IQ Motif and SEC7 Domain-containing Protein 3 (IQSEC3 ... · GCC ACG ACA TTA-3′) into the XhoI...

Role of IQSEC3 in inhibitory synapse formation

1

IQ Motif and SEC7 Domain-containing Protein 3 (IQSEC3) interacts

with gephyrin to promote inhibitory synapse formation

Ji Won Um1,2

, Gayoung Choii1, Dongseok Park

2, Dongwook Kim

2, Sangmin Jeon

1, Hyeyeon

Kang2, Takuma Mori

3, Theofilos Papadopoulos

4, Taesun Yoo

5, Yeunkum Lee

5, Eunjoon Kim

5,6,

Katsuhiko Tabuchi3,7

and Jaewon Ko1, *

From the 1Department of Biochemistry, College of Life Science and Biotechnology, Yonsei

University, Seoul 120-749, Republic of Korea;

2Department of Physiology and BK21 PLUS Project for Medical Science, Yonsei University

College of Medicine, Seoul 120-751, Republic of Korea;

3Shinshu University School of Medicine, Matsumoto 390-8621, Japan;

4Department of Molecular Biology, University Medicine Göttingen, Göttingen 37075,

Germany;

5Department of Biological Sciences, Korea Advanced Institute of Science and Technology

(KAIST), Daejeon 305-701, Korea;

6Center for Synaptic Brain Dysfunctions, Institute for Basic Science (IBS), Daejeon 305-701,

Republic of Korea;

7PRESTO, Japan Science and Technology Agency (JST), Kawaguchi 332-0012, Japan.

Running Title: Role of IQSEC3 in inhibitory synapse formation

*To whom correspondence should be addressed: Jaewon Ko, Department of Biochemistry,

College of Life Science and Biotechnology, Yonsei University, 134 Shinchon-dong,

Seodaemun-gu, Science Research Center S421, Seoul 120-749, Republic of Korea. Tel: +82-2-

2123-5699. Fax: +82-2-362-9897. E-mail: [email protected]

Key words: IQSEC3, BRAG3, SynARFGEF, gephyrin, inhibitory synapse organization

http://www.jbc.org/cgi/doi/10.1074/jbc.M115.712893The latest version is at JBC Papers in Press. Published on March 21, 2016 as Manuscript M115.712893

Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on June 15, 2020http://w

ww

.jbc.org/D

ownloaded from

mailto:[email protected]

http://www.jbc.org/cgi/doi/10.1074/jbc.M115.712893

http://www.jbc.org/


2

ABSTRACT

Gephyrin is a central scaffold protein that

mediates development, function and

plasticity of mammalian inhibitory

synapses by interacting with various

inhibitory synaptic proteins. Here, we

show that IQSEC3, a guanine nucleotide

exchange factor (GEF) for ARF6, directly

interacts with gephyrin, an interaction

that is critical for the inhibitory synapse

localization of IQSEC3. Overexpression of

IQSEC3 increases inhibitory, but not

excitatory, synapse density in a GEF

activity-dependent manner. Conversely,

knockdown (KD) of IQSEC3 decreases

size of gephyrin cluster without altering

gephyin puncta density. Collectively, these

data reveal that IQSEC3 acts together

with gephyrin to regulate inhibitory

synapse development.

INTRODUCTION

Postsynaptic scaffolding proteins

organize functional synapses and promote

reliable synaptic transmission by ensuring the

accurate accumulation of postsynaptic

receptors in precise apposition to presynaptic

release sites. They also provide platforms for

postsynaptic receptors and regulate

downstream signaling cascades to adjust the

molecular composition of the postsynaptic

machineries that enable postsynaptic

plasticity (1,2). The most extensively studied

proteins at inhibitory synapses are arguably

gephyrin and its notable binding protein,

collybistin (3,4). However, although

significant progress has been made,

integrated principles that would allow a

comprehensive understanding of inhibitory

synapse organization and development,

particularly at molecular levels, remain to be

established (5,6).

Gephyrin forms a hexagonal lattice

beneath the postsynaptic membrane at

inhibitory synapses and anchors GABAA (-

aminobutyric acid) and glycine receptors

(4,6-8). Gephyrin interacts with numerous

other proteins whose functions at inhibitory

synapses, with the exception of collybistin

and neuroligin-2 (NL-2), are largely

undefined (6,9). Collybistin is required for

gephyrin clustering, and their absence in

mice compromises GABAergic synaptic

transmission and spatial learning (10,11).

NL-2 is an established inhibitory synaptic-

adhesion molecule essential for inhibitory

synaptic transmission that trans-synaptically

interacts with presynaptic neurexins and

intracellularly binds to gephyrin (12).

Through its tripartite interactions with

gephyrin and collybistin, NL-2 nucleates the

postsynaptic apparatus, locally inducing

gephyrin clustering and promoting its

submembrane targeting (9).

IQSEC3 (also known as BRAG3 or

SynArfGEF), together with IQSEC1 and


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


3

IQSEC2, constitute a family of brefeldin A-

resistant ARF guanine nucleotide exchange

factors (GEFs) (13). IQSEC family members

exhibit distinct synaptic localization in mouse

retina (14). IQSEC2/BRAG1 and

IQSEC1/BRAG2 directly interact with PSD-

95 and are involved in α-amino-3-hydroxy-5-

methyl-4-isoxazolepropionic acid (AMPA)-

type glutamate receptor trafficking and long-

term synaptic depression at excitatory

synapses (15,16). IQSEC1 is involved in

signaling pathways that induce breast cancer

invasion (17), and IQSEC2 mutations are

associated with non-syndromic X-linked

intellectual disability (XLID) (18,19). By

contrast, IQSEC3 is exclusively localized to

inhibitory synapses (13,14). However, it

remains to be determined whether IQSEC3 is

functionally important at inhibitory synapses,

and if so, how it orchestrates inhibitory

synapse development and function.

Here, we show that IQSEC3 directly

binds to gephyrin to promote inhibitory

synapse formation in an Arf-GEF activity-

dependent manner in cultured hippocampal

neurons. Gephyrin is required for inhibitory

synapse localization of IQSEC3, which is

critical for clustering of gephyrin in cultured

hippocampal neurons. Moreover, IQSEC3 is

important for maintenance of gephyrin

cluster size. Our results suggest a novel

molecular mechanism of inhibitory synapse

formation that may link ARF activity to the

IQSEC3-gephyrin complex and further imply

that IQSEC3 is critical for mediating

neuronal inhibition, possibly hinting at its

crucial roles in organizing inhibitory neural

circuit properties.

EXPERIMENTAL PROCEDURES

Yeast Two Hybrid Screens – Yeast two-

hybrid screening was performed as

previously described (20) using the PBN204

yeast strain harboring URA3, ADE2, and β-

gal as reporter genes. Full-length gephyrin

(aa 2-736) was subcloned into pGBKT (Gal4

fusion vector; Clontech) and used to screen

~1.0 106 clones from a human brain cDNA

library (Clontech) constructed in pACT2

(Gal4 activation domain vector; Clontech).

All of the prey clones were verified by

nucleotide sequencing.

Construction of Expression Vectors – 1.

IQSEC3. Expression plasmids for fragments

of rat IQSEC3 (Genbank accession number,

NM_207617.1) were prepared by amplifying

the corresponding region of the gene by PCR

and subcloning into the pCAGGS-FLAG

vector at EcoRI/EcoRV sites. Fragments

corresponding to the following amino acid

(aa) regions were prepared: 1-350, 1-315,

336-655, 1-995, 336-995, 336-1194, 636-

1194, 1-100, 101-200, 996-1194, 996-1095,

and 1-1190. cDNA encoding full-length rat

IQSEC3 (aa 1-1194) was PCR-amplified and

subcloned into the pcDNA3.1 myc vector

(Invitrogen) at EcoRI/EcoRV sites. The Arf-

GEF-inactive mutant E749A was generated

by QuikChange site-directed mutagenesis

(Stratagene) using pcDNA3.1 myc-IQSEC3


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


4

as a template. The shRNA lentiviral

expression vector against Iqsec3 was

constructed by annealing, phosphorylating,

and cloning oligonucleotides targeting rat

Iqsec3 (5’-GAG CTG GTG GTA GGC TCT

ATG AAA-3’) into the XhoI and XbaI sites

of a single KD vector (L-309; see (21) for a

schematic diagram of L-309) immediately

downstream of the human H1 promoter. For

the IQSEC3 rescue vector, three nucleotides

(underlined) in the

GAGCTAGTGGTCGGCTCTACGAAA

sequence of pcDNA3.1 myc-IQSEC3 or

pCAGGS-FLAG-IQSEC3 were mutated to

render them shRNA-resistant (see Fig. 9H).

IQSEC3 cDNA fragments corresponding to

amino acids 1-185 and 311-645 were cloned

into the BamHI and EcoRI sites of the

pGEX4T-1 vector (GE Healthcare). 2.

Gephyrin. Expression plasmids for fragments

of rat gephyrin (Genbank accession number,

NM_022865) were prepared by PCR-

amplifying and subcloning the corresponding

region of the gene into the pcDNA3.1 myc

vector (Invitrogen) at HindIII/XhoI sites.

Fragments corresponding to the following aa

regions were prepared: 1-185 (Gephyrin-G),

aa 166-322 (Gephyrin-C), and aa 303-736

(Gephyrin-E). The shRNA lentiviral

expression vector against gephyrin was

constructed by annealing, phosphorylating,

and cloning oligonucleotides targeting rat

gephyrin (5′-ACA TCA GAC CCA TCG

GCC ACG ACA TTA-3′) into the XhoI and

XbaI sites of the L-309 vector. A cDNA

fragment of gephyrin (corresponding to aa 1-

185) was cloned into the BamHI and EcoRI

sites of the pRSETA vector (Thermo Fisher

Scientific). 3. Previously published reagents.

The following constructs were as previously

described: Myc-gephyrin (26); pCAGGS-

FLAG-IQSEC1, pCAGGS-FLAG-IQSEC2,

and pCAGGS-FLAG-IQSEC3 (a gift from

Hiroyuki Sakagami) (13).

Antibodies – Fusion proteins of

glutathione-S-transferase (GST) and rat

IQSEC3 (aa 1-185) were produced in BL21 E.

coli and purified on a glutathione-Sepharose

column (GE Healthcare). Following

immunization of rabbits with this

immunogen, the IQSEC3-specific antibody

JK079 was affinity-purified using a Sulfolink

column (Pierce) on which the same GST-

fused IQSEC3 protein was immobilized. The

following commercially available antibodies

were used: mouse monoclonal anti-HA

(clone HA-7; Covance), mouse monoclonal

anti-FLAG (clone M1; Sigma), mouse

monoclonal anti-myc (clone 9E10; Santa

Cruz Biotechnology), goat polyclonal anti-

EGFP (Rockland), mouse monoclonal anti-

NL-1 (clone N97A/31; NeuroMab), rabbit

polyclonal anti-NL-2 (Synaptic Systems),

guinea pig polyclonal anti-VGLUT1

(Millipore), mouse monoclonal anti-GAD67

(clone 1G10.2; Millipore), mouse

monoclonal anti-PSD-95 (clone K28/43;

Thermo Scientific), mouse monoclonal anti-

α-tubulin (clone DM1A; Sigma), mouse

monoclonal anti-gephyrin (clone 3B11;

Synaptic Systems), mouse monoclonal anti-

gephyrin (clone mAb7a; Synaptic Systems),


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


5

rabbit polyclonal anti-collybistin (Synaptic

Systems), and mouse monoclonal anti-

GABARγ2 (clone 331A12; Synaptic

Systems). The following antibodies were

previously described: anti-S-SCAM (1146)

(28), and anti-IgSF9b (1913) (29).

Co-immunoprecipitation Assays – Rat

brain homogenates from P42 rats were

incubated with anti-IQSEC3 antibody

(JK079) overnight at 4 °C, after which 30 μl

of a 1:1 suspension of protein A-Sepharose

(Incospharm Corporation) was added, and the

mixture was incubated for 2 h at 4 °C with

gentle rotation. In detail, rat brains (2 g) were

homogenized in 10 ml ice-cold

homogenization buffer consisting of 320 mM

sucrose, 5 mM HEPES-NaOH (pH 7.5), 1

mM EDTA, 0.2 mM PMSF, 1 μg/ml

aprotinin, 1 μg/ml leupeptin, 1 μg/ml

pepstatin, and 1 mM Na3VO4. The

homogenized tissue was centrifuged at 2000

g for 15 min, and then the supernatant was

centrifuged at 100,000 x g for 1 h. The pellets

were homogenized in buffer consisting of 20

mM Hepes-NaOH (pH 7.5), 0.15 M NaCl, 2

mM CaCl2, 2 mM MgCl2, 0.2 mM PMSF, 1

μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml

pepstatin, and 1 mM Na3VO4. Triton X-100

was added to a final concentration of 1 %

(w/v) and dissolved with constant stirring at

4 °C for 1 h. Supernatants obtained after

centrifugation at 100,000 g for 1 h were

used for co-immunoprecipitation assays. The

beads were pelleted and washed three times

with lysis buffer (20 mM HEPES-NaOH (pH

7.5), 0.15 M NaCl, 2 mM CaCl2, 2 mM

MgCl2, 1 % Triton X-100, 0.2 mM PMSF, 1


pepstatin, and 1 mM Na3VO4). Immune

complexes were then resolved by SDS-PAGE

and immunoblotted with anti-gephyrin, anti-

NL-1, anti-NL-2, anti-S-SCAM, anti-

collybistin, or anti-IQSEC3 antibodies. For

Figs. 2A, 3B and 3D, human embryonic

kidney 293T (HEK293T) cells were

maintained in Dulbecco’s Modified Eagle’s

medium (DMEM) containing 10% fetal

bovine serum (FBS) and 100 U/ml of

penicillin-streptomycin. HEK293T cells were

then transfected with the indicated

combination of plasmids. After 48 h, the

transfected HEK293T cells were rinsed with

ice-cold phosphate-buffered saline (PBS) and

solubilized in lysis buffer (20 mM Tris (pH

7.4), 1.0 % Triton X-100, 0.1% SDS, 150

mM NaCl, 10% glycerol, 0.2 mM PMSF, 1


pepstatin, and 1 mM Na3VO4). After

centrifugation at 20,000 x g, the supernatants

were incubated with 1 μg of the appropriate

antibody overnight at 4 °C. Thereafter, 30 μl

of a 1:1 suspension of protein A-Sepharose

(Incospharm Corporation) was added, and the

mixture was incubated for 2 h at 4 °C with

gentle rotation. Immune complexes were then

resolved by SDS-PAGE and immunoblotted

with the indicated antibodies.

Coimmunoprecipitation experiments were

repeated at least three times, and quantified

results are expressed as the amount of protein

co-precipitated relative to input amount.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


6

Representative immunoblot images are

presented in the indicated figures.

Quantitative Reverse Transcription PCR

– Cultured rat cortical neurons were infected

with recombinant lentiviruses at DIV3 and

harvested at DIV10 for quantitative real-time

PCR using SYBR green qPCR master mix

(Takara). Total RNA was extracted from rat

cortical neurons using the TRIzol reagent

(Invitrogen) according to the manufacturer’s

protocol. Briefly, one well of a 12-well plate

of cultured neurons was harvested and

incubated with 500 μl of TRIzol reagent at

room temperature for 5 min. After phenol-

chloroform separation, RNA in the upper

aqueous phase was precipitated. cDNA was

synthesized from 500 ng of RNA by reverse

transcription using a ReverTra Ace-α-kit

(Toyobo). qPCR was performed with 1 μl of

cDNA using CFX96 Touch Real-Time PCR

(BioRad). The ubiquitously expressed

glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) was used as an endogenous

control. The sequences of the primer pairs

used are as follows: IQSEC1, 5’-TGC CAT

CAT CCT CCT CAA-3’ (forward) and 5’-

CGA TGA GCT TCT CAA CCT TCT-3’

(reverse); IQSEC2, 5’-TGC CAT CAT CCT

CCT CAA-3’ (forward) and 5’-CAC CAT

TGT CAA CTC CTC TC-3’ (reverse); and

IQSEC3, 5’-GGA GCA GAT TCG GAT

AGA ATG G-3’ (forward) and 5’-GGG TGA

TCC TTG CTT TGA CT-3’ (reverse).

Neuron Culture, Transfections, Imaging,

and Quantitation – Cultured hippocampal

neurons were prepared from E18 rat brains,

as previously described (30), cultured on

coverslips coated with poly-L-lysine, and

grown in Neurobasal medium supplemented

with B-27 (Invitrogen), 0.5% fetal bovine

serum, 0.5 mM Glutamax (Invitrogen), and

sodium pyruvate (Invitrogen). For

overexpression of IQSEC3 in cultured

neurons, hippocampal neurons were

transfected with pCAGG-FLAG-IQSEC3 or

its various derivatives, as indicated in the

individual figures, or with EGFP (Control)

using a CalPhos Kit (Clontech) at DIV10 and

immunostained at DIV14. For KD of

IQSEC3 in cultured neurons, hippocampal

neurons were transfected with L-309 alone

(Control), L-309 sh-IQSEC3 (B3; IQSEC3-

KD), or cotransfected with IQSEC3-KD and

shRNA-resistant myc-IQSEC3 using a

CalPhos Kit (Clontech) at DIV8 and

immunostained at DIV14. For

immunocytochemistry, cultured neurons

were fixed with 4% paraformaldehyde/4%

sucrose, permeabilized with 0.2% Triton X-

100 in PBS, immunostained with primary

antibodies as indicated, and detected with

Cy3- and fluorescein isothiocyanate (FITC)-

conjugated secondary antibodies (Jackson

ImmunoResearch). Images were acquired

using a confocal microscope (LSM710, Carl

Zeiss) with a 63x objective lenses; all image

setting were kept constant. Z-stacked images

were converted to maximal projection and

analyzed to obtain the size, intensity, and

density of puncta immunoreactivities derived

from marker proteins. Quantification was


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


7

performed in a blind manner using

MetaMorph software (Molecular Devices).

Statistics – All data are expressed as

means ± SEM. All experiments were

repeated using at least three independent

cultures, and data were statistically evaluated

using Student’s t-test or ANOVA with

Tukey’s test.

RESULTS

Identification of IQSEC3 as a novel

gephyrin-interacting protein – To identify

additional gephyrin-binding proteins, we

screened a human brain DNA library by yeast

two hybrid assay using full-length gephyrin

(aa 2-735) as bait. Of ~1 106 yeast colonies,

60 positive clones were selected using three

independent reporters, 27 of which were

found to be genuine positives (Fig. 1A).

Among these clones, eight encoded

previously known gephyrin-binding proteins

(six for collybistins and two for RAFT-1)

(Fig. 1A). Intriguingly, nine encoded a partial

cDNA for IQSEC2/BRAG1 and three

encoded a cDNA fragment for

IQSEC3/BRAG3 covering an N-terminal

region between a CC1 (coiled-coil 1) domain

and an IQ motif (Fig. 1B).

Co-immunoprecipitation assays in

HEK293T cells expressing FLAG-tagged

IQSEC1, IQSEC2 or IQSEC3, and Myc-

tagged gephyrin confirmed these results,

showing that IQSEC2 and IQSEC3, but not

IQSEC1, co-immunoprecipitated with

gephyrin (Fig. 2A). Quantitative analyses

revealed that gephyrin more strongly

interacted (~4.3 fold) with IQSEC3 than with

IQSEC2 (Fig. 2A). Thus, despite structural

similarities among IQSEC family proteins,

only IQSEC2 and IQSEC3 were found to

bind gephyrin. For this study, we focused on

IQSEC3 because it is exclusively localized to

inhibitory postsynaptic specializations in

brains (13,14). Next, we found that IQSEC3

immunoprecipitated from crude synaptosome

lysates of adult rat brains with IQSEC3

antibodies (JK079; see below)

coimmunoprecipitated significant amounts of

gephyrin as well as NL-2 and S-SCAM, but

not NL-1 or collybistin (Fig. 2B).

Immunocytochemistry analyses performed

using an IQSEC3-specific antibody (JK079)

generated in our laboratory (Fig. 2C, 2D, 2E

and 2F) revealed strong colocalization of

IQSEC3 and gephyrin in mature hippocampal

neurons (63% ± 4% of gephyrin puncta were

positive for IQSEC3) (Fig. 2D and E). This

antibody specifically recognized a single

band in HEK293T cells expressing IQSEC3

and two distinct bands of ~150–170 kDa in

brain crude synaptosomes (Fig. 2C). Taken

together, these results indicate that IQSEC3

shares similar biochemical and expression

properties, and forms specific complexes

with gephyrin in rat brains, in accordance

with the previous reports of strong

colocalization of IQSEC3 with gephyrin in

brains and cultured neurons (13,14).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


8

Minimal binding domains of gephyrin-

IQSEC3 interaction – To determine the

minimal regions responsible for the

interaction, we generated a series of gephyrin

and IQSEC3 deletion variants and performed

co-immunoprecipitation assays in HEK293T

cells. We found that the G-domain, but not

other domains, of gephyrin interacted with

IQSEC3 (Fig. 3A and B). Intriguingly, the

minimal gephyrin-binding region in IQSEC3

was mapped to two parts: an N-terminal

region (aa 101-200) and a C-terminal region

(aa 636-1194). Both bound to gephyrin,

although their individual binding strengths

were weaker than that of full-length IQSEC3

(aa 1-1194) (Fig. 3C and D). In addition, His-

gephyrin G-domain brought down GST-fused

IQSEC3 aa 1-185, but not GST-IQSEC3 aa

311-645, suggesting that the G-domain of

gephyrin directly binds to IQSEC3 (Fig. 4).

Quantitative analyses showed that the N-

terminal binding site had a stronger binding

affinity for gephyrin than the C-terminal

binding site (Fig. 3C and D). These results

suggest that two parts of the IQSEC3

molecule interact with the G-domain of

gephyrin.

Overexpression of IQSEC3 promotes

inhibitory synapse formation through its Arf-

GEF activity – Next, to determine whether

IQSEC3 affects inhibitory synapse

development, we cotransfected cultured

hippocampal neurons at 10 d in vitro

(DIV10) with expression vectors encoding

EGFP alone (Control), or EGFP with FLAG-

tagged IQSEC1, IQSEC2, or IQSEC3, and

immunostained transfected neurons for the

excitatory synaptic marker VGLUT1

(vesicular glutamate transporter 1), the

inhibitory presynaptic marker GAD67

(glutamic acid decarboxylase 67 kDa

(GAD67), or the inhibitory postsynaptic

marker gephyrin at DIV14. Because certain

inhibitory synaptic proteins preferentially act

in specific subcellular domains of specific

neuron types (9,34), we analyzed GAD67-

and gephyrin-positive puncta in both

dendrites and soma. Overexpression of

IQSEC3 did not alter excitatory synapse

density, labelled as VGLUT1-positive puncta

(Fig. 5A and B). However, overexpression of

IQSEC3, but not IQSEC1 or IQSEC2, caused

an increase in GAD67-positive or gephyrin-

positive puncta density in both dendritic and

perisomatic regions (Fig. 5C-F; data not

shown), suggesting that IQSEC3 specifically

fosters inhibitory synapse formation in both

subcellular compartments. Consistent with

this, overexpression of IQSEC3 led to an

increase in the number of GABAAγ2 puncta

in dendrites or soma of the transfected

neurons (data not shown).

To determine whether inhibitory

synaptic localization of IQSEC3 depends on

the presence of gephyrin in cultured neurons,

we knocked down endogenous gephyrin

proteins at DIV8 and examined whether

localization or stability of endogenous

IQSEC3 protein was altered at DIV14 (Fig.

6). Notably, gephyrin knockdown (KD) with

short hairpin RNA (shRNA) significantly

decreased the number of endogenous


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


9

IQSEC3 puncta, but not GAD67 puncta,

suggesting that the maintenance of IQSEC3

at inhibitory synapses is dependent on

gephyrin and that this interaction contributes

to IQSEC3-dependent inhibitory synapse

formation (Fig. 6; see Fig. 9F and G for

gephyrin KD characterization).

To corroborate this notion, we

overexpressed a series of IQSEC3 deletion

constructs with gephyrin-binding activity or

lacking gephyrin-binding activity (Fig. 7).

We found that overexpression of IQSEC3 aa

1-995 and IQSEC3 aa 336-1094 increased

the number of gephyrin-positive synaptic

puncta, whereas IQSEC3 aa 1-315 or

IQSEC3 aa 336-995 did not (Fig. 7). To

probe the reason for the failure of IQSEC3 aa

1-315 to increase gephyrin-positive synaptic

puncta, we generated an additional set of

IQSEC3 constructs that either lacked the

ability to bind to PDZ-containing proteins

(e.g., S-SCAM) or abolished its ARF-GEF

activity (IQSEC3 E749A) (Fig. 8). We found

that the IQSEC3 ΔPDZ C-tail (aa 1-1190),

but not the dominant-negative (DN) E749A

construct (although retaining the ability to

interact with gephyrin; Fig. 8C), still boosted

inhibitory synapse formation (Fig. 8). These

results suggest that IQSEC3 promotes

inhibitory synapse development through its

Arf-GEF activity, not through its C-terminal

PDZ-domain-binding. In addition, our results

support the conclusion that gephyrin-binding

activity alone does not dictate the inhibitory

synapse-promoting property of IQSEC3,

because IQSEC3 aa 1-315 was able to bind

well to gephyrin, but lacked the SEC7

domain, which is critical for ARF-GEF

activity.

Knockdown of IQSEC3 decreases

gephyrin puncta size in cultured neurons –

To address whether IQSEC3 is required for

inhibitory synapse formation and function,

we tested the effect of IQSEC3 KD using

shRNAs targeting rat Iqsec3. Preliminary

tests in HEK293T cells showed that, of the

four designed shRNAs (B1-B4), only the B3

construct was effective, suppressing the level

of co-expressed FLAG-IQSEC3 by ~80%

(Fig. 9A and B). The efficacy of the B3

construct against endogenous IQSEC3 was

further confirmed in cultured rat cortical

neurons infected with lentiviruses expressing

B3 or control vector (Control). Quantitative

real-time RT-PCR and semi-quantitative

immunoblotting showed that B3 specifically

decreased Iqsec3 mRNA levels by ~70%

(Fig. 9C) and protein levels by ~80%,

without affecting gephyrin or PSD-95 protein

levels (Fig. 9D and E).

Next, to determine whether IQSEC3 KD

alters synaptic morphology parameter, we

transfected cultured neurons at DIV8 with

lentiviral expression vectors for EGFP only

(Control), or EGFP together with Iqsec3-

shRNA, or Iqsec3-shRNA and shRNA-

resistant full-length IQSEC3 expression

vector (see Fig. 9H), and immunostained

transfected neurons at DIV14 for gephyrin.

Surprisingly, in contrast to the robust effects

of IQSEC3 gain-of-function (Fig. 5),


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


10

IQSEC3 KD had no effect on gephyrin-

positive inhibitory synapse numbers in

dendrites or soma (Fig. 10A and B). However,

IQSEC3 KD significantly reduced gephyrin

puncta size in dendrites and soma.

Importantly, coexpression of an shRNA-

resistant form of IQSEC3 (+rescue)

completely abolished the deficits in gephyrin

puncta size observed with IQSEC3 KD (Fig.

10A and B), confirming that the observed

phenotypes are not derived from off-target

effects. Note that coexpression of the

IQSEC3 rescue vector leads to IQSEC3 gain-

of-function phenotypes (i.e., increased

gephyrin puncta density). These results may

appear to indicate that the expression level of

the IQSEC3 rescue vector is too high to

induce an IQSEC3-overexpression effect

(data not shown). However, we rejected this

interpretation because parallel experiments

showed no increase in gephyrin puncta size

(Fig. 10A and B). Overall, our data are

consistent with the idea that IQSEC3 is

required for gephyrin clustering (Fig. 10A

and B).

DISCUSSION

The presumption that inhibitory

postsynaptic specialization is less elaborate

than excitatory PSD has been increasingly

challenged by rapid progress in our

understanding of inhibitory synapse

organization. Because inhibitory synapses are

fundamentally different from excitatory

synapses with regard to the nature of

neurotransmitter receptors and associated

proteins, the organizing principles underlying

inhibitory synapse development are also

likely different. Gephyrin is a key scaffolding

molecule at inhibitory synapses, and its

interaction with collybistin plays a major role

in clustering of gephyrin and modulating

GABAA receptor functions (4). Gephyrin also

directly interacts with NL-2, which

specifically drives postsynaptic assembly at

perisomatic inhibitory synapses (9). However,

in light of the molecular heterogeneity of

inhibitory synapses, efficient inhibition

requires that various inhibitory synaptic

components, including gephyrin, are

functionally and physically coupled. In the

present study, we identified a direct

molecular interaction of gephyrin with

IQSEC3, a previously unexplored inhibitory

synaptic protein, and investigated its synaptic

functions using various experimental

approaches, including biochemistry, cell

biology, and knockdown-and-rescue. We

made two principal findings:

First, IQSEC3 is a new gephyrin-

binding protein (Figs. 1, 3, and 4). The

IQSEC family of Arf-GEF proteins is

composed of three members, IQSEC1,

IQSEC2, and IQSEC3, and both IQSEC2 and

IQSEC3, but not IQSEC1, interact with

gephyrin in heterologous cells. We mapped

the binding sites in IQSEC3 to N- and C-

terminal sites, and in gephyrin to the G-

domain. To the best of our knowledge,

IQSEC3 is the first protein identified that

binds to the G-domain of gephyrin; most


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


11

other gephyrin-interacting proteins bind to

either C- or E-domains. Therefore, it is

tempting to speculate that IQSEC3 may

regulate G-domain-mediated trimer

formation by gephyrin (35). Indeed,

knockdown of IQSEC3 markedly decreased

gephyrin puncta size, an indicative of

reduced clustering. In addition, our

observation that gephyrin binds to two

distinct sites in IQSEC3 suggests a possible

U-shaped conformation of IQSEC3 when

associated with gephyrin, a result reminiscent

of cytohesin 2 (another Arf-GEF), which

exhibits a coiled-coil intramolecular

interaction and shows full Arf-GEF activity

only when in the open conformation (36).

Second, our gain- and loss-of-function

experiments in cultured hippocampal neurons

clearly establish the significance of IQSEC3

in inhibitory synapse development (Fig. 5

and 10). Overexpression of IQSEC3

strikingly increased inhibitory synapse

density, whereas KD of IQSEC3 caused a

decrease in gephyrin puncta size with no

alternation in gephyrin puncta density.

Intriguingly, we found that IQSEC3-

mediated inhibitory synapse promotion is

independent of PDZ protein binding, but

instead relies on its Arf-GEF activity (Fig. 8).

It is also notable that overexpression of

IQSEC3-E749A abolished the inhibitory

synapse-promoting activity of IQSEC3, an

effect that was not observed following

IQSEC3 KD (Fig. 8). Incomplete KD of

IQSEC3 protein expression cannot wholly

account for this phenotypic discrepancy

because IQSEC3 KD significantly reduced

inhibitory synaptic transmission (J.W.U. and

J.K., unpublished observation). Instead, these

data support the interpretation that the

phenotype derived from IQSEC3-E749A

substitution is a gain-of-function phenotype

because it was not observed with IQSEC3

loss-of-function. Although further details

remain to be determined, it is likely that

residual IQSEC3 proteins remaining after KD

are still functionally capable of activating Arf

proteins that coordinate inhibitory synapse

development. Regardless of the precise

mechanisms, our data suggest that IQSEC3

employs diverse molecular mechanisms at

inhibitory synapses and that Arf6 may also be

involved in inhibitory synapse development.

At excitatory synapses, IQSEC2, together

with Arf6 signaling pathways, regulates

synaptic activity-dependent removal of

AMPA receptors (15). Moreover, Arf6 and

its Arf-GEF, EFA6A, coordinate excitatory

synapse development (22), implying that

actions of Arf6 signaling pathways operate in

both excitatory and inhibitory synapses.

Furthermore, it should be rigorously

determined whether IQSEC3 acts as a GEF

for specific Arf proteins and whether Arfs

function in inhibitory synapse development

together with IQSEC3.

In summary, our study illustrates the

significance of the gephyrin/IQSEC3

complex in organizing inhibitory synapse

development and provides compelling

evidence to corroborate the idea that IQSEC3

is a critical factor in governing inhibitory


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


12

synapse formation. The immediate goals of

future studies should be to validate the

postulated roles of IQSEC3 presented here

using conditional KO and/or knock-in mice

lacking gephyrin interactions to address

whether IQSEC3 broadly mediates inhibition

at specific synapse types in specific neuron

types, and by extension, associated neural

circuits.

Acknowledgment: We are grateful to Hiroyuki Sakagami (Kitasato University, Japan) and

Shiva Tyagarajan (University of Zurich, Switzerland) for reagents. This work was supported by

grants from the National Research Foundation of Korea (NRF) funded by the Ministry of

Science and Future Planning (2014051826 to J.K.; NRF-2015R1C1A2A01052176 to J.W.U.),

the Yonsei University Future-leading Research Initiative of 2014 (to J.K.), the Yonsei

University Future-leading Research Initiative of 2015 (to J.W.U.), NRF funded by the Ministry

of Education, Science and Technology (NRF-2013R1A6A3A04061338 to J.W.U.), DFG grant

PA2087/1-3 (to T.P.), the Institute for Basic Science (IBS-R002-D1 to E.K.), JST PRESTO (to

K.T.), and in part by the Brain Korea 21 (BK21) PLUS program. G.C., D.P., S.J., and H.K. are

fellowship awardee by BK21 PLUS program.

Conflict of interest: The authors declare that they have no conflict of interest with the contents

of this article.

Author contributions: The authors have made the following declarations about their

contributions: Conceived and designed the experiment: JWU, TP, EK, KT and JK. Performed

the experiments: JWU, GC, DP, DK, HK, TP, TY and YL. Analyzed the data: JWU, GC, SJ,

TM, TP, EK, KT and JK. Contributed reagents/materials/analysis tools: SJ and JK. Wrote the

paper: JWU and JK.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


13

REFERENCES

1. Fritschy, J. M., Panzanelli, P., and Tyagarajan, S. K. (2012) Molecular and functional

heterogeneity of GABAergic synapses. Cell Mol Life Sci 69, 2485-2499

2. Arancibia-Carcamo, I. L., and Moss, S. J. (2006) Molecular organization and assembly

of the central inhibitory postsynapse. Results Probl Cell Differ 43, 25-47

3. Papadopoulos, T., and Soykan, T. (2011) The role of collybistin in gephyrin clustering

at inhibitory synapses: facts and open questions. Front Cell Neurosci 5, 11

4. Choii, G., and Ko, J. (2015) Gephyrin: a central GABAergic synapse organizer. Exp

Mol Med 47, e158

5. West, A. E., and Greenberg, M. E. (2011) Neuronal activity-regulated gene

transcription in synapse development and cognitive function. Cold Spring Harb

Perspect Biol 3

6. Ko, J., Choii, G., and Um, J. W. (2015) The balancing act of GABAergic synapse

organizers. Trends Mol Med 21, 256-268

7. Tyagarajan, S. K., and Fritschy, J. M. (2014) Gephyrin: a master regulator of neuronal

function? Nat Rev Neurosci 15, 141-156

8. Specht, C. G., Izeddin, I., Rodriguez, P. C., El Beheiry, M., Rostaing, P., Darzacq, X.,

Dahan, M., and Triller, A. (2013) Quantitative nanoscopy of inhibitory synapses:

counting gephyrin molecules and receptor binding sites. Neuron 79, 308-321

9. Poulopoulos, A., Aramuni, G., Meyer, G., Soykan, T., Hoon, M., Papadopoulos, T.,

Zhang, M., Paarmann, I., Fuchs, C., Harvey, K., Jedlicka, P., Schwarzacher, S. W., Betz,

H., Harvey, R. J., Brose, N., Zhang, W., and Varoqueaux, F. (2009) Neuroligin 2 drives

postsynaptic assembly at perisomatic inhibitory synapses through gephyrin and

collybistin. Neuron 63, 628-642

10. Papadopoulos, T., Korte, M., Eulenburg, V., Kubota, H., Retiounskaia, M., Harvey, R.

J., Harvey, K., O'Sullivan, G. A., Laube, B., Hulsmann, S., Geiger, J. R., and Betz, H.

(2007) Impaired GABAergic transmission and altered hippocampal synaptic plasticity

in collybistin-deficient mice. EMBO J 26, 3888-3899

11. Papadopoulos, T., Eulenburg, V., Reddy-Alla, S., Mansuy, I. M., Li, Y., and Betz, H.

(2008) Collybistin is required for both the formation and maintenance of GABAergic

postsynapses in the hippocampus. Mol Cell Neurosci 39, 161-169

12. Krueger, D. D., Tuffy, L. P., Papadopoulos, T., and Brose, N. (2012) The role of

neurexins and neuroligins in the formation, maturation, and function of vertebrate

synapses. Curr Opin Neurobiol 22, 412-422

13. Fukaya, M., Kamata, A., Hara, Y., Tamaki, H., Katsumata, O., Ito, N., Takeda, S., Hata,

Y., Suzuki, T., Watanabe, M., Harvey, R. J., and Sakagami, H. (2011) SynArfGEF is a


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


14

guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic

specializations of inhibitory synapses. J Neurochem 116, 1122-1137

14. Sakagami, H., Katsumata, O., Hara, Y., Tamaki, H., Watanabe, M., Harvey, R. J., and

Fukaya, M. (2013) Distinct synaptic localization patterns of brefeldin A-resistant

guanine nucleotide exchange factors BRAG2 and BRAG3 in the mouse retina. J Comp

Neurol 521, 860-876

15. Myers, K. R., Wang, G., Sheng, Y., Conger, K. K., Casanova, J. E., and Zhu, J. J.

(2012) Arf6-GEF BRAG1 regulates JNK-mediated synaptic removal of GluA1-

containing AMPA receptors: a new mechanism for nonsyndromic X-linked mental

disorder. J Neurosci 32, 11716-11726

16. Scholz, R., Berberich, S., Rathgeber, L., Kolleker, A., Kohr, G., and Kornau, H. C.

(2010) AMPA receptor signaling through BRAG2 and Arf6 critical for long-term

synaptic depression. Neuron 66, 768-780

17. Morishige, M., Hashimoto, S., Ogawa, E., Toda, Y., Kotani, H., Hirose, M., Wei, S.,

Hashimoto, A., Yamada, A., Yano, H., Mazaki, Y., Kodama, H., Nio, Y., Manabe, T.,

Wada, H., Kobayashi, H., and Sabe, H. (2008) GEP100 links epidermal growth factor

receptor signalling to Arf6 activation to induce breast cancer invasion. Nat Cell Biol 10,

85-92

18. Tarpey, P. S., Smith, R., Pleasance, E., Whibley, A., Edkins, S., Hardy, C., O'Meara, S.,

Latimer, C., Dicks, E., Menzies, A., Stephens, P., Blow, M., Greenman, C., Xue, Y.,

Tyler-Smith, C., Thompson, D., Gray, K., Andrews, J., Barthorpe, S., Buck, G., Cole, J.,

Dunmore, R., Jones, D., Maddison, M., Mironenko, T., Turner, R., Turrell, K., Varian,

J., West, S., Widaa, S., Wray, P., Teague, J., Butler, A., Jenkinson, A., Jia, M.,

Richardson, D., Shepherd, R., Wooster, R., Tejada, M. I., Martinez, F., Carvill, G.,

Goliath, R., de Brouwer, A. P., van Bokhoven, H., Van Esch, H., Chelly, J., Raynaud,

M., Ropers, H. H., Abidi, F. E., Srivastava, A. K., Cox, J., Luo, Y., Mallya, U., Moon,

J., Parnau, J., Mohammed, S., Tolmie, J. L., Shoubridge, C., Corbett, M., Gardner, A.,

Haan, E., Rujirabanjerd, S., Shaw, M., Vandeleur, L., Fullston, T., Easton, D. F., Boyle,

J., Partington, M., Hackett, A., Field, M., Skinner, C., Stevenson, R. E., Bobrow, M.,

Turner, G., Schwartz, C. E., Gecz, J., Raymond, F. L., Futreal, P. A., and Stratton, M. R.

(2009) A systematic, large-scale resequencing screen of X-chromosome coding exons

in mental retardation. Nat Genet 41, 535-543

19. Shoubridge, C., Tarpey, P. S., Abidi, F., Ramsden, S. L., Rujirabanjerd, S., Murphy, J.

A., Boyle, J., Shaw, M., Gardner, A., Proos, A., Puusepp, H., Raymond, F. L., Schwartz,

C. E., Stevenson, R. E., Turner, G., Field, M., Walikonis, R. S., Harvey, R. J., Hackett,

A., Futreal, P. A., Stratton, M. R., and Gecz, J. (2010) Mutations in the guanine


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


15

nucleotide exchange factor gene IQSEC2 cause nonsyndromic intellectual disability.

Nat Genet 42, 486-488

20. Ko, J., Kim, S., Valtschanoff, J. G., Shin, H., Lee, J. R., Sheng, M., Premont, R. T.,

Weinberg, R. J., and Kim, E. (2003) Interaction between liprin-alpha and GIT1 is

required for AMPA receptor targeting. J Neurosci 23, 1667-1677

21. Ko, J., Soler-Llavina, G. J., Fuccillo, M. V., Malenka, R. C., and Sudhof, T. C. (2011)

Neuroligins/LRRTMs prevent activity- and Ca2+/calmodulin-dependent synapse

elimination in cultured neurons. J Cell Biol 194, 323-334

22. Choi, S., Ko, J., Lee, J. R., Lee, H. W., Kim, K., Chung, H. S., Kim, H., and Kim, E.

(2006) ARF6 and EFA6A regulate the development and maintenance of dendritic

spines. J Neurosci 26, 4811-4819

23. Mayer, S., Kumar, R., Jaiswal, M., Soykan, T., Ahmadian, M. R., Brose, N., Betz, H.,

Rhee, J. S., and Papadopoulos, T. (2013) Collybistin activation by GTP-TC10 enhances

postsynaptic gephyrin clustering and hippocampal GABAergic neurotransmission. Proc

Natl Acad Sci U S A 110, 20795-20800

24. Soykan, T., Schneeberger, D., Tria, G., Buechner, C., Bader, N., Svergun, D., Tessmer,

I., Poulopoulos, A., Papadopoulos, T., Varoqueaux, F., Schindelin, H., and Brose, N.

(2014) A conformational switch in collybistin determines the differentiation of

inhibitory postsynapses. EMBO J 33, 2113-2133

25. Harvey, K., Duguid, I. C., Alldred, M. J., Beatty, S. E., Ward, H., Keep, N. H.,

Lingenfelter, S. E., Pearce, B. R., Lundgren, J., Owen, M. J., Smart, T. G., Luscher, B.,

Rees, M. I., and Harvey, R. J. (2004) The GDP-GTP exchange factor collybistin: an

essential determinant of neuronal gephyrin clustering. J Neurosci 24, 5816-5826

26. Kins, S., Betz, H., and Kirsch, J. (2000) Collybistin, a newly identified brain-specific

GEF, induces submembrane clustering of gephyrin. Nat Neurosci 3, 22-29

27. Fuhrmann, J. C., Kins, S., Rostaing, P., El Far, O., Kirsch, J., Sheng, M., Triller, A.,

Betz, H., and Kneussel, M. (2002) Gephyrin interacts with Dynein light chains 1 and 2,

components of motor protein complexes. J Neurosci 22, 5393-5402

28. Mok, H., Shin, H., Kim, S., Lee, J. R., Yoon, J., and Kim, E. (2002) Association of the

kinesin superfamily motor protein KIF1Balpha with postsynaptic density-95 (PSD-95),

synapse-associated protein-97, and synaptic scaffolding molecule PSD-95/discs

large/zona occludens-1 proteins. J Neurosci 22, 5253-5258

29. Woo, J., Kwon, S. K., Nam, J., Choi, S., Takahashi, H., Krueger, D., Park, J., Lee, Y.,

Bae, J. Y., Lee, D., Ko, J., Kim, H., Kim, M. H., Bae, Y. C., Chang, S., Craig, A. M.,

and Kim, E. (2013) The adhesion protein IgSF9b is coupled to neuroligin 2 via S-

SCAM to promote inhibitory synapse development. J Cell Biol 201, 929-944


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


16

30. Ko, J., Kim, S., Chung, H. S., Kim, K., Han, K., Kim, H., Jun, H., Kaang, B. K., and

Kim, E. (2006) SALM synaptic cell adhesion-like molecules regulate the differentiation

of excitatory synapses. Neuron 50, 233-245

31. Bates, M., Huang, B., Dempsey, G. T., and Zhuang, X. (2007) Multicolor super-

resolution imaging with photo-switchable fluorescent probes. Science 317, 1749-1753

32. Dani, A., Huang, B., Bergan, J., Dulac, C., and Zhuang, X. (2010) Superresolution

imaging of chemical synapses in the brain. Neuron 68, 843-856

33. Sumita, K., Sato, Y., Iida, J., Kawata, A., Hamano, M., Hirabayashi, S., Ohno, K., Peles,

E., and Hata, Y. (2007) Synaptic scaffolding molecule (S-SCAM) membrane-associated

guanylate kinase with inverted organization (MAGI)-2 is associated with cell adhesion

molecules at inhibitory synapses in rat hippocampal neurons. J Neurochem 100, 154-

166

34. Gibson, J. R., Huber, K. M., and Sudhof, T. C. (2009) Neuroligin-2 deletion selectively

decreases inhibitory synaptic transmission originating from fast-spiking but not from

somatostatin-positive interneurons. J Neurosci 29, 13883-13897

35. Saiyed, T., Paarmann, I., Schmitt, B., Haeger, S., Sola, M., Schmalzing, G.,

Weissenhorn, W., and Betz, H. (2007) Molecular basis of gephyrin clustering at

inhibitory synapses: role of G- and E-domain interactions. J Biol Chem 282, 5625-5632

36. Hiester, K. G., and Santy, L. C. (2013) The cytohesin coiled-coil domain interacts with

threonine 276 to control membrane association. PLoS One 8, e82084

ABBREVIATIONS

The following abbreviations are used: GAD, glutamic acid decarboxylase; HEK, human

embryonic kidney; IQSEC, IQ motif and Sec7 domain;; KD, knockdown; NL, neuroligin;

VGLUT, vesicular glutamate transporter.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


17

FIGURE LEGENDS

Figure 1. A yeast two-hybrid screen identifies IQSEC3 as a gephyrin-binding protein. A,

Overview of the yeast two-hybrid screen of a human brain cDNA library using gephyrin full-

length cDNA (aa 2-753) cloned into the pGBKT vector. SD-LWU, selective medium lacking

leucine, tryptophan, and uracil; SD-LWA, selective medium lacking leucine, tryptophan, and

alanine; SD-LW, selective medium lacking leucine and tryptophan. B, Human brain cDNA

clones isolated from the yeast two-hybrid screen (A) are shown aligned below a schematic of

IQSEC2 and IQSEC3 protein (drawn to scale). The prey clones are labelled “AD”. CC1, coiled-

coil motif 1; IQ, calcium/calmodulin-binding IQ motif; SEC, Sec7 domain; PH, pleckstrin

homology domain; CC2, coiled-coil motif 2; PBM, PDZ-binding motif.

Figure 2. Interaction of IQSEC3 with gephyrin in HEK293T cells, formation of an

IQSEC3-gephyrin complex in rat brains, and characterization of IQSEC3 antibodies. A,

Co-immunoprecipitation experiment demonstrating that IQSEC2 and IQSEC3, but not IQSEC1,

interact with gephyrin. HEK293T cells were transfected with FLAG-tagged IQSEC1 (FLAG-

IQSEC1), FLAG-tagged IQSEC2 (FLAG-IQSEC2), or FLAG-tagged IQSEC3 (FLAG-

IQSEC3) alone or together with Myc-tagged gephyrin (Myc-Gephyrin), and co-

immunoprecipitation of IQSECs with gephyrin was assayed. A representative immunoblot

visualized by ECL (left) and quantitative bar graphs (right) analyzing coimmunoprecipitation

efficiency are shown. Note that maximum coimmunoprecipitation efficiency could not be

achieved. Input, 5%. B, Co-immunoprecipitation experiment in rat brains demonstrating that

IQSEC3 forms complexes with gephyrin, NL-2 and S-SCAM, but not with NL-1 or collybistin.

Crude synaptosomal fractions of adult mouse brains were immunoprecipitated with anti-

IQSEC3 antibody (JK079) and immunoblotted with the indicated antibodies. Equal amounts of

rabbit IgG (IgG) were used as a negative control. Input, 5%. C, Biochemical characterization of

the anti-IQSEC3 antibody used in this study. Immunoblot analyses of the anti-IQSEC3 antibody,

JK079, using rat brain crude synaptosomes (P2) and lysates from HEK293T cells transfected

with a FLAG-tagged IQSEC3 expression vector [Trans.]. The expression of FLAG-tagged

IQSEC3 was confirmed by immunoblotting with an anti-FLAG antibody. Br., brain; P2, crude

synaptosomes; Unt., untransfected HEK293T cell lysates. D, Colocalization of IQSEC3 puncta

with gephyrin puncta in rat cultured hippocampal neurons. Cultured neurons at DIV14 were

immunostained with anti-IQSEC3 (JK079; red) and anti-gephyrin antibodies (green), and

detected with Cy3- or FITC-conjugated secondary antibodies. Scale bar, 10 μm (applies to all

images). E, Validation of the in-house IQSEC3 antibody (JK079; red) by immunocytochemistry

using DIV14 rat primary hippocampal cultured neurons. JK079-immunoreactive puncta


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


18

colocalize with those obtained using previously published antibodies (old). Scale bar, 10 μm

(applies to all images). F-G, Cross-reactivity of the anti-IQSEC3 antibody. HEK293T cells,

untransfected or transfected with the indicated IQSEC expression vectors, were immunoblotted

or immunostained with anti-IQSEC3 (JK079) and anti-FLAG antibodies. JK079 is specific for

IQSEC3. An anti-α-tubulin antibody was used for normalization. Scale bar, 10 μm (applies to

all images).

Figure 3. Determination of minimal binding regions of gephyrin and IQSEC3. A,

Schematic diagrams of a series of gephyrin deletion constructs. G, G-domain; C, C-domain; E,

E-domain. B, Co-immunoprecipitation experiment showing that the G-domain of gephyrin is

sufficient for interaction with IQSEC3. HEK293T cells were transfected with Myc-tagged

gephyrin (Myc-Gephyrin) alone or together with FLAG-tagged IQSEC3 (FLAG-IQSEC3), and

co-immunoprecipitation of IQSEC3 with gephyrin constructs was assayed. Asterisks denote

nonspecific bands. Input, 5%. C, Schematic diagrams of a series of IQSEC3 deletion constructs.

CC1, coiled-coil motif 1; IQ, calcium/calmodulin-binding IQ motif; SEC, Sec7 domain; PH,

pleckstrin homology domain; CC2, coiled-coil motif 2; and PBM, PDZ-binding motif. D, Co-

immunoprecipitation experiment showing that two regions (aa 101-200 and 636-1195) of

IQSEC3 are required for interaction with gephyrin. HEK293T cells were transfected with Myc-

Gephyrin alone or together with FLAG-IQSEC3 constructs, and co-immunoprecipitation of

IQSEC3 deletion variants with gephyrin construct was assayed and quantified as the percentage

of coimmunoprecipitation efficiency (red). Input, 5%.

Figure 4. Direct interaction of gephyrin with IQSEC3. A, Purified recombinant His-tagged

gephyrin or GST-tagged IQSEC3 proteins produced in E. coli are analyzed by SDS-PAGE and

Coomassie Brilliant Blue (CBB) staining. An asterisk indicates the position of purified GST-

IQSEC3 aa 1-185. B, Direct interaction of IQSEC3 with gephyrin in vitro. GST-IQSEC3 (aa 1-

185 or aa 331-645) or GST alone was incubated with H6-gephyrin. The precipitates were

analyzed by immunoblotting with GST antibodies. Asterisks denote the position of purified

GST-IQSEC3 (aa 1-185) bands. Input, 5%.

Figure 5. IQSEC3 promotes inhibitory synapse formation in cultured hippocampal

neurons. A, Representative images of cultured hippocampal neurons transfected at DIV10 with

EGFP alone (Control) or together with IQSEC3 constructs (IQSEC1, IQSEC2, or IQSEC3).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


19

Neurons were analyzed by double-immunofluorescence labelling for VGLUT1 (red) and EGFP

(blue; pseudo-colored) at DIV14. Scale bar, 10 μm (applies to all images). B, Summary graphs

of the effects of IQSEC overexpression in neurons on excitatory synapse density (left),

excitatory synapse size (middle) and excitatory synapse strength (right), as measured using

VGLUT1 as an excitatory presynaptic marker. At least five to eight dendrites per transfected

neuron were analyzed and group-averaged. Data are presented as means ± SEMs. C, Same as

(A), except that the transfected neurons were analyzed by double-immunofluorescence labelling

for GAD67 (red) and EGFP (blue) at DIV14. Scale bar, 10 μm (applies to all images). D,

Summary graphs of the effects of IQSEC overexpression in neurons on GAD67-positive

inhibitory presynapse density (left), inhibitory presynapse size (middle), and inhibitory

presynapse strength (right). Data are presented as means ± SEMs (*p < 0.05; ANOVA with

Tukey’s test). E, Same as (A), except that dendrites and soma of transfected neurons were

analyzed by double-immunofluorescence labelling for gephyrin (red) and EGFP (blue) at

DIV14. Scale bar, 10 μm (applies to all images). F, Summary graphs of the effects of IQSEC

overexpression in neurons on gephyrin-positive inhibitory postsynapse density (left), inhibitory

postsynapse size (middle), and inhibitory postsynapse strength (right). At least five to eight

dendrites or one soma per transfected neuron were analyzed and group-averaged. Data are

presented as means ± SEMs (*p < 0.05; 3*p < 0.001; ANOVA with Tukey’s test). More than

800 gephyrin puncta were quantified in an individual image.

Figure 6. Gephyrin is required for inhibitory synaptic localization of IQSEC3 in cultured

neurons. A, Representative images of cultured hippocampal neurons transfected at DIV8 with

lentiviral constructs expressing EGFP alone (Control) or co-expressing EGFP with shRNAs

against gephyrin (Gephyrin KD). Neurons were analyzed by triple-immunofluorescence

labelling for IQSEC3 (red), GAD67 (green), and EGFP (blue) at DIV14. Scale bar, 10 μm

(applies to all images). B, Summary graphs of the effects of gephyrin KD in neurons on density

(left), size (middle), and intensity (right) of IQSEC3 puncta. At least five to eight dendrites per

transfected neuron were analyzed and group-averaged. Data are presented as means ± SEMs

(3*p < 0.001; Student’s t-test). C, Same as (B), except that density (left), size (middle), and

intensity (right) of GAD67 puncta were quantified. Data are presented as means ± SEMs

(2*p < 0.01; Student’s t-test).

Figure 7. Gephyrin-IQSEC3 interaction is required for IQSEC3-mediated promotion of

inhibitory synapse formation. A, Representative images of cultured hippocampal neurons


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


20

transfected at DIV10 with EGFP alone (Control) or together with the indicated IQSEC3 deletion

constructs. Neurons were analyzed by double-immunofluorescence labelling for gephyrin (red)

and EGFP (blue) at DIV14. Scale bar, 10 μm (applies to all images). For detailed information

regarding IQSEC3 deletion constructs, please see Fig. 3. B, Summary graphs showing the

effects of overexpressing IQSEC3 deletion constructs in neurons on gephyrin puncta density

(left), gephyrin puncta size (middle) and gephyrin puncta intensity (right), measured using

gephyrin as an inhibitory postsynaptic marker. At least five to eight dendrites per transfected

neuron were analyzed and group-averaged. Data are presented as means ± SEMs (*p < 0.05, 2*p

< 0.01, 3*p < 0.001 vs. control group; ANOVA Tukey’s test). More than 500 gephyrin puncta

were quantified in an individual image.

Figure 8. IQSEC3 promotes inhibitory synapse formation that depends on its ARF-GEF

activity, but is independent of its PDZ-binding property. A, Representative images of

cultured hippocampal neurons transfected at DIV10 with EGFP alone (Control) or together with

the indicated IQSEC3 constructs. Neurons were analyzed by double-immunofluorescence

labelling for gephyrin (red) and EGFP (blue) at DIV14. Scale bar, 10 μm (applies to all images).

B, Summary graphs of the effect of overexpressing IQSEC3 constructs in neurons on gephyrin

puncta density (left), gephyrin puncta size (middle) and gephyrin puncta intensity (right),

measured using gephyrin as an inhibitory postsynaptic marker. At least five to eight dendrites a

per transfected neuron were analyzed and group-averaged. More than 500 gephyrin puncta were

quantified in an individual image. Data are presented as means ± SEMs (2*

p < 0.01, 3*

p < 0.001

vs. control group; Student’s t-test). C, Co-immunoprecipitation experiment demonstrating that

IQSEC3-E749A interacts with gephyrin. HEK293T cells were transfected with Myc-IQSEC3 or

its indicated mutants alone or together with FLAG-gephyrin, and co-immunoprecipitation of

gephyrin with IQSEC3 was assayed. A representative immunoblot visualized by ECL is shown.

Input, 5 %.

Figure 9. Characterization of an Iqsec3 shRNA construct in HEK293T cells and cultured

neurons. A, KD efficacies of shRNAs. Levels of IQSEC3 were measured by Western blotting

in HEK293T cells cotransfected with FLAG-IQSEC3 and the indicated shRNA constructs. B,

Quantification of IQSEC3 levels from (A) normalized to control. Data are presented as means ±

SEMs of three experiments. C, Specificity of IQSEC3-KD construct, B3. Levels of mRNA

(Iqsec1-3) were measured by qRT-PCR in cultured cortical neurons infected at DIV3 with

lentiviruses expressing Iqsec3 shRNA (B3). mRNA was prepared at DIV10. Note that Iqsec3


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/


21

mRNA was specifically reduced. D, Cultured cortical neurons were infected with lentiviruses

expressing Iqsec3 shRNA (B3) at DIV3 and subjected to immunoblotting with the indicated

antibodies at DIV10. E, Quantification of IQSEC3, gephyrin, and PSD-95 levels from (D),

normalized to control. Data are presented as means ± SEMs of three experiments. F, Cultured

cortical neurons were infected with lentiviruses expressing gephyrin shRNA at DIV3 and

subjected to immunoblotting with the indicated antibodies at DIV10. G, Quantification of

IQSEC3, gephyrin, and PSD-95 levels from (F), normalized to control. Data are presented as

means ± SEMs of three experiments. H, Validation of the IQSEC3 shRNA-resistant construct.

Levels of IQSEC3 were measured by Western blotting in HEK293T cells transfected with WT

IQSEC3 or an IQSEC3 shRNA-resistant mutant construct (Mut.) alone or together with Iqsec3

shRNA (B3). Box at right indicates the shRNA target sequences in WT Iqsec3 and shRNA-

resistant sequences in mutated Iqsec3.

Figure 10. IQSEC3 is required for maintenance of gephyrin clusters in cultured neurons.

A, Representative images of both dendrites and soma of cultured hippocampal neurons

transfected at DIV8 with lentiviral constructs expressing EGFP alone (Control), co-expressing

EGFP with shRNAs against Iqsec3 (IQSEC3-KD), or cotransfected with IQSEC3-KD and the

shRNA-resistant IQSEC3 full-length vector (+IQSEC3 rescue). Neurons were analyzed by

double-immunofluorescence labelling for gephyrin (red) and EGFP (blue) at DIV14. Scale bar,

10 μm (applies to all images). B, Summary graphs of the effects of IQSEC3 KD in neurons on

gephyrin puncta density (left), gephyrin puncta size (middle), and gephyrin puncta intensity

(right). At least five to eight dendrites or one soma per transfected neuron were analyzed and

group-averaged. Data are presented as means ± SEMs (*p < 0.05,

3*p < 0.001; ANOVA with

Tukey’s test). More than 850 gephyrin puncta were quantified in an individual image.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

With bait

A

B

Filter assay

lacZ expression

SD-LWU

URA3 expression

SD-LWA

ADE2 expression Master plate

SD-LW

1

1

3 2

+ - 2 With vector

Bait: gephyrin (aa 2-735)Vector: pGBKT alone

1: IQSEC2 (clone AD1)2: IQSEC3 (clone AD3)3: pACT2 alone (prey vector)+: positive control-: negative control

CC IQ SEC PH PBM

Number of Positive Colonies

119114

116Real Positives 27 out of 60 U+A+ Z+candidates

Reporter I (lacZ)Reporter II (URA3)Reporter III (ADE2)

IQSEC2

IQSEC3CC1 IQ SEC PH PBMCC2

AD1, AD4, AD6, AD7, AD8, AD9, AD10, AD11AD12

AD3, AD19, AD20

FIGURE 1

Identity of real positives

26

7IQSEC2 and IQSEC3

Collybistin

RAFT1

House-keeping genes

12


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

C D Gephyrin

IQSEC3 (JK079) MergedIQSEC3 (old)E

kDa

JK079

Unt.

293T Br

Tran

s.

FLAG

250150100

75

IQSEC3 (JK079)

IQSEC3 (old) = previously published Ab

Merged

FLAG-IQSEC2

FLAG-IQSEC1

FLAG-IQSEC3

FLAG JK079G

150

100FLAG

kDa

α-tubulin50

IQSE

C2IQ

SEC1

IQSE

C3

Unt.

HEK293T

100

150

IQSEC3 (JK079)

F

Myc-GephyrinInput IP: FLAG

Myc-gephyrin

kDa

AFLAG-IQSEC2FLAG-IQSEC1FLAG-IQSEC3

FLAG-IQSECs

100

100

150

250

+ + + + + + + ++

++

++

+

B

IQSEC3

Gephyrin

S-SCAM

NL1NL2

Collybistin

100

IP

kDa

75

100100

150

150

50

Inpu

tIg

GIQ

SEC3

HEK293T Coimmunoprecipitation

FIGURE 2

5

0

*4

3

2

1

2

FLAG-IQ

SEC2FLA

G-IQSEC3

P2

5037

25

Unt.

293T Br

Tran

s.

P2

FLAG-IQ

SEC1

n.d.

% o

f coi

mm

unop

reci

piat

ed


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

CC1 IQ SEC PH

A

Gephyrin full

Gephyrin-G

Gephyrin-C

Gephyrin-E

Schematic of Gephyrin constructs

C

B

DSchematic of IQSEC3 constructs

G C E

1-350

1-315

336-655

336-995

336-1194

636-1194

101-200

1-1190

996-1194

996-1095

1-100

150 75

Myc-Gephyrin

7550

7550

7550

7575

75100

7575

7520

7525

7520

75150

PBM1-1194

kDa Myc-Gephyrin

InputFLAG-IQSEC3+ +

+

FLAG-IQSEC3

IP: Myc+ +

+

Input+ +

+

IP: Myc+ +

+

kDaMyc-Gephyrin

Input

FLAG-IQSEC3+ +

+

IP: FLAG+ +

+

Input+ +

+

IP: FLAG+ +

+

FLAG-IQSEC3MYC-Gephyrin

CC2

7520

150

150

150

15075

25

25

50

FIGURE 3

166 3181 736

4.70% ± 0.30 %

4.95% ± 0.65 %

5.20% ± 0.30 %

2.50% ± 0.65 %

1.90% ± 0.36 %

0.85% ± 0.40 %

4.85% ± 0.10 %

150 751-995 3.63 % ± 0.34 %


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

755037

2520

IB: GST

Input

GST

alo

ne1-

185

311-

645

kDa

His-pulldown

GST

alo

ne1-

185

311-

645

GST-IQSEC3 311-645

* * GST-IQSEC3 1-185

GST alone

FIGURE 4

CBB

755037

2520

kDa

*

GST

alo

ne1-

185

311-

645

Input

755037

2520

kDa

His-GephyrinG-domain

CBB

CBB Staining His-pulldown assaysA B


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Control IQSEC2IQSEC1 IQSEC3

VGLUT1

EGFP

Merged

A

B

Control IQSEC3

GAD67

EGFP

Gephyrin

EGFP

Merged

Control IQSEC3E

F

IQSEC1 IQSEC2

Gephyrin (soma)

400

80120160

Punc

ta in

tens

ity (A

U)

Gephyrin (soma)Ar

ea (µ

m2 )

Syn

apse

s/µm

2 D

endr

ite

Gephyrin (dendrite)

0.8

1.6*

0.01

0

0.02 *0

0

0.4

0.8

0

0.4

0.8

Merged

C

VGLUT1 (dendrite)

S

ynap

ses/

10µm

Den

drite

Punc

ta d

ensi

ty

Area

(µm

2 )

Punc

ta in

tens

ity (A

U)

Control

IQSEC2

IQSEC1

IQSEC3

40

80

120

Punc

ta s

ize

Punc

ta in

tens

ity

2

4

0

0.5

1.0

0 0

D GAD67 (dendrite)

Area

(µm

2 )

Punc

ta in

tens

ity (A

U)

40

80

120

0.41.0

0 0 0

0.82.0 *

IQSEC2IQSEC1

Control

IQSEC2

IQSEC1

IQSEC3

Control

IQSEC2

IQSEC1

IQSEC3

S

ynap

ses/

10µm

Den

drite

S

ynap

ses/

10µm

Den

drite

FIGURE 5

160

3 1.2

400

80120160


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Syna

pses

/10

µm D

endr

ite

Control Gephyrin KD

EGFP

Merged

GAD67

IQSEC3

0.40.8

0.8

0.4

0

0

40

80

120

160

0

40

80

120

0

1.62.0

0

0.8

1.2

0

Area

(µm

2 )Ar

ea (µ

m2 )

Punc

ta in

tens

ity (A

U)

Punc

ta in

tens

ity (A

U)

IQSEC3 (dendrite)

Punc

ta d

ensi

ty

Punc

ta s

ize

Punc

ta in

tens

ity

Punc

ta d

ensi

ty

Punc

ta s

ize

Punc

ta in

tens

ity

A

GAD67 (dendrite)

*3 *3

*

B

C Control Gephyrin KD

Control Gephyrin KDSy

naps

es/1

0 µm

Den

drite

FIGURE 6

1.2

0.4

0.4

20.8


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

FIGURE 7

Puncta intensityPuncta sizeB Puncta density

Syna

pses

/10

µm D

endr

ite

0.5

1.5

Area

(µm

2 )

0

0.4

0.8

Punc

ta in

tens

ity (A

U)

0

40

80

120*

A

0

160

Contro

lWT

336-1

194

336-9

95

IQSEC3 variants

1.0

*3*2

1-3151-9

95

Contro

lWT

336-1

194

336-9

95

IQSEC3 variants

1-3151-9

95

Contro

lWT

336-1

194

336-9

95

IQSEC3 variants

1-3151-9

95

Gep

hyrin

pun

cta

Gephyrin

EGFP

Merged

Control IQSEC3 WT

IQSEC3 336-1194IQSEC3 336-995

IQSEC3 1-315

IQSEC3 1-995

Gephyrin

EGFP

Merged


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

IQSEC3 E749AControl IQSEC3 WTA

Puncta intensityPuncta sizeB

Gephyrin

EGFP

Merged

Puncta density

Syna

pses

/10

µm D

endr

ite

0.5

0

1.5

Area

(µm

2 )

0

0.2

0.4

0.6

0.8Pu

ncta

inte

nsity

(AU

)

0

40

80

120

Contro

lWT

Contro

lWT

Contro

lWT

E749A

E749A

E749A

*2

IQSEC3 ΔPDZ-tail

*

ΔPDZΔPDZ

ΔPDZ

FIGURE 8

C

150 75

75150

kDa FLAG-Gephyrin

InputMyc-IQSEC3 constructs+ +

++ +

+

Input+ +

+

IP: FLAG+ +

+

IP: FLAG

Myc-IQSEC3 WT

Myc-IQSEC3 E749A

FLAG-GephyrinMyc-IQSEC3

3

1.0

2.0


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

100

80

60

40

20

0

IQSEC3

E

Pro

tein

leve

ls (%

)(n

orm

aliz

ed to

con

trol)

Gephy

rin

PSD-95

shRNAs(B3)

Cont

rol

B3

IQSEC3 (JK079)

α-tubulin

D

Gephyrin

PSD-95

50

10075

100150kDa

100

80

60

40

20

0

Pro

tein

leve

ls (%

)(n

orm

aliz

ed to

con

trol)

B1shRNAs B2 B3 B4

IQSEC3BA C

mR

NA

leve

ls (%

)(n

orm

aliz

ed to

con

trol)

shRNAs(B3)

80

60

40

20

0

IQSEC1

IQSEC2

IQSEC3

kDa Cont

rol

B1 B2 B3 B4

HEK293T cells

IQSEC3

α-tubulin

Cotransfection of FLAG-IQSEC3with candidate constructs (B1-B4)

150

50

100

80

60

40

20

0

IQSEC3

G

Pro

tein

leve

ls (%

)(n

orm

aliz

ed to

con

trol)

Gephy

rin

PSD-95

shRNAs(sh-Geph)

Cont

rol

IQSEC3 (JK079)

α-tubulin

F

Gephyrin

PSD-95

50

10075

100150kDa sh

-Gep

h

H

kDa Untra

ns.

Cont

rol

B3

HEK293T cells

IQSEC3

α-tubulin150

50

Cont

rol

B3

Mut.WTWT = rat IQSEC3 wild-typeGAGCTGGTGGTAGGCATCTATGAAA

Mut. = rat IQSEC3 mutant (shRNA-resistant)GAGCTAGTGGTCGGCATCTACGAAA

FIGURE 9


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Control IQSEC3 KD

EGFP

Gephyrin

Merged

IQSEC3 KD (+rescue)A

Area

(µm

2 )

Punc

ta in

tens

ity (A

U)

Syna

pses

/10

µm D

endr

ite

Punc

ta d

ensi

ty

Punc

ta s

ize

Punc

ta in

tens

ity

B Gephyrin (dendrite)

0

0.2

0.6

0.4

0.8

0.8

1.6

0 04080

160

0

40

80

120

160

0.1

0.30.2

0.50.4

00

0.04

0.08

0.12

0.16

Syna

pses

/µm

2 D

endr

ite

Area

(µm

2 )

Punc

ta in

tens

ity (A

U)

Gephyrin (soma)

Punc

ta d

ensi

ty

Punc

ta s

ize

Punc

ta in

tens

ity

Control IQSEC3 KD IQSEC3 KD (+rescue)

Soma(Gephyrin)

*

*

*

*

FIGURE 10

3

120


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Kim, Katsuhiko Tabuchi and Jaewon KoKang, Takuma Mori, Theofilos Papadopoulos, Taesun Yoo, Yeunkum Lee, Eunjoon

Ji Won Um, Gayoung Choii, Dongseok Park, Dongwook Kim, Sangmin Jeon, Hyeyeongephyrin to promote inhibitory synapse formation

IQ Motif and SEC7 Domain-containing Protein 3 (IQSEC3) interacts with

published online March 21, 2016J. Biol. Chem.

10.1074/jbc.M115.712893Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M115.712893

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M115.712893v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2016/03/21/jbc.M115.712893

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2016/03/21/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2016/03/21/jbc.M115.712893

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

IQ Motif and SEC7 Domain-containing Protein 3 (IQSEC3 ... · GCC ACG ACA TTA-3′) into the XhoI...

Documents

Transcript of IQ Motif and SEC7 Domain-containing Protein 3 (IQSEC3 ... · GCC ACG ACA TTA-3′) into the XhoI...