2
CDH11
HLA-C
ANOS1
ITGA6
L1CAM
ITGA3
CEMIP2
PTK7
ATP1B3
SEPTIN2
CEMIP
ENO1TFRCHSPG2

exPPI of PTK7 with EGFR was increased by geﬁtinib treatment. A large
number of PLA signals were detected in PTK7, and the PLA signal was
also reduced by EGF+geﬁtinib treatment compared with that by EGF
treatment. (Fig. 6e). The PLA signal values and biotinylation of PTK7
were mismatch. This is probably because the PLA signal of PTK7 was sohigh even before treatment with ligand or drug that it exceeded thethreshold for the PLA method to detect interaction changes upon

Nature Communications | (2023)14:8301

ATP5F1A

EEF1A1MICA

TACSTD2

SLC3A2ADGRG6
DSG2

ALPP
HLA-B

EZR

KRT1
MELTF

Directly connected
Connected via one nodeConnected via two nodes
Connected via three nodes
Not connected

addition of drug or ligand. In addition, not all interaction detections
coincided because EGFR-FabID and PLA have different distances ofdetectable interaction. In contrast, the proximity exPPI of INSR was
increased by EGF and EGF+geﬁtinib (Fig. 6b, A431 cell panel), and the
PLA signal showed a signiﬁcant increase (INSR in Fig. 6e). Furthermore,
the proximity of exPPI of ADAM17 with EGFR also increased by EGR+geﬁtinib treatment (Fig. 6b, A431 cell panel), and the PLA signal also10Articlehttps://doi.org/10.1038/s41467-023-43931-7

Fig. 5 | Total proximity extracellular biotinylation analysis of EGFR using EGFRFabID in A431 and NCI-H226 cells with or without EGF ligand and/or geﬁtinib.
a Workﬂow of PPI analysis targeting A431 cells. The same procedure as that in Fig. 3
was used to narrow down the peptides to extracellular peptides. As a result, 216
peptides in A431 cells and 347 peptides in NCI-H226 cells were derived from cellsurface peptides. In A431 cells, 15 molecules were known to interact with EGFR, and29 molecules were unknown interacting proteins. In NCI-H226 cells, 16 molecules
were known to interact with EGFR, and 50 molecules were unknown interacting

proteins. b Venn diagram of proteins predominantly biotinylated in EGFRoverexpressing Expi293F, A431, and NCI-H226 cells. The numbers represent the
number of proteins in the region (EGFR-FabID/AGIA-FabID ratios >1 and P < 0.05).
Signiﬁcant changes in the volcano plots and heat map were calculated by Student’s
two-sided t test and the false discovery rate (FDR)-adjusted P-values calculatedusing Benjamini–Hochberg method are shown in the Supplementary Data 3, 4.
c Pathway analysis of 44 proteins in A431 cells and 66 proteins in NCI-H226 cells,
obtained from workﬂow shown in Fig. 5a.

increased in the EGF+geﬁtinib treatment (ADAM17 in Fig. 6e). These
results indicated that PLA signals were similar to biotinylation signals
from EGFR-FabID proximity exPPIs with EGFR. Taken together, theseresults strongly suggest that EGF ligand and a kinase inhibitor provide
dynamic exPPIs of EGFR on the cell membrane, and the FabID system
can detect them via biotinylation.

REV38,39 and indicated that proximity biotinylation occurred in theextracellular region. The analysis, therefore, provided proximity
exPPIs with EGFR and found many well-known EGFR interactors, as wellas unknown proximity exPPI candidate proteins (Fig. 5). Individual
analyses also indicated direct exPPIs with the extracellular region of
EGFR (Fig. 6). Because other conventional methods have not identiﬁed
biotinylation sites, there is no information on the proximal region of
the identiﬁed proteins. These results indicate that biotinylation sites
provide important information for interactome analysis of membraneproteins.
The FabID system showed that exPPI with EGFR changes dynamically with the addition of EGF ligand and geﬁtinib (Fig. 6 and Supplementary Fig. 8). EGF ligands induce dimerisation of EGFR along withother conformational changes36. The EGFR-FabID method detected achange in biotinylation sites on EGFR with or without EGF (Fig. 4d, e,
and Supplementary Fig. 3), contributing to the dynamic inﬂuence ofproximity exPPIs with EGFR on the cell membrane. As EGF induces
conformational changes in the extracellular region of EGFR, it is consistent that EGF affects the interaction of exPPIs with EGFR. In addition,
geﬁtinib inhibits tyrosine kinase activity, resulting in complete loss ofphospho-tyrosine sites45. Since phospho-tyrosine is used as a scaffold
site for the formation of the EGF signal complex54, geﬁtinib may also
inhibit its complex formation. The results of EGF+geﬁtinib treatment in
EGFR-FabID, therefore, suggest that the inhibition of EGFR complex
formation in the intracellular region affects the interaction withextracellular regions of EGFR. Taken together, FabID provides a toolfor extracellular interaction analysis, as it can capture not only theinteraction analysis of extracellular regions but also interacting proteins altered by ligands and drugs.
The EGFR-FabID method revealed direct interactions in theextracellular domain of INSR and EGFR, which interact during intracellular signalling48,49. This ﬁnding may indicate that extracellular signalling interactions occur between heterogeneous receptors on themembrane. Further, the exPPI analysis of various RTKs using FabID on
the cell membrane may provide new insights into signalling cooperation in the extracellular region. Furthermore, since they can changewith drug treatment, FabID-based exPPIs may provide a new perspective on drug development for RTKs.
In conclusion, the FabID system for the analysis of extracellularinteractions can capture not only the interaction analysis of extracellular regions but also interacting proteins altered by ligands anddrugs, thus delivering a powerful tool for the study of membraneproteins.

Discussion
In this study, we developed the EGFR-FabID and EGFR-mAbID probes,
and then EGFR-FabID was used to study the exPPI activity of EGFR. The
structural model suggested that the distance from FabID to the antigen
was ~10 nm (Fig. 2d) and that from mAbID to the antigen would be~14 nm (Fig. 2e), resulting in a higher biotinylation activity of EGFRFabID than that of mAbID (Fig. 2c), although the in vitro biotinylation
activity was almost the same (Fig. 2b). These results suggest that the
distance between the probe and target protein is a key factor inproximity biotinylation of exPPI partners. Recently, EMARS52,53 andµMap10 were reported to detect proximal interactions with target
membrane proteins. These methods used a full-size IgG antibody andnot the Fab form. Furthermore, horseradish peroxidase (HRP) and achemical molecule, ({Ir[dF(CF3)ppy]2(dtbbpy)}PF6), are used as biotinylation catalysts in EMARS and µMap, respectively. Biotinylation via
HRP with biotin phenol occurs in the range of 200–300 nm, whileµMap has a biotinylation range of 50–100 nm11. In addition, the chemocatalytic molecule used in µMap is conjugated on a secondary
antibody but not the primary antibody10, suggesting that the distance
between the biotinylation catalyst and target membrane protein iseven further. FabID conjugates the AirID enzyme directly to the
C-terminus of Fab. Taken together, the FabID system can efﬁciently
biotinylate interacting proteins in the proximity of membrane proteins, thus making it the ideal method for the analysis of exPPIs on thecell membrane.
BioID-based exPPI analysis has been performed by expressing
proteins with BioID genetic insertion of BioID enzyme between thedomains of the extracellular regions of membrane proteins26.
Although this approach has revealed important exPPI results, it has
several drawbacks. Many membrane proteins have a signal peptide attheir N-terminus that is required for membrane-spanning and prevents
insertion of the BioID enzyme to the N-terminus. This necessitates that
membrane proteins with an N-terminus exposed to the extracellularspace integrate BioID between domains in the extracellular region.
However, the integration of BioID enzymes between the domains ofmembrane proteins is likely to affect the exPPI of these proteins due tostructural effects. In addition, BioID-fused proteins do not allow for
exPPI analysis of endogenously expressed membrane proteins in theirnative form. In this regard, FabID could be used to analyze the endogenously expressed membrane protein exPPI, although it requires
well-characterised antibodies against the target protein. EGFR-FabID
was used to analyze the exPPIs of endogenously expressed EGFR
(Fig. 5). The advantage of the FabID method is that it can analyse theproximity exPPI of endogenous membrane proteins, which are difﬁcult
to analyse using conventional BioID enzyme-based methods.
Using an EGFR-FabID system based on AirID to analyse exPPIs of
the EGFR protein in living cell lines (both adherent and nonadherent
cells), we revealed biotinylation sites by LC-MS/MS using Tamavidin2-

Nature Communications | (2023)14:8301

Methods
Reagents
Geﬁtinib (#078-06561, Lot. SKP4099, FUJIFILM Wako) was dissolved in
DMSO (#13445-74, Nacalai tesque) at 2 mM and stored at −20 °C as
stock solutions. EGF (#059-07873, FUJIFILM Wako) was dissolved inphosphate-buffered saline (PBS) at 100 μg/mL and stored at −20 °C as
stock solutions. Streptavidin Sepharose High Performance(#90100484, Cytiva) was stored at 4 °C as stock solutions. StreptavidinHRP (#ab7403, abcam) (IB 1:10000) was stored at 4 °C as stock solutions. Dynasore (#S8047, Selleck) was dissolved in DMSO (#13445–74,
Nacalai tesque) at 80 mM and stored at −20 °C as stock solutions.
11Articlehttps://doi.org/10.1038/s41467-023-43931-7

5

10

-10

0

5

10

8

15

50 11
39

-Log10(p-value)4-5

Log2(EGFR-FabID/AGIA-FabID)

31 12
19

6

6
4

2

p=0.05

0
-5

0

5

0
-10

10

Log2(EGFR-FabID/AGIA-FabID)

4

2

0

5

10

15

P=0.05

35 12
23

8

-Log10(p-value)

6-5

Log2(EGFR-FabID/AGIA-FabID)

33 10
23

8

P=0.052

64
2

P=0.05

0

0
-5

0

5

-10

10

-5

0

5

1.98

3.37

0.67

3.13

1.40

4.22

4.31

5.79

1.90

1.91

1.03

3.91

3.52

4.56

3.31

2.23

2.13

3.18

3.60

3.04

3.65

3.21

2.57

1.23

1.98

2.25

0.45

0.81

0.24

1.75

2.07

2.09

0.24

0.40

1.17

1.41

1.24

1.01

1.64

1.56

0.87

1.80

2.90

2.36

2.74

2.85

1.83

1.29

2.37

3.43

0.43

1.31

0.57

1.16

0.77

0.69

0.05

0.17

0.02

1.08

0.05

-0.15

0.82

1.33

1.12

0.50

0.74

0.02

0.90

1.37

1.07

0.69

0.60

0.22

1.30

1.42

0.98

0.33

-0.37

-0.04

0.90

0.78

1.18

0.01

0.08

1.56

1.90

1.56

2.03

-0.24

-0.26

0.47

0.80

0.96

0.86

-0.72

-0.54

0.08

0.17

0.70

0.55

-0.31

-0.58

-0.67

0.65

0.60

0.68

-0.84

-1.04

-0.01

0.74

0.35

0.73

-1.51

-0.97

-0.51

0.55

1.05

0.89

0

2

-1

4

0

1

2

3

Log2(EGFR-FabID/AGIA-FabID)

15

Log2(EGFR-FabID/AGIA-FabID)

Log2(EGFR-FabID/AGIA-FabID)

d150000Nanobit signalcAlphaScreen Signal

10

0.72

20000
15000
10000

300000Nanobit signal-Log10(p-value)

P=0.05EGF +gefitinib0

Log2(EGFR-FabID/AGIA-FabID)-Log10(p-value)

2

Protein
Name
EGFR
PTK7
GPC1
MICA
ICAM1
PLXNB2
DCBLD2INSR
ADAM17
ENO1
CA2
EFNB1
ANXA2
EEF1A1
DSG2
CD44
ITGA6
GAPDH
SLC3A2
HLA-A

0
-5

EGF +
gefitinib

44 12
32

NCI-H226
EGF

p=0.05

0

EGF

4

6

A431
EGF +
gefitinib2-Log10(p-value)-Log10(p-value)DMSO(Control)

8
22
New

30

4

b

8

DMSO

NCI-H226
MembraneKnown
proteins

EGF

A431
6

DMSO

a

10000050000

200000100000

5000

0

0

17

DHFR-LgBit
EGFR-LgBit

+

-

+

-

-

+

-

+

A

D

A

M

7
K

SR

IN

H
D

PT

FR

0

e

INSR-SmBit

PTK7-SmBit

A431
INSR

PTK7

Plasmids
pcDNA3.1(+) and pcDNA3.4 vectors were purchased from Invitrogen/
Thermo Fisher Scientiﬁc and RIKEN, respectively. The pEU vector forwheat cell-free protein synthesis was constructed in our laboratory as
previously described55. pBiT1.1-C[TK/LgBiT] (# N2014, Promega) and
pBiT2.1-C[TK/SmBiT](#N2014, Promega) were purchased from Promega. pcDNA3.4-TEV-His, pEU-bls-MCS, and pEU-FLAG-MCS plasmids

Nature Communications | (2023)14:8301

15

10

ns

5

ADAM17
20

15

10

ns
5

EG
F
EG
ge F+
fit
in
ib

M
D

M
SO

SO

0

0

D

0

Dot numbers on PLA/nucleus

5

20

EG
F
EG
ge F+
fit
in
ib

D

10

EG
F
EG
ge F+
fit
in
ib

SO
EG
F
EG
ge F+
fit
in
ib

0

15

SO

ns
5

ns

M

ns

10

20

D

15

Dot numbers on PLA/nucleus

20

M

Dot numbers on PLA /nucleus

ns

Dot numbers on PLA/nucleus

Control

were constructed by polymerase chain reaction (PCR) using the InFusion system (Takara Bio) or PCR and restriction enzymes. AirID waswas constructed in our laboratory, as previously described24. EGFR andMDM2 were purchased from the Kazusa Clone collection56 using pEUFLAG-GST-EGFR. EGF, PTK7, OTULIN, ADAM17 and p53 were purchasedfrom Mammalian Gene Collection (MGC). The SHB and LHB genes (Sprotein of hepatitis B virus) were kindly provided by Prof. Y. Matsuura
12Articlehttps://doi.org/10.1038/s41467-023-43931-7

Fig. 6 | Individual analysis of exPPI response to EGFR in A431 and NCI-H226 cells
in the presence of EGF and geﬁtinib. a Volcano plot of DMSO (Control) EGF- or
EGF+geﬁtinib-treated zones, each detected as biotinylated peptides by LC-MS/MS.
Peptides derived from plasma membrane proteins are indicated by red dots.
Numbers in black indicate the number of proteins known to interact with EGFR.
Numbers in blue indicate the number of proteins that are unknown interactors with
EGFR. Signiﬁcant changes in the volcano plots and heat map were calculated byStudent’s two-sided t test and the false discovery rate (FDR)-adjusted P-values
calculated using Benjamini–Hochberg method are shown in the Supplementary
Data 3, 4. b Heat map showing exPPI changes upon addition of EGF or geﬁtinib forcell surface proteins that were commonly biotinylated in A431 and NCI-H226 cells.
Each cell was assigned an average value. c EGFR interactions were detected using
AlphaScreen (independent experiments, n = 3). AlphaScreen with the EGFR

extracellular domain and various FLAG fusion proteins. Statistical signiﬁcance wasdetermined using one-way ANOVA with Dunnett’s multiple comparisons test(****P ≤ 0.0001, ***P ≤ 0.0002, **P ≤ 0.0021). Error bars represent standard deviations. d Conﬁrmation of live cell interactions using the NanoBiT system. DHFRLgBit was used as the control. Statistical signiﬁcance was determined using two
sided t test (three biological reprecates, ****P ≤ 0.0001, ***P ≤ 0.0002). Error bars
represent standard deviations. The cells used were HEK293T cells. e Proximity
ligation assay (PLA) performed on A431 cells. Bar graph depicting the number of
PLA-positive foci compared to each treatment group calculated with Andy’s PLA
algorithm and quantiﬁed using GraphPad Prism 9 software (n = 9 per group, Oneway ANOVA with Tukey’s post-hoc test, ****P ≤ 0.0001, ***P ≤ 0.0002, **P ≤ 0.0021,*P ≤ 0.0332). Error bars represent standard deviations. Source data are provided as
a Source data ﬁle.

of the Osaka University, Center for Infectious Diseases Education andResearch. The pEU-FLAG-GST-DRD1 CTD was constructed in ourlaboratory, as previously described28. The restriction enzyme sites
were added to EGFR, EGFR extracellular domain, PTK7, OTULIN, SHB
and ADAM17 by PCR. The EGFR extracellular domain was cloned into
pEU-MCS and pEU-bls-MCS. EGFR was cloned into pcDNA3.1( + )-MCS
and pBiT1.1-C [TK/LgBiT] plasmids. OTULIN and SHB were cloned intopEU-AGIA-MCS. PTK7 and INSR [28–758aa] were cloned into pEU-FLAGMCS. PTK7 and INSR was cloned into pBiT2.1 [TK/SmBiT], respectively.
ADAM17 and LHBs were cloned into pEU-FLAG-MCS. MDM2, and p53
were cloned into pEU-FLAG-GST or pEU-AGIA vectors using the Gateway cloning system (Thermo Fisher Scientiﬁc). cDNAs for the anti-AGIA
antibody light chains and EMab-134 light chain were cloned into the
pcDNA3.4 expression vector using PCR and In-Fusion Reaction. The
anti-AGIA heavy chain Fab fragment or EMab-134 heavy chain Fabfragment was cloned into pcDNA3.4-TEV-His using the In-Fusion HD
Cloning Kit (#639649, Takara Bio) together with the AirID or TurboID
fragment to generate pcDNA3.4-anti-AGIA Heavy Fab-AirID-TEV-His
and pcDNA3.4-EMab-134 Heavy chain-AirID-TEV-His, respectively.

anti-EGFR (#4267, Cell Signalling Technology, IF 1:100, RRID:AB_2864406), anti-ADAM17 (#3976, Cell Signalling Technology, IF
1:100, PLA 1:100, RRID:AB_2242380), anti-ADAM17 (#sc-390859,
SANTA CRUZ, IF 1:100), anti-P-EGFR-Tyr1173 (#4407, Cell SignallingTechnology, IB 1:1000, RRID:AB_331795), anti-biotin (#5597, Cell Signalling Technology, IB 1:1000, IF 1:100, RRID:AB_10828011), anti-insulin
receptor alpha (#74118, Cell Signalling Technology, PLA 1:100, RRID:AB_2799850), anti-INSR (#A19067, Abclonal, IF 1:100, RRID:AB_2862559), anti-STAT3 (#9132, Cell Signalling Technology, IB
1:1000, RRID:AB_331588), anti-P-STAT3-Y705 (#9145, Cell SignallingTechnology, IB 1:1000, RRID:AB_2491009), anti-EEF1A1 (#sc-21758,
SANTA CRUZ, IF 1:50, RRID:AB_309663) and anti-PTK7 (#17799-1-AP,
proteintech, IF 1:100, PLA 1:100, RRID:AB_2878442). Anti-rabbit IgG(#7074, HRP-conjugated, Cell Signalling Technology, IB 1:10000, RRID:AB_2099233), anti-mouse IgG (#7076, HRP-conjugated, Cell Signalling Technology, IB 1:10000, RRID:AB_330924), F(ab’)2-Goat antiRabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor
555 (#A21431, Thermo Fisher Scientiﬁc, IF 1:1000, RRID:AB_2535852),
and goat anti-mouse IgG (H + L) Cross-Adsorbed Secondary Antibody,
Alexa Fluor™ 488 (#A11001, Thermo Fisher Scientiﬁc, IF 1:1000, RRID:AB_2534069) were used as secondary antibodies.

Cell culture and transfection
Expi293F cells (Gibco/Thermo Fisher Scientiﬁc, RRID:CVCL_D615) wereshaken at 125 ± 5 rpm at 37 °C under 8% CO2 in Expi293F medium(#A1435101, Gibco/Thermo Fisher Scientiﬁc) supplemented with
100 U/mL penicillin and 100 µg/mL streptomycin (#15140122, Gibco/
Thermo Fisher Scientiﬁc). A431 (JCRB Cell Bank, RRID:CVCL_0037)
cells were cultured in high-glucose DMEM (#043-30085, FUJIFILM
Wako) supplemented with 10% foetal bovine serum (FBS; FUJIFILM
Wako), 100 U/mL penicillin, and 100 µg/mL streptomycin (#15140122,
Gibco/Thermo Fisher Scientiﬁc) at 37 °C under 5% CO2. HeLa-S3 cells(JCRB Cell Bank, RRID:CVCL_0058) were shaken at 125 ± 5 rpm at 37 °C
under 8% CO2 in Ham’s F-12 (#087-08335, FUJIFILM Wako) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin(#15140122, Gibco/Thermo Fisher Scientiﬁc). NCI-H1975 (ATCC,
RRID:CVCL_1511) and NCI-H226 (ATCC, RRID:CVCL_1544) were cultured in RPMI160 GlutaMAX medium (#72400047, Gibco/Thermo
Fisher Scientiﬁc) supplemented with 10% foetal bovine serum (FUJIFILM Wako), 100 U/mL penicillin, and 100 µg/mL streptomycin(#15140122, Gibco/Thermo Fisher Scientiﬁc) at 37 °C under 5% CO2.
Expi293F cells were transiently transfected using Expi Fectamine 293transfection kit (#A14524, Gibco/Thermo Fisher Scientiﬁc).

Antibodies
The following horseradish peroxidase (HRP)-conjugated antibodies
were used in this study: anti-FLAG (#A8592,Sigma-Aldrich, IB 1:5000,
RRID:AB_439702), anti-AGIA (produced in our laboratory, IB
1:10000)28, anti-tubulin (#PM054-7, MBL, IB 1:5000, RRID:AB_10695326), anti-His (#sc-8036, Santa Cruz, IB 1:1000, RRID:AB_627727), and biotin (#7075, Cell Signalling Technology, IB
1:1000, RRID:AB_10696897). The following primary antibodies wereused: anti-EGFR (clone EMab-134, IB 1:1000, PLA 1:100, IF 1:100)34,35,

Nature Communications | (2023)14:8301

Preparation of FabID and mAbID
FabID and mAbID were expressed using the Expi293F Expression
System (Thermo Fisher Scientiﬁc), according to the manufacturer’s
instructions. The culture medium was puriﬁed using protein Ni
Sepharose Excel (#GE17371201, Cytiva). The supernatants were added
to Ni Sepharose Excel and incubated for 3 h at 4 °C. The mixture wasthen washed with three-column volumes of wash buffer (20 mM
sodium phosphate, 300 mM NaCl, and 10 mM imidazole). Proteins
were eluted in 500 µL fractions with elution buffer (20 mM sodium
phosphate, 300 mM NaCl, and 500 mM imidazole). The fractions weredialysed against PBS. Puriﬁed FabID and mAbID were frozen and storedat −80 °C.

Immunoblot analysis
Protein samples were separated using SDS-PAGE and transferred onto
polyvinylidene diﬂuoride (PVDF) membranes (#IPVH00010, Millipore).
The membranes were blocked using 5% skimmed milk (#4273437,
Megmilk Snow Brand) in TBST (20 mM Tris-HCl [pH 7.5], 150 mM NaCl,and 0.05% Tween20) at 27 °C for 1 h and then treated with the appropriate antibodies. Immobilon (#WBKLS0500 Merck), ImmunoStar LD(#296–69901, FUJIFILM Wako), or EzWestLumi plus (#WSE-7120, Atto)
were used as substrates for HRP, and the luminescence signal was
detected using an ImageQuant LAS 4000 mini (GE Healthcare). In someblots, the membrane was stripped with a stripping solution (#19316375, FUJIFILM Wako) and reprobed with other antibodies.

Wheat cell-free protein synthesis
The recombinant protein was synthesised using a wheat cell-free system. In vitro transcription and wheat cell-free protein synthesis were13

Article
performed using the WEPRO1240 Expression Kit (Cell-Free Sciences)or Disulﬁde Bond PLUS Expression Kit (#CFS-EDX-DB, Cell-free Sciences). Transcription was performed using SP6 RNA polymerase, withplasmids or DNA fragments as templates. The translation reaction was
performed in bilayer mode using the WEPRO1240 expression kit (CellFree Sciences) or Disulﬁde Bond PLUS Expression Kit (Cell-Free Sciences), according to the manufacturer’s protocol. For biotin labellingof the bls-EGFR extracellular domain, cell-free synthesised crude biotin
ligase (BirA) produced using the wheat cell-free expression system wasadded to the bottom layer, and 0.5 µM (ﬁnal concentration) of d-biotin(#04822-91, Nacalai Tesque) was added to both the upper and lower
layers8.

In vitro biotinylation assay with FabID and mAbID
Wheat cell-free expression system synthesised each protein weremixed with FabID or mAbID (Fin 80 ng/µL), added to the reaction
mixture, and incubated for 1 h at 26 °C. Next, d-biotin (#04822-91,
Nacalai Tesque) was added at a concentration of 500 nM and incubated at 26 °C for 3 h. After the reaction, biotinylated proteins wereanalysed using SDS-PAGE and immunoblotting.

Biotinylation of the protein complex by AGIA-FabID
Wheat cell-free expression system synthesised proteins were mixed
and incubated for 1 h at 26 °C. In addition, AGIA-FabID (Fin 80 ng/µL)
was added to the reaction mixture and incubated for 1 h at 26 °C. Next,
d-biotin (#04822-91, Nacalai Tesque) was added at a concentration of
500 nM and incubated at 26 °C for 5 h. After the reaction, biotinylated
proteins were analysed using SDS-PAGE and immunoblotting.

AGIA-FabID pH resistance analysis
One hundred microlitres of protein synthesised using the wheat cellfree protein synthesis system was dialysed in 10 mL of buffer at eachpH (100 mM HEPES-NaOH buffer pH = 7.0, 7.2, 7.4, 7.6, and 7.8;
200 mM Tris-HCl buffer pH = 8.0). After 24 h of dialysis, an in vitro
biotinylation assay was performed.

Molecular docking of EGFR-FabID and EGFR-mAbID with EGFR
extracellular domains
The complex model of EGFR-FabID and EGFR-mAbID was predictedusing AlphaFold2 (AF2)57. The predictive antigen-binding region of
EGFR-FabID was manually adjusted to dock into epitope regions of the
EGFR extracellular domains (Protein Data Bank (PDB) code: 1IVO
[https://doi.org/10.2210/pdb1ivo/pdb])). The light and heavy chains of
EGFR-FabID were superimposed onto the structure of the anti-canine
lymphoma monoclonal antibody (Mab231 and PDB code: 1IGT [https://doi.org/10.2210/pdb1igt/pdb]), and the predictive antigen-binding
region of EGFR-mAbID was also manually docked into epitope
regions of EGFR extracellular domains (PDB code: 1IVO [https://doi.
org/10.2210/pdb1ivo/pdb])). All the molecular structures were generated using PyMOL (Schrödinger).

Stable cell line
EGFR-overexpressing Expi293F cells were transfected with pcDNA3.1EGFR in Expi293F cells (5 × 106 cells). After 24 h of infection, the culture
medium was exchanged, and 1 mg/mL Geneticin G418 (Sigma Aldrich)selection was started 24 h after exchanging the culture medium.

Cell biotinylation assay with FabID
Biotinylation of EGFR on the plasma membrane was performed using
EGFR-overexpressing Expi293F cells transfected with pcDNA3.1-EGFR.
Expi293F cells (5 × 106 cells) were transfected with pcDNA3.1-EGFR(2 µg) and incubated for 24 h. Next, 385 µL Expi293F serum-free medium (10 mM HEPES) and 115 µL FabID suspension cells reaction buffer[355.5 µg/mL EGFR-FabID or AGIA-FabID, 22.2 mM ATP, 66.7 mM
MgCl2, and 333.3 µM d-biotin] were added and incubated at 37 °C for
Nature Communications | (2023)14:8301

https://doi.org/10.1038/s41467-023-43931-7

2 h with rotation. Cells were collected by centrifugation and washedtwice with 1 mL of PBS. Cells were lysed with 500 µL RIPA buffer +protease inhibitor (Sigma-Aldrich), followed by sonication and streptavidin pull-down assays.
Expi293F steady EGFR expression cells (1.0 × 107 cells) and HeLa
S3 cells (5 × 106 cells) were resuspended in 385 µL Expi293F medium(serum-free, +10 mM HEPES) or low-glucose D-MEM (#041-29775,
FUJIFILM Wako) (serum-free, 10 mM HEPES). Next, 115 µL FabID suspend cell reaction buffer was added and incubated at 37 °C for 2 h withrotation. Cells were collected by centrifugation and washed twice with
1 mL of PBS. Cells were lysed with 500 µL RIPA buffer + protease inhibitor (#P8340, Sigma-Aldrich), followed by sonication and streptavidin
pull-down assays.
For A431, NCI-H226, and NCI-H1975 cells, a 10 cm conﬂuent dishwas washed once with 2 mL PBS, after which 4.2 mL of high-glucose DMEM (serum-free, 10 mM HEPES) or RPMI1640 (serum-free, 20 mM
HEPES), 800 µL FabID adherent cells reaction buffer (177.8 µg/mL
EGFR-FabID or AGIA-FabID, 22.2 mM ATP, 66.7 mM MgCl2, and333.3 µM d-biotin) were added and incubated at 37 °C for 2 or 6 h. Afterthe reaction, the cells were washed with PBS (2.5 mL) and dissolved in
500 µL of RIPA buffer + protease inhibitor (#P8340, Sigma-Aldrich).
After sonication, streptavidin pull-down assays were performed.

Streptavidin pull-down assay (STA-PDA) using suspension or
adherent cells
Streptavidin Sepharose (20 µL/L sample) was washed three times inwash buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1% SDS), resuspended in 200 µL of wash buffer, and added to the sample. The mixture was then rotated at room temperature for 1 h, and the supernatant
was removed by centrifugation. Streptavidin Sepharose was washedthree times with 1 mL of wash buffer and boiled in 2× sample buffer(40 µL) at 100 °C for 10 min. The supernatant was recovered bycentrifugation.

Kinase assay
A431 (3.0 × 105 cells/mL) cells were seeded in 24-well plates. After 24 h,
the cells media was replaced with serum-free media, and cells weretreated with DMSO (ﬁnal 0.1%) or 2 µM geﬁtinib for 2 h. Next, EGF(100 ng/mL) was added for 5 min, and cells were treated with 80 ng/µL
EGFR-FabID. After 15 min, the cells were collected and lysed in 100 µL
of 2× sample buffer + 10% mercaptoethanol. Phosphorylation of EGFR
and STAT3 activated by EGF stimulation was conﬁrmed by immunoblotting using speciﬁc antibodies.

Flow cytometry
Expi293F cells stably expressing EGFR were biotinylated using EGFRFabID for 2 h as described in this section above (Cell biotinylation assay
with FabID). The cells without EGFR-FabID or Biotin were added PBS
instead. After biotinylation of cells by EGFR-FabID, cells were washedthree times with 1 mL of PBS. The cells were ﬁxed with 4% paraformaldehyde (#163–20145; FUJIFILM Wako). The cells were thenstained with Alexa Fluor 488-conjugated Streptavidin (#S32354,
Thermo Fisher Scientiﬁc, 1:100, RRID: AB_2315383) suspended in PBS
with 5% BSA for 1 h at 4 °C. The stained cells were washed three times
with 1 mL of PBS and were replaced with FACS buffer (PBS with 2% FBS,
2 mM EDTA and 0.01% NaN3). Flow cytometry was performed using BD
FACSLyricTM Flow Cytometer (BD Biosciences) and the results wereanalyzed using FlowJo software (Tree Star, Ashland, OR, USA). The
ﬁlters used for BD FACSLyricTM Flow Cytometer were FITC (excitation
wavelength 488 nm, emission wavelength 530/30 nm).

MTS assay
A431 (3.0 × 105 cells/mL) cells were seeded in 96-well plates. After 24 h,
the cell medium were replaced with 80 µL serum-free media, and thecells were treated with 20 µL FabID adherent cell reaction buffer14

Article(177.8 µg/mL EGFR-FabID, AGIA-FabID, or PBS: 22.2 mM ATP, 66.7 mM
MgCl2, and 333.3 µM d-biotin). After 2 h, cell viability was measured
using the CellTiter 96® AQueous One Solution Cell Proliferation Assay(#G3582, Promega), according to the manufacturer’s protocol. The
absorbance at 490 nm was measured using SpectraMaxTM iD3 Multimode microplate reader (Molecular Devices).

Biotinylation of EGFR by EGFR-FabID during inhibition of
endocytosis
NCI-H226 cells in 6 cm conﬂuent dish was washed once with 2 mL PBS,
and 2.1 mL of RPMI1640 (serum-free, 20 mM HEPES) were added followed by treatment with DMSO or 50 µM Dynasore (#S8047, Selleck)for 1 h. Next, 400 µL FabID adherent cell reaction buffer was added tothe medium and incubated at 37 °C for 3 h. After the reaction, the cells
were washed three times with 1 mL of PBS and dissolved in 500 µL of
RIPA buffer + protease inhibitor (#P8340, Sigma-Aldrich). Biotinylated
cell lysates were analysed by SDS-PAGE and immunoblotting.

Preparation of cell lysates treated with EGFR-FabID for theenrichment of biotinylated peptides
EGFR biotinylation on the plasma membrane of pcDNA3.1-EGFRtransfected EGFR-overexpressing, Expi293F cells was performed using
EGFR-FabID in three independent wells (three biological replicates;n = 3). Expi293F cells (5 × 106 cells) transfected with pcDNA3.1-EGFR for
24 h. Next, 385 µL Expi293F medium (10 mM HEPES) and 115 µL FabID
suspend cell reaction buffer were added and incubated at 37 °C for 2 hwith rotation. Cells were collected by centrifugation and washed twicewith 1 mL HEPES-Saline (20 mM HEPES-NaOH, pH 7.5, 137 mM NaCl).
The cells were then lysed in 500 µL Gdm-TCEP buffer (6 M guanidineHCl, 100 mM HEPES-NaOH pH 7.5, 10 mM TCEP, and 40 mM chloroacetamide). After lysing the cells, three independent lysates werepooled in one tube and divided into three tubes of 500 µL each. Eachtube was subjected to enrichment of biotinylated peptides followed bymass spectrometry.
Biotinylation of EGFR on plasma membrane using Expi293F
steady EGFR expression cells was performed with EGFR-FabID in three
independent wells (three biological replicates; n = 3). Expi293F steady
EGFR expression cells (1.0 × 107 cells) were resuspended in 385 µL
Expi293F medium (serum-free, +10 mM HEPES). Next, 115 µL FabID
suspend cell reaction buffer was added and incubated at 37 °C for 2 hwith rotation. Cells were collected by centrifugation and washed twicewith 1 mL HEPES-Saline (20 mM HEPES-NaOH, pH 7.5, 137 mM NaCl).
The cells were then lysed in 500 µL Gdm-TCEP buffer (6 M guanidineHCl, 100 mM HEPES-NaOH pH 7.5, 10 mM TCEP, and 40 mM chloroacetamide). After lysing the cells, three independent lysates werepooled in one tube and divided into three tubes of 500 µL each. Eachtube was analysed by mass spectrometry.
Biotinylation of A431 cells by EGFR-FabID was performed in three
independent 10 cm dishes. A 10 cm dish conﬂuent with A431 cells inthree biological replicates was washed once with 1 mL of PBS. Then,
2.1 mL of DMEM (serum-free, 10 mM HEPES) was added, and cells weretreated with DMSO or 2 µM geﬁtinib (DMSO ﬁnal 0.1%) for 2 h, followed
by treatment with 100 ng/mL EGF. Next, 800 µL FabID adherent cell
reaction buffer was added to the medium and incubated at 37 °C for
2 h. After the reaction, cells were washed with 5 mL Hepes-Saline(20 mM HEPES-NaOH, pH 7.5, 137 mM NaCl) and lysed in 500 µL GdmTCEP buffer (6 M guanidine-HCl, 100 mM HEPES-NaOH pH 7.5, 10 mM
TCEP, and 40 mM chloroacetamide). Cell lysates were pooled in one
tube and divided into three tubes of 500 µL each. Each tube was subjected to enrichment of biotinylated peptides followed by mass
spectrometry.
Biotinylation of NCI-H226 cells by EGFR-FabID was performed in
ﬁve independent 10 cm dishes. Conﬂuent NCI-H226 cells (10 cm dish,ﬁve biological replicates) were washed once with 1 mL PBS. Then,

Nature Communications | (2023)14:8301

https://doi.org/10.1038/s41467-023-43931-7

2.1 mL of RPMI1640 (serum-free, 20 mM HEPES) was added, and cells
were treated with DMSO or 2 µM geﬁtinib (DMSO ﬁnal 0.1%) for 1 h,followed by treatment with 5 µL EGF (100 µg/µL). Next, 800 µL FabID
adherent cell reaction buffer was added to the medium and incubatedat 37 °C for 3 h. After the reaction, cells were washed with 5 mL HepesSaline (20 mM HEPES-NaOH, pH 7.5, 137 mM NaCl) and lysed in 300 µL
Gdm-TCEP buffer (6 M guanidine-HCl, 100 mM HEPES-NaOH pH 7.5,
10 mM TCEP, and 40 mM chloroacetamide). Cell lysates were pooled in
one tube and divided into three tubes of 500 µL each. Each tube wassubjected to enrichment of biotinylated peptides followed by mass
spectrometry.

Enrichment of biotinylated peptides using Tamavidin 2-REV
All experiments were performed in triplicate for each treatment. The
cell lysates in Gdm-TCEP buffer were dissolved by heating and sonication and then centrifuged at 20,000 × g for 15 min at 4 °C. The
supernatants were recovered and proteins were puriﬁed bymethanol–chloroform precipitation and solubilised using PTS buffer(12 mM SDC, 12 mM SLS, 100 mM Tris-HCl, pH8.0). After sonicationand heating, the protein solution was diluted 5-fold with 100 mM TrisHCl, pH8.0 and digested with trypsin (MS grade, Thermo Fisher Scientiﬁc) at 37 °C overnight. The resulting peptide solutions were diluted
5-fold with TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl). Biotinylated
peptides were captured on a 15 µL slurry of MagCapture HP Tamavidin
2-REV magnetic beads (#133-18611, FUJIFILM Wako) after incubationfor 3 h at 4 °C. After washing with TBS ﬁve times, the biotinylated
peptides were eluted with 100 µL of 1 mM biotin in TBS for 15 min at
37 °C twice. The combined eluates were desalted using GL-Tip SDB(#7820-11200, GL Sciences), evaporated in a SpeedVac concentrator(Thermo Fisher Scientiﬁc), and re-dissolved in 0.1% TFA and 3%
acetonitrile (ACN).

Data-dependent LC-MS/MS analysis
LC-MS/MS analysis of the resultant peptides was performed on an
EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer using a nanoelectrospray ion source (Thermo Fisher Scientiﬁc). The peptides were separated on a 150-mm C18 reversed-phasecolumn with an inner diameter of 75 µm (Nikkyo Technos) using alinear 4–32% ACN gradient for 0–60 min, followed by an increase to
80% ACN for 10 min. The mass spectrometer was operated in datadependent acquisition mode with a maximum duty cycle of 3 s. The
MS1 spectra were measured with a resolution of 120,000, an automatic
gain control (AGC) target of 4 × 105, and a mass range of 375–1500 m/z.
HCD MS/MS spectra were acquired in a linear ion trap with an AGC
target of 1 × 104, an isolation window of 1.6 m/z, a maximum injection
time of 200 ms, and a normalised collision energy of 30. Dynamic
exclusion was set to 10 s. Raw data were directly analysed against the
Swiss-Prot database restricted to Homo sapiens using Proteome Discoverer version 2.4 (Thermo Fisher Scientiﬁc) with the Sequest HT
search engine. The search parameters were as follows: (a) trypsin as an
enzyme with up to two missed cleavages, (b) precursor mass toleranceof 10 ppm, (c) fragment mass tolerance of 0.6 Da; (d) carbamidomethylation of cysteine as a ﬁxed modiﬁcation, and (e) acetylation ofprotein N-terminus, oxidation of methionine, and biotinylation oflysine as variable modiﬁcations. Peptides were ﬁltered at a false discovery rate (FDR) of 1% using the Percolator node. Label-free quantiﬁcation was performed based on the intensities of the precursor ionsusing a precursor ion quantiﬁer node. Normalisation was performed
such that the total sum of the abundance values for each sample overall peptides was the same. For statistical analyses of the MS data, the Pvalues in each volcano plot were calculated using Student’s t tests byusing Microsoft Excel (version 16.78.3). The adjusted P-values were
calculated by controlling the FDR by using Microsoft Excel (version
16.78.3) and are shown in the Supplementary Data. Proteins with

15

Articlestatistically signiﬁcant modiﬁcation levels were identiﬁed using theStudent’s t test (P < 0.05) between EGFR-FabID triplicates and AGIAFabID control triplicates. Additionally, a protein was determinedenriched only when it showed at least a 1-fold change over the AGIAFabID control.

GST pull-down assay
pcDNA3.4-FLAG-GST-TEV-His-AGIA-GS linker-EGF (FLAG-GST-EGF)
was constructed in the form of a TEV-His-AGIA-GS linker inserted
between
FLAG-GST
and
EGF.
The
secretedsignal(MGILPSPGMPALLSLVSLLSVLLLMGCVAETG)58 was fused before FLAGGST. The FLAG-GST-EGF plasmid was transfected into Expi293F cells
stably expressing EGFR (5 × 106 cells) in 2 mL Expi293F medium. After
24 h of transfection, enhancer of Expi293F Transfection Kit (#A14525,
Thermo Fisher Scientiﬁc) was added. After 24 h of enhancer addition,
200 µL EGFR-FabID or PBS and 200 µL biotin mix buffer (5 mM HEPES,
500 µM ATP, 5 µM MgCl2 and 50 µM d-Biotin) or PBS were added andincubated at 37 °C for 2 h with rotation. Cells and cell culture medium
were collected by centrifugation. Supernatant (40 µL) was collected as
sup section. Then NaCl (ﬁnal 150 mM) and DTT (ﬁnal 10 mM) are added
to the culture medium. A 30 µL slurry of Glutathione SepharoseTM 4B(#17-0756-01, Cytiva) washed with PBS and suspended in 100 µL PBS is
added to the culture medium. The beads were washed 3 times with
1 mL PBS after 3 h rotation at 27 °C. After boiling with 2×sample buffer(40 µL), biotinylation of eluted proteins was analyzed byimmunoblotting.

AlphaScreen-based biochemical assays using recombinant
proteins
The bls-EGFR extracellular domain and FLAG fusion proteins weresynthesised using a Disulﬁde Bond PLUS Expression Kit (#CFS-EDX-DB,
CellFree Science). Proteins synthesised using the wheat cell-free
expression system were mixed in 15 µL of reaction buffer (100 mM
Tris-HCl pH 8.0, 1 mg/mL BSA, 100 mM NaCl, and 0.1% Tween20) andincubated at 26 °C for 1 h. Then, 10 µL of the detection mixture [1 mg/
mL BSA, 100 mM NaCl, and 0.1% Tween20, 0.04 mg/mL protein A
acceptor beads (PerkinElmer) and 0.04 mg/mL streptavidin donorbeads (PerkinElmer)] in the reaction buffer were mixed. After incubation at 26 °C for 1 h, the chemiluminescence signal was detected using
an Envision microplate reader (PerkinElmer). The Envision microplate
reader ﬁlter detected light between 520 and 620 nm at an excitation
wavelength of 680 nm.

Immunoﬂuorescent staining
A431 cells were cultured on 13 mm poly L-lysine-coated glass slides(#C1110, Matsunami) in 24-well plates. After culturing A431 for 24 h, 24well plates were washed once with 200 µL PBS, after which 380 µL of
high-glucose D-MEM (serum-free, 10 mM HEPES) and 120 µL FabID
adherent cell reaction buffer (177.8 µg/mL EGFR-FabID, 22.2 mM ATP,66.7 mM MgCl2, and 333.3 µM d-biotin) were added to the A431 cells
and incubated at 37 °C for 2 h. After one wash with 500 µL PBS,
A431 cells were ﬁxed with 4% paraformaldehyde in PBS for 15 min atroom temperature. Cells were incubated with anti-EGFR (clone EMab13434,35), anti-biotin, and 4’,6-diamidino-2-phenylindole (DAPI) overnight at 4 °C after blocking with 0.5% CS in TBST overnight. Afterwashing with TBST at 27 °C for 15 min, cells were incubated in F(ab’)2Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody,
Alexa Fluor™ 555 and Goat anti-Mouse IgG (H + L) Cross-Adsorbed
Secondary Antibody, and Alexa Fluor™ 488 at 27 °C for 1 h. Afterwashing with TBST at room temperature for 15 min, the stained cells
were mounted using ProLong™ Gold Antifade Mountant with DAPI(#P36935, Invitrogen/Thermo Fisher Scientiﬁc) and observed under aBZ-X810 Microscope (Keyence, Osaka, Japan).
A431 and NCI-H226 cells were cultured on 13 mm poly L-lysinecoated glass slides (#C1110, Matsunami) in 24-well plates. After 24 hNature Communications | (2023)14:8301

https://doi.org/10.1038/s41467-023-43931-7

of culture, cells were washed twice with PBS. Cells were ﬁxed with
4% paraformaldehyde in PBS for 15 min at room temperature andpermeabilised with 0.01% digitonin in PBS at 27 °C for 15 min. For
the EEF1A1 treatment section, no permeability treatment was performed. Then, the cells were incubated with 0.5% cattle serum (CS)in TBST at 4 °C for 1 h. A431 cells were incubated with anti-ADAM17(#sc-390859, SANTA CRUZ, 1:100), anti-EGFR (Cell SignallingTechnology, #4267, 1:100), or anti-PTK7 (Proteintech, 17799-1-AP,
1:100), and anti-EGFR (clone EMab-13434,35, 1:100) antibodies. NCIH226 cells were incubated with anti-INSR (#A19067, Abclonal,
1:100) or anti-EEF1A1 (#sc-21758, SANTA CRUZ, 1:50) and anti-EGFR(clone EMab-134; produced in our laboratory, 1:100) antibodies oranti-EGFR (Cell Signalling Technology, #4267, 1:100). After washingwith TBST at 27 °C for 15 min, the cells were incubated in DAPI,
F(ab’)2-Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary
Antibody, Alexa Fluor™ 555 and Goat anti-Mouse IgG (H + L) CrossAdsorbed Secondary Antibody, and Alexa Fluor™ 488 at room
temperature for 1 h. After washing with TBST at room temperaturefor 15 min, the stained cells were mounted using ProLong™ Gold
Antifade Mountant with DAPI (#P36935, Invitrogen/Thermo Fisher
Scientiﬁc) and observed under a BZ-X810 Microscope (Keyence,
Osaka, Japan). The ﬁlters used for the BZ-X810 Microscope were BZX Filter GFP (excitation wavelength 470/40 nm, emission wavelength 525/50 nm), BZ-X Filter TRITC (excitation wavelength 545/
25 nm, emission wavelength 605/70 nm), and BZ-X Filter DAPI
(excitation wavelength 360/40 nm, emission wavelength 460/
50 nm) (Keyence).

NanoBiT PPI assay
Plasmids (25 ng of pBiT2.1-PTK7-SmBiT or pBiT2.1-INSR-SmBiT together with 25 ng of pBiT1.1-DHFR-LgBiT or pBiT1.1-EGFR-LgBiT) and50 ng of pcDNA3.1-(+) vectors were transfected into HEK293T cells in
96 well-plate using polyethyleneimine (PEI) Max (MW 40,000) (PolyScience, Inc.) After 24 h, the culture medium was replaced with 80 µL
Opti-MEM reduced serum medium (Gibco). Then, 20 µL Nano-Glo Live
Cell Reagent (Promega) was added and measured NanoBiT signal using
the GloMAX Discover System (Promega). Three wells of cells cultured
and transfected in independent wells were tested. (three biologicalreplicates; n = 3)

Proximity ligation assay (PLA)
A431 cells were cultured on 13 mm poly L-lysine-coated glass slides(#C1110, Matsunami) in 24-well plates. After culturing for 24 h, the cells
were washed two times with PBS. Cells were ﬁxed with 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilised with 0.1% Triton X-100 in PBS (PTK7 and ADAM17); INSR was not
amenable to permeabilisation. The cells were then washed twice with
PBS and blocked with Duolink block solution for 1 h at 37 °C. Next, thecells were stained overnight at 4 °C with primary antibodies diluted in
Duolink antibody dilution buffer. Then, the cells were washed twicewith wash buffer A (10 mM Tris, pH-7.4, 150 mM NaCl, and 0.05%
Tween) and incubated with 20 μL of secondary antibody [4 μL antimouse PLUS antibody (#DUO82001, Sigma-Aldrich) + 4 μL anti-rabbit
MINUS antibody(#DUO82005, Sigma-Aldrich) + 12 μL Duolink® dilution buffer] at 37 °C for 1 h. The secondary antibody mix was gently
aspirated and washed twice with wash buffer A. Fifteen microliters ofligation mix was added to each sample and incubated at 37 °C for
30 min. Slides were washed twice with wash buffer A for 2 min each.
Fifteen microliters of the polymerase mix was added to each sample
and incubated for 100 min at 37 °C. The ampliﬁcation mix was aspirated and washed twice with wash buffer B (200 mM Tris, pH-7.5,
100 mM NaCl) for 10 min each. The slides were washed once with 0.01×
buffer B for 1 min. After aspirating buffer B, slides were then mounted
using Duolink® in situ mounting medium with DAPI (#DUO82040,
Sigma-Aldrich) and observed under a BZ-X810 Microscope (Keyence).
16

Article
The ﬁlters used for the BZ-X810 Microscope were BZ-X Filter GFP(excitation wavelength 470/40 nm, emission wavelength 525/50 nm)
and BZ-X Filter DAPI (excitation wavelength 360/40 nm, emission
wavelength 460/50 nm) (Keyence). PLA foci and the number of nuclei
were measured using Andy’s PLA algorithm59. The number of PLA fociwas measured in nine separate photographs using Andy’s PLA algorithm (biological replicates, n = 9). A table of the optimised image
parameters used for the PLA image analysis is provided in thesource data.

Bioinformatics analyses
Proteins predominantly biotinylated were identiﬁed using EGFRFabID. To determine the subcellular localisation of the biotinylated
proteins, the TMHMM (https://services.healthtech.dtu.dk/service.
php?TMHMM-2.0) software and the UniProt database were
employed. If a protein has transmembrane helices or is annotated asmembrane localised, it was considered as a membrane located protein.
If the biotinylation site of the membrane located protein was predicted
as extracellular region, it was considered to be located on extracellularregion. The known protein-protein interaction data were downloaded
from the IntAct database (https://www.ebi.ac.uk/intact). Pathway diagrams of protein-protein interaction network based on data analysedby DTX (https://harrier.nagahama-i-bio.ac.jp/dtx/) were generatedusing the Cytoscape software (https://cytoscape.org).

Statistical analyses
Signiﬁcant changes were analysed by Student’s t tests using Microsoft
Excel spreadsheets with a basic statistical programme or one-way
ANOVA followed by Tukey’s post-hoc test using GraphPad Prism
9 software (GraphPad, Inc.). Signiﬁcant changes in the volcano plots
were calculated by Student’s two-sided t test and the false discovery
rate (FDR)-adjusted P-values calculated using Benjamini–Hochberg
method by Microsoft Excel (Microsoft) are shown in the Supplementary Data. For all tests, P < 0.05 was considered statistically signiﬁcant.
Immunoblot analyses and streptavidin pull-down assays were repeatedmore than twice, with similar results.

Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.

Data availability
The MS proteomics data have been provided in Supplementary
Data 1–8 and deposited to the ProteomeXchange Consortium via thejPOST partner repository with the dataset identiﬁers PXD039449(Proximity biotinylation of Expi293F cells overexpressing EGFR by
EGFR-FabID), PXD043525 (Proximity biotinylation of Expi293F cells
stably expressing EGFR by EGFR-FabID), PXD039450 (Proximity biotinylation of A431 cells by EGFR-FabID), PXD039451 (Proximity biotinylation of NCI-H226 cells by EGFR-FabID), PXD043526 (Proximity
biotinylation of A431 cells in the geﬁtinib alone treatment by EGFRFabID), and PXD043527 (Proximity biotinylation of NCI-H226 cells in
the geﬁtinib alone treatment by EGFR-FabID). Source data are provided with this paper.

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Acknowledgements
The authors thank for Y. Adachi technical assistance and the Applied
Protein Research Laboratory of Ehime University. We thank Prof. Y.
Matsuura (Osaka University) for providing LHB and SHB genes. We
would like to thank the Applied Protein Research Laboratory of Ehime
University. This work was partially supported by the Platform Projectfor Supporting Drug Discovery and Life Science Research [Basis for
Supporting Innovative Drug Discovery and Life Science Research(BINDS)] from the Japan Agency for Medical Research and Development (AMED) under Grant Number JP21am0101077 (T.Sawasaki.),
22ama121010j0001 (T.S.), 23ama121010j0002 (T.S.), JP22ama121008(Y.K.). This work was also partially supported by JSPS KAKENHI(19H03218 and JP21K19230 for T.S.), Joint Usage and Joint Research
Programs of the Institute of Advanced Medical Sciences, Tokushima
University (H.K., T.S.), and Takeda Science Foundation. We would liketo thank Editage (www.editage.com) for English language editing.

Author contributions
K.Y. performed cloning and characterisation of AGIA-mAbID, EGFRmAbID and EGFR-FabID, cell-based assays, and biotinylation assay; R.S.
performed cloning and analysis of AGIA-FabID; K.N. and H.K. performedenrichment of biotinylated peptides and LC-MS/MS analyses; H.F. performed structural modelling; M.K.K. and Y.K. cloned antibodies; A.H. andT.Shirai. performed bioinformatic analyses of biotinylated sites andproteins; K.Y. and T.Sawasaki. analysed the data and wrote the draught
paper; T.Sawasaki. conceived the research and designed the study; H.K.
and T.Sawasaki. designed the experiments, wrote the paper, and all
authors revised the manuscript.

Competing interests
The authors declare no competing interests.

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Additional information
Supplementary information The online version contains
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