Gain-of-function glutamate receptor interacting
protein 1 variants alter GluA2 recycling and
surface distribution in patients with autism
Rebeca Mejiasa, Abby Adamczyka, Victor Anggonob,c, Tejasvi Niranjana,d, Gareth M. Thomasb,c, Kamal Sharmab,c,
Cindy Skinnere, Charles E. Schwartze, Roger E. Stevensone, M. Daniele Fallinf, Walter Kaufmanng,h,
Mikhail Pletnikovb,h, David Vallea, Richard L. Huganirb,c,1, and Tao Wanga,1
aMcKusick-Nathans Institute of Genetic Medicine and Department of Pediatrics,bDepartment of Neuroscience,cThe Howard Hughes Medical Institute,
dPredoctoral Training Program in Human Genetics, andhDepartment of Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine,
Baltimore, MD 21205;eGreenwood Genetic Center, Greenwood, SC 29646;fDepartment of Epidemiology, The Johns Hopkins University School of Public
Health, Baltimore, MD 21205; andgThe Kennedy Krieger Institute, Baltimore, MD 21205
Contributed by Richard L. Huganir, February 14, 2011 (sent for review December 13, 2010)
Glutamate receptor interacting protein 1 (GRIP1) is a neuronal
scaffolding protein that interacts directly with the C termini of
glutamate receptors 2/3(GluA2/3)viaits PDZdomains 4 to6(PDZ4–
6). We found an association (P < 0.05) of a SNP within the PDZ4-6
genomic region with autism by genotyping autistic patients (n =
480) and matched controls (n = 480). Parallel sequencing identified
five rare missense variants within or near PDZ4–6 only in the au-
tism cohort, resulting in a higher cumulative mutation load (P =
0.032). Two variants correlated with a more severe deficit in re-
ciprocal social interaction in affected sibling pairs from proband
families. These variants were associated with altered interactions
with GluA2/3 and faster recycling and increased surface distribu-
tion of GluA2 in neurons, suggesting gain-of-function because
GRIP1/2 deficiency showed opposite phenotypes. Grip1/2 knockout
mice exhibited increased sociability and impaired prepulse inhibi-
tion. These results support a role for GRIP in social behavior and
implicate GRIP1 variants in modulating autistic phenotype.
monozygotic twins (1, 2). Rare deleterious mutations of large
effect are increasingly recognized as contributing to genetic
predisposition in autism (3, 4). Point mutations, de novo dele-
tions, and duplications associated with autism were found in
genes controlling glutamate receptor clustering, synaptogenesis,
axon guidance, and dendritic development (5–8). These results
support the hypothesis that autism may be caused by an accu-
mulation of individually rare mutations in multiple genes leading
to disturbances in shared neurodevelopmental pathways and
consequent behavioral phenotype. Identifying these genes and
understanding the consequences of their mutations will shed
light on the disease mechanism in autism (9).
Previous studies have implicated impaired glutamate signaling
activities of glutamic acid decarboxylase—the rate-limiting en-
zyme that converts glutamate to GABA (11)—were lower in
brains from individuals with autism. Abnormally increased tran-
scripts of AMPA glutamate receptor 1, glial glutamate trans-
porter 1, and glutamate receptor interacting protein 1 (GRIP1)
were also found in the cerebellum of autistic patients (12). Mul-
tiple de novo deletions and duplications involving genes in the
glutamate signaling pathway were identified in large autism
cohorts (6, 13) These results highlight glutamate-signaling genes
as promising candidates for genetic predisposition in autism.
GRIP1 was mapped to 12q14.3, a region that was implicated in
autism in a study using Affymetrix GeneChip analysis (14).
GRIP1 plays an important role in receptor trafficking, synaptic
organization, and transmission in glutamatergic and GABAergic
synapses (15–19). Interaction of PDZ domains 4 to 6 (PDZ4–6)
with AMPA glutamate receptors, GluA2/3, is essential for re-
utism spectrum disorders are a group of highly heritable,
heterogeneous disorders with 70% to 90% concordance in
ceptor targeting and localization to the postsynaptic membrane
and for activity-dependent synaptic reorganization of AMPA
receptors (16, 20).
We identified an association (P = 0.048) of a common SNP
(rs7397862) in intron 15 of GRIP1 within the region encoding
PDZ4–6 (exons 12–16) in a case-control study of autism (n =
480) and ethnically matched normal controls (n = 480) (Tables
S1 and S2). We built on this initial finding to perform parallel
sequencing of all 25 exons and ∼50-bp flanking introns of GRIP1
in both cohorts. This analysis identified five missense variants
involving highly conserved amino acid residues within or near
PDZ4–6 only in autistic patients (Fig. 1A) (21). All five are
transmitting variants and each was found in a single family
(Table S3). The cumulative allele frequency of the five variants
was higher (P = 0.032) in autism compared with ethnically
matched controls (Table S2). Additionally, one missense variant,
V54I in PDZ1, was found in two patients and two missense
variants, Q821E and R869Q near PDZ7, were found in both
autism and controls (Fig. 1A). Noncoding variants of interest
within the immediate flanking intronic regions of exons 12 to 18
encoding PDZ4–6 include a single base-substitution (T > C) at
position −3 of intron 13 in one patient, a three base (TTG)
deletion at positions −10 to −12 of intron 11 in three patients
and one control, and two common SNPs at positions +13
(rs7397862) and +34 (rs7397861) of intron 15 in both autism
patients and controls (Fig. S1).
In particular, two variants, A625T and M794R, were found in
families with two affected brothers who had different genotypes
for respected GRIP1 variants. This finding permitted a correla-
tion study of the presence of these variants with the severity of
autism phenotype, as defined primarily by Autism Diagnostic
Interview-Revised (ADI-R) scores (Table 1). In the first family,
the affected boy, who was heterozygous for A625T, had relatively
(with respect to other components of these instruments) more
impairment in ADI-R’s Reciprocal Social Interaction, the Au-
Author contributions: R.M., D.V., R.L.H., and T.W. designed research; R.M., A.A., V.A., and
T.N. performed research; V.A., G.M.T., K.S., C.S., C.E.S., R.E.S., and M.D.F. contributed new
reagents/analytic tools; R.M., A.A., V.A., T.N., G.M.T., K.S., W.K., M.P., and T.W. analyzed
data; and R.M., A.A., D.V., R.L.H., and T.W. wrote the paper.
Conflict of interest statement: Under a licensing agreement between Millipore Corpora-
tion and The Johns Hopkins University, R.L.H. is entitled to a share of royalties received by
the University on sales of products described in this article. R.L.H. is a paid consultant to
Millipore Corporation. The terms of this arrangement are being managed by The Johns
Hopkins University in accordance with its conflict-of-interest policies.
1To whom correspondence may be addressed. E-mail: email@example.com or rhuganir@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| March 22, 2011
| vol. 108
| no. 12www.pnas.org/cgi/doi/10.1073/pnas.1102233108
tism Diagnostic Observation Schedule’s Reciprocal Social In-
teraction domain, and Vineland Adaptive Behavior Scales’ So-
cialization scale, compared with his affected brother, who did not
carry this variant. In the second family, the boy who was ho-
mozygous for M794R also had relatively worse scores on ADI-
R’s Reciprocal Social Interaction domain, compared with his
affected brother, who was heterozygous for this variant. In-
terestingly, their mother, who was also heterozygous for M794R
but not diagnosed with autism, had a history of restricted
interests, repetitive behavior, poor eye contact, and eccentricity.
interacting proteins with PDZ4–6. (A) PDZ domain structure, locations of missense variants in both controls and autism, and alignment of nine species for the
amino acid residues involved. PDZ4–6 domains interact with proteins involved in excitatory neurotransmission (GluA2 and GluA3, AMPA glutamate receptor 2
and 3 subunits; PICK1, protein interacting with protein kinase C, Liprin-α; KIF5, kinesin superfamily protein 5; NEEP21, neuron-enriched endosomal protein
21). (B) PDZ4-5 NMR structure overlapping with corresponding peptide sequence of rat, mouse, and human GRIP1 (modified from ref. 21). The nature and
locations of the variants within PDZ4-5 domains are indicated by arrows.
Missense variants of GRIP1-PDZ4-6 in autism, evolutionary conservation, locations in an NMR structure of PDZ domains 4 and 5 (PDZ4–5), and known
Table 1.Neurological and behavioral phenotype of sibling pairs with different genotype in two families with GRIP1 variants
Wild-type HeterozygousHeterozygous Homozygous
SubjectAffected with Autism
Age (years at interview)
Reciprocal Social Interaction
Repetitive and Stereotypic Behaviors
Heterozygous for the variant */+
Homozygous for the variant **
Wild-type allele, no variant +/+
Open symbol, unaffected
Filled symbol, affected with autism
Squares filled with black indicate individuals with a clinical diagnosis of autism and unfilled symbols (circles or squares) indicate individuals without
a clinical diagnosis of autism; all individuals shown in the pedigrees were genotyped for respected GRIP1 variants. Abbreviations: PPVT: Peabody Picture
Vocabulary Test; ADI-R: Autism Diagnostic Interview-Revised. # , scores on ADI-R Nonverbal Communication component; */+, heterozygous for the indicated
GRIP1 allele; **, homozygous for the indicated GRIP1 allele; +/+, WT allele.
Mejias et al.PNAS
| March 22, 2011
| vol. 108
| no. 12
The two aforementioned socially more-impaired subjects also
had overall higher ADI-R scores and lower cognitive—but not
motor—skills than their brothers.
The five GRIP1 variants (I549V, T550M, I586L, A625T,
M794R), clustered around PDZ4–6 and found only in autism
subjects, were further studied because these domains are known
to directly interact with the C terminus of GluA2/3. The amino
acid residues I549 and T550 within PDZ4, and I586 and A625
within PDZ5, are identical in a nine-species alignment, and the
aliphatic/nonpolar nature of the amino acid at M794 is also
conserved (Fig. 1A). Mapping the residues to a published NMR
structure of PDZ4/5 (21) revealed that I549 and T550 are lo-
cated within a six-amino acid motif (DSSITS) of the αB-βF loop
in PDZ4, which is essential for stabilizing the PDZ4/5 super-
module. Residue I586 is part of the four-amino acid motif
(ELGI) that directly binds the C terminus of GluA2/3, and A625
is a highly conserved residue within a βB loop in PDZ5 (Fig. 1B).
We hypothesized that these GRIP1 variants would display al-
tered interactions with GluA2/3 and would affect GluA2/3
function and distribution in neurons.
To address the first possibility, we determined the in vitro
interaction of the five variants with C-terminal domains of
GluA2/3 (GluA2C and GluA3C) using a yeast two-hybrid assay.
Deletion of the C-terminal four amino acids of GluA2/3 (R3-
CΔ4) or artificial mutations, K577A_K578A, of GRIP1 are
known to completely abolish GRIP1-GluA2/3 interaction. As
three variants, I549V, I550M, and I586L with GluA2C, as reflec-
ted by β-galactosidase activity (WT,100.0%; I549V, 120.5 ± 4.2%,
0.001; A625T, 80.2 ± 5.9%, P = 0.006; M794R, 110.3 ± 2.0%;
K577A_K578A,0.0± 0.0%;n = 6 yeastclones)and GluA3C (WT
100.0%; I549V, 114.6 ± 5.7%; I550M, 114.0 ± 4.0; I586L, 133.7 ±
11.0% P = 0,001; A625T, 71.5 ± 2.6% P = 0,01; M794R, 91.7 ±
3.2%; K577A_K578A, 0.0 ± 0.0%; R3-CΔ4, 0.0 ± 0.0%; n = 6).
The other two variants, A625T and M794R, showed moderately
reduced interaction. Interestingly, I586L showed the most signif-
icant increase in the interaction, which is consistent with the NMR
structure of PDZ4/5, in which I586 directly binds GluA2C and
GRIP1 was recently reported to modulate the activity-
dependent trafficking of a pH-sensitive pHluorin-GluA2 fusion
protein (pH-GluA2) (22). We therefore examined the effects of
altered GRIP1-GluA2/3 interaction of three GRIP1 variants
(T550M, I586L, A625T) on pH-GluA2 internalization and
recycling in live, transfected hippocampal neurons (23). Ex-
pression of both WT and GRIP1 variants in neurons mimics the
heterozygous state of these variants in individuals with autism.
Compared with WT GRIP1, all three variants showed compa-
rable amplitude of fluorescence changes, reflecting the rate of
pHluorin-GluA2 internalization following NMDA stimulation
(Fig. 3 A, B, and D) (amplitude, WT 0.70 ± 0.03; T550M 0.67 ±
0.03; I586L, 0.72 ± 0.02; A625T, 0.63 ± 0.03) but a faster recy-
cling of pHluorin-GluA2 as reflected by shorter T1/2, the time
for recovery of 50% of fluorescence. The differences in two
variants, I586 and T550M, reached statistical significance (Fig. 3
A–C) [t1/2(min), WT, 6.66 ± 0.64; T550M, 5.37 ± 0.64, P = 0.174;
I586L, 3.97 ± 0.38, P = 0.003; A625T, 3.97 ± 0.83, P = 0.026;
n = 6–13 neurons]. Consistent with the yeast two-hybrid data,
I586L showed the most significant effect on GluA2 recycling.
Because Grip1/2-deficient hippocampal neurons display slower
a yeast two-hybrid assay. GluA2C or GluA3C (C-terminal 50 amino acids) and
expression constructs for individual GRIP1 variants (containing PDZ4–6) were
cotransformed into Y190. K577A-K578A: K577_K578 of GRIP1 were changed
to A577_A578; R3-CΔ4: deletion of the last four amino acids at the C ter-
minus of GluA3; both completely block the interaction between GRIP1 and
GluA2/3. The Mean and SEM of β-galactosidase enzyme activities are shown.
ANOVA followed by Student–Newman-Keuls method (n = 6 yeast clones).
*P < 0.05, **P < 0.01.
Interaction of GRIP1 variants with the C terminus of GluA2/3 in
pocampal neurons. (A) Images from pHluorin assay of hip-
pocampal neurons (d in vitro 16) transfected with wt GRIP1
and I586L GRIP1 variant during NMDA (3-min treatment: 11–
14 min) perfusion/washout experiments. (Scale bar, 10 μm.)
(B) Average pHluorin-GluA2 fluorescence time course from
WT GRIP1 and T550M, I586L and A625T GRIP1 variants in
response to 3-min NMDA treatment. (C), Histogram of T1/2
from WT GRIP1 and T550M, I586L, and A625T GRIP1 variants
after NMDA washout. (D) Histogram of pHluorin fluores-
cence change amplitude in response to NMDA treatment.
Data presented as mean ± SEM (n = 6–13 neurons). Student’s
t test, *P < 0.05.
GRIP1 variants alter GluA2 recycling process in hip-
| www.pnas.org/cgi/doi/10.1073/pnas.1102233108Mejias et al.
GluA2 recycling in the same assay (22), these results suggest a
gain of GRIP1 function for these variants on GluA2 recycling.
Finally, we determined the relative abundance of surface
versus total GluA2 in hippocampal neurons transfected with in-
dividual GRIP1 variants. Among the five variants, three (I586L,
A625T, M794R) increased the relative abundance of surface
GluA2 (Fig. 4). The differences in two variants, I586L and
A625T, reached statistical significance compared with WT
GRIP1 (surface/total GluA2 (%), WT, 100.0; I549V, 100.6 ± 9.9;
T550M, 106.0 ± 8.7; I586L, 163.9 ± 22.7, P = 0.006; A625T,
141.9 ± 10.8, P < 0.001; M794R, 123.3 ± 14.5; n = 11–22 neu-
rons). These data are consistent with that from the pHluorin-
GluA2 recycling studies (Fig. 3).
GRIP1 and GRIP2 are highly homologous proteins that are
known to complement each other for certain neuronal functions
(16). To understand the role of GRIP1/2 in modulating social
behaviors, we studied the loss-of-function phenotype of Grip1/2
double-knockout (DKO) mice. DKO mice were generated by
crossing Grip2 conventional KO mice with conditional Grip1
(neuron-specific deletion via Nestin-Cre expression) KO mice on
C57BL6/129 strain background (17) to circumvent the embryonic
lethality of conventional Grip1 KO mice (24), and Grip1 deletion
was then achieved by crossing with Nestin-Cre mice. Cre-
dependent Grip1 deletion was verified in brain lysates by West-
ern blot analysis. Control mice (WT) were matched for age, sex,
and strain background with DKO mice. A standard battery of
behavioral tests, including an open-field, elevated plus maze, Y-
maze, intruder-resident test, novel object recognition, sociability
toward stranger mice, and prepulse inhibition (PPI) were used.
Compared with WT, Grip1/2 DKO mice showed a selective,
significant increase in sociability toward stranger mice (empty
cage in seconds: WT, 98 ± 9; DKO, 112 ± 11; with stranger mice
in seconds: WT, 116 ± 10; DKO, 192 ± 23, P = 0.012; n = 10
mice), impaired PPI (percent of inhibition; WT, 54 ± 25 and
DKO, 84 ± 18 at p74; WT, 27 ± 8 and DKO, 79 ± 18 at p78; P =
0.012; WT, 21 ± 5 and DKO, 59 ± 17 at p82; P = 0.035; WT,
16 ± 3 and DKO, 50 ± 15 at p86; P = 0.034; WT, 17 ± 4 and
DKO, 41 ± 11 at p90, P = 0.041; n = 10 mice), and impairment
in recognizing a novel object (time spent interacting with object
in seconds: WT with familiar object, 7.1 ± 1.2; DKO with fa-
miliar object, 12.0 ± 1.9, P = 0.043; WT with novel object, 12.0 ±
2.4; DKO with novel object 12.8 ± 2.0) (Fig. 5). Lack of differ-
ence for other behavioral tests is depicted in Fig. S2. These
results support a role for GRIP1/2 proteins in the modulation of
social behaviors in mammals.
We extended a previous linkage to chromosome 12q14 by iden-
tifying an association of a common SNP within the genomic re-
gion encoding PDZ4–6 of GRIP1 with autism. Sequencing of
GRIP1 in autism and matched control cohorts identified five rare
missense variants located within or near PDZ4–6 only in the
autism cohort. Importantly, these variants showed a higher cu-
mulative mutation load in autism and were found to alter GRIP1
interaction with GluA2/3 in in vitro and neuron-based assays. A
positive correlation of two variants with greater autistic behav-
ioral severity, particularly of the core social-interaction domain,
as measured by ADI-R, was found in families with affected
sibling pairs. More severe genotypes in these families were also
linked to greater cognitive impairment. Although three of the
four parents in these two families are heterozygous for the re-
spective variants, none of them was diagnosed with autism.
Limited behavioral data were available only for the mother who
was heterozygous for M794R and was noted having a history of
hippocampal neurons. (A) Representative microscope images of surface
GluA2 (Top), total GluA2 (Middle), and GRIP1 (Bottom) immunofluorescence
in hippocampal neurons (d in vitro 16) cotransfected with myc-WT GRIP1 or
myc-I586L GRIP1 variant and GFP-GluA2 (for 48 h). (Scale bar, 10 μm.) (B)
Average fluorescence (expressed in percentage of the WT control) coming
from surface GluA2 immunostaining normalized by total amount of trans-
fected GluA2 receptor in dendritic areas of neurons cotransfected with myc-
WT GRIP1 or myc-GRIP1 variants (T550M, I586L, A625T, or M794R) and GFP-
GluA2. Fluorescent intensities were quantified using ImageJ software (NIH)
in 11 to 22 neurons (coming from two independent experiments) per con-
dition using one to six dendritic areas per neuron analyzed. Data presented
as mean ± SEM (n = 11–22 neurons). Student’s t test, **P < 0.01.
GRIP1 variants increase GluA2 surface expression in transfected
Grip2 DKO mice. Grip1/Grip2 DKO were generated as described previously
(17), and were subjected to a battery of behavioral tests following proto-
cols from the Johns Hopkins Behavioral Core (www.brainscienceinstitute.
org/index.php/cores/). (A) Mouse sociability testing for WT and Grip1/Grip2
DKO mice with age-, and sex-matched WT controls (n = 10, 3–6 mo) revealed
an increase in the time that DKO spent interacting with a reference mouse.
(B), Time spent with a novel object compared with a familial object in the
Object Recognition test. (C) Decrease in PPI of acoustic startle response in
Grip1/Grip2 DKO mice with regards to WT controls (n = 10, 3–6 mo). Data
presented as mean ± SEM (n = 10 mice). Student’s t test, *P < 0.05.
Increased sociability and decreased prepulse inhibition of Grip1/
Mejias et al. PNAS
| March 22, 2011
| vol. 108
| no. 12
restricted interests, repetitive behavior, poor eye contact, and
eccentricity. Together, these results suggest that GRIP1 variants
may modify the severity of the deficits in social interactions and
cognitive function in autistic patients, but are not sufficient by
themselves to cause autism.
Because most patients carrying GRIP1 variants in this study
are heterozygotes, we speculate that these variants affect neu-
ronal and behavioral phenotype through a dominant mechanism.
Consistent with this hypothesis, we found that three variants
(I549V, T550M, and I586L) increased interaction with GluA2,
two variants (I586L and A625T) are associated with a faster
recycling of GluA2, and three variants (I586L, A625T, and
M794R) increased surface GluA2 in hippocampal neurons. Be-
cause loss of GRIP1/2 function was associated with reduced in-
teraction and slower GluA2 recycling (22), our data support a
gain of GRIP1 function in these variants. Although disruption of
GluA2-GRIP interactions in neurons also alters synaptic function
and plasticity (19, 25), the effect of GRIP1 variants on these
readouts remains to be elucidated.
Among the five variants, I586L most strongly affected GRIP1
interaction with GluA2/3 in a yeast two-hybrid assay, GluA2
recycling, and surface distribution. This finding is consistent with
the NMR structure of PDZ4/5, in which I586 is one of the four
amino acid residues that bind directly to GluA2/3 (Fig. 1B). We
recognize that not all variants exhibited the same profile of
changes in different assay systems. This finding suggests that ad-
ditional factors may be involved, which may not be satisfactorily
explained based on a simple model of one-on-one interaction
between GRIP1 and GluA2/3. These contributing factors may
include the following: (i) PDZ4 and -5 form a protein super-
module that interacts with GluA2/3, but individual PDZ domains
play different roles: I549 and T550 are located in PDZ4, which
stabilizes the supermodule; I586L and A625T are located in
PDZ5, which mediates the direct interaction of GRIP1 with
GluA2/3; (ii) multimerizations of GRIP1 with GRIP1, and GRIP1
with GRIP2 likely affect GRIP function in neurons; (iii) PDZ4–6
are known to interact with several other proteins in the gluta-
matergic and GABAergic signaling pathways. It is possible that
some effects of GRIP1 variants are exerted via interactions with
one or more of these proteins. Studies of neurons from knock-in
mice carrying specific Grip1 variants will be needed to further
understand the underlying neuronal mechanisms.
The behavioral studies of Grip1/2 DKO mice also suggest a
role for GRIP proteins in the modulation of social behavior and
possibly cognitive or memory function. Grip1/2 DKO mice
exhibited increased sociability, impaired PPI, and object recog-
nition. Deficits in sociability are one of the core features of au-
tism. Impaired PPI was observed in a small group of adult
patients with autism (26) and in mice deficient in neurexin-1α,
a gene associated with autism (27). Although we recognize that
human autistic phenotypes consist of complex behaviors that
may not be fully replicated or interpreted in mouse models,
studies of knock-in mice carrying GRIP1 variants may help to
elucidate how GRIP1 regulates social behavior and cognitive and
Identification of rare functional variants is important in iden-
tifying molecular pathways that are disturbed in autism. GRIP1
binds key proteins that regulate glutamatergic signaling, including
liprin-α (28), PICK1 (29), KIF5 (30), and NEEP21 (31), and also
uses the same PDZ4–6 to bind the GABA receptor-associated
protein, gephyrin (32) (Fig. 1A). We speculate that functional
GRIP1 variants in PDZ4–6 may alter not only glutamatergic but
also GABAergic synaptic function, leading to an imbalance in
selected neuronal circuits in autism (33). A role for synaptic
imbalance in autism is supported by findings in mice carrying an
autism-associated mutation (i.e., R451C) (5) in Neuroligin-3.
These mice were found to have increased inhibitory synaptic
markers, gephyrin, and vGABA transporter, and in the size of
inhibitory synaptic responses and spontaneous inhibitory event
frequency, suggesting an enhanced inhibitory synaptic trans-
mission affecting a subset of inhibitory interneurons in forebrain
through a gain of function mechanism (34).
Materials and Methods
Samples. DNA samples from patients with autism (n = 480) were obtained
from the Autism Genetic Research Exchange (AGRE) and the South Carolina
Autism Project (SCAP). Ethnically matched normal controls (n = 480), without
the diagnoses of pervasive developmental disorder or autistic disorder, were
obtained from Greenwood Genetic Center. An informed consent was
obtained from each enrolled family at the respective institutions. All en-
rolled patients met Diagnostic and Statistical Manual of Mental Disorders,
4thedition diagnostic criteria for autistic disorder, which was supported by
the ADI-R, and documented by clinical and behavioral phenotype. DNA
samples from parents and other affected and unaffected relatives of the
probands were available for the majority of the families from AGRE and
SCAP. The study was approved by Institutional Review Board at the Johns
Hopkins Medical Institutions.
Genotyping. SNP Genotyping was conducted by Sanger sequencing of in-
dividual samples from the autism and control cohorts. The SNP, rs7397862
located at +13 of intron 15 was selected for (i) its location near the center of
the genomic region encoding PDZ4–6 (exons 12–18), (ii) on the same SNP
linkage disequilibrium block encompassing exons 12 to 25 of GRIP1, and (iii)
high minor-allele frequencies for all ethnic groups in the study cohorts with
average heterozygosity of 0.458 ± 0.139 (mean ± STE) (dbSNP Build 132).
Sequencing. A Multiplexing Sample Preparation Oligonucleotide Kit (Illu-
mina; PE-400-1001) was used to generate indexed libraries following the
manufacturer’s instructions. Sequencing was performed using a Genomic
Analyzer II (Illumina) at the Johns Hopkins High-Throughput Sequencing
Facility. Standard Sanger sequencing for variant detection and validation
was conducted using a BigDye Terminator v3.1 Cycle Sequencing Kit and run
using an ABI3100 automatic DNA analyzer (Applied Biosystems) following
the manufacturer’s instructions.
Yeast Two-Hybrid Assay. Yeast vectors containing PDZ4–6 domains from
mutant GRIP1 (I549V, T550M, I586L, A625T, M794R, or K577A-K578A PDZ4–
6), in fusion with the GAL4 DNA binding domain, were generated by site-
directed mutagenesis using pPC97-wt GRIP1-PDZ4-6 as a template. The
C-terminal 50 amino acids from GluA2C or GluA3C were cloned into yeast
vector pPC86 in fusion with the GAL4 activation domain (15). Next, pPC86-
GluA2, pPC86-GluA3, or pPC86-GluA3-CΔ4 (with deletion of the last four
amino acids at the C terminus of GluA3) were transformed into yeast strain
Y190, using the lithium acetate method, together with either pPC97-GRIP1-
WT or individual pPC97-GRIP1 variants. Positive yeast clones from cotrans-
formation were selected on triple-deficient plates (Leu−, Trp−, His−) con-
taining 50 mM 3-aminotriazole and ONPG (o-nitrophenyl-β-D-galactoside).
Yeast clones that grew on triple-selection plates and with a blue color, were
selected to be cultured in triple-selection (Leu−, Trp−, His−) liquid medium.
β-Galactosidase activity was determined for six clones of each trans-
formation following a standard protocol (Clontech).
Neuron Culture and Transfection. Low-density primary hippocampal cultures
were prepared from embryonic day 18 (E18) rat brains, and maintained in
serum-free medium, as described previously (22). Cells were fed twice per
week and used for experiments after 14 to 18 d in vitro. Hippocampal
neurons were transfected at 14 d in vitro using Lipofectamine 2000 (Invi-
trogen), following the manufacturer’s instructions. Cells were used for
experiments 48 h after transfection. Plasmids used for transfection were pH-
GluA2 (23), pRK5-myc full length GRIP1 WT, or pRK5-myc full-length GRIP1
variants (generated by mutagenesis on pRK5-myc GRIP1 WT and verified by
sequencing), and pmCherry vector (Clontech).
Immunocytochemistry and Surface GluA2 Quantification. Cultured neurons
were incubatedwith GFPantibody(JH4030, 1:2,000) inartificial cerebrospinal
fluid buffer (containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2,
25 mM Hepes, 30 mM glucose, pH = 7.4) for 30 min. Neurons were fixed
by Parafix (4% paraformaldehyde + 4% sucrose in PBS) for 15 min and
permeabilized using 0.25% Triton in PBS for 10 min. Subsequently, neurons
were incubated with myc primary antibody (9E10 clone, mouse monoclonal,
1:1,000), and fluorophore-conjugated secondary antibodies (Alexa Fluorgoat
anti-rabbit 555, and goat anti-mouse 647; Invitrogen). Coverslips were
| www.pnas.org/cgi/doi/10.1073/pnas.1102233108Mejias et al.
mounted on slides using Fluoromount-G Media (SouthernBiotech). The cells Download full-text
were then observed using epifluorescence microscopy (Axiovert 200; Zeiss),
and images were collected using a charge-coupled device camera (Hama-
matsu Photonics) with axiovision (Zeiss) analysis software. Fluorescent in-
tensities were quantified using ImageJ software (NIH, National Institutes of
Health) in 11 to 22 neurons per condition using one to six dendritic areas per
pHluorin-GluA2 Recycling. Taking advantage of the pH difference between
extracellular space (pH = 7.4) and intracellular vesicles (pH < 6.0), this assay
use a pH-sensitive green fluorescent protein, pHluorin fused to the N ter-
minus of GluA2 (pH-GluA2) as a reporter to visualize the dynamic process of
GluA internalization and recycling (22, 23). Full details are in SI Materials
Immunoblotting Analysis. Brain tissues were homogenized in lysis buffer
(0.5% Triton, 5 mM EDTA in PBS) supplemented with protease inhibitor
mixture tablets (Roche). Homogenates were incubated on ice for 30 to 45 min
and centrifuged at 14,200 × g for 30 min at 4 °C. Supernatants were collected
and 20 μg of total proteins were transferred to membranes and incubated
with primary antibodies [GRIP1 mouse monoclonal antibody, 1:1,000 di-
lution (BD Transduction Laboratories) or GRIP2 rabbit polyclonal antibody,
1:1,000 dilution (16)], followed by horseradish peroxidase-conjugated sec-
ondary antibodies. The target proteins were visualized using Amersham ECL
Western Blotting Detection Kit (GE Healthcare) and quantified using NIH
Animal Breeding and Genotyping. All experimental procedures with mice and
rats were approved by the Animal Care and Use Committee of the Johns
HopkinsUniversity School ofMedicine,andweredoneincompliancewiththe
relevant laws and institutional guidelines. The housing room was maintained
at 23 °C on a 12-h light/dark cycle. Animals were provided with standard
mouse chow and free access to water ad libitum. Grip2 conventional
knockout mice were bred to Grip1 flox/flox mice (17). Mice were then
crossed to Nestin-CRE transgenic mice (kindly provided by Paul Worley,
Johns Hopkins University) for neuron-specific deletion of Grip1. Grip1/2 DKO
mice used for this study were Grip1 flox/KO with Grip2 KO/KO (conventional
KO), and Nestin CRE-positive heterozygous. Control animals were wt for
Grip1 and Grip2, and Nestin CRE-positive heterozygous. Grip1/2 DKO mice
and WT controls were in mixed 129 × C57BL/6 background (17). Full details
of genotyping are in SI Materials and Methods.
MouseBehavioralTesting.Mice were subjected toa battery ofbehavioral tests
following Behavioral Core User Manual of the Johns Hopkins Behavioral Core
(www.brainscienceinstitute.org/index.php/cores/). Age- and sex-matched WT
(n = 10) and Grip1/2 DKO mice (n = 10) were generated as above, and tested
between 3 and 6 mo of age. Full details are in SI Materials and Methods.
Statistical Analysis. Groups were first tested for normality and variance ho-
way ANOVA test for pair-wise multiple comparisons (Student-Newman-Keuls
Method). SigmaStat software (SPSS) was used for data analyses. Data were
presented as mean ± SEM and P < 0.05 was considered statistically significant.
ACKNOWLEDGMENTS. We thank Dr. Paul Worley for the Nestin-CRE trans-
genic mice, Jennifer Yocum for help with animal behavioral testing, Yilin Yu
for help with animal genotyping, and Eva Andres-Mateos for help with the
figures; and the resources provided by the South Carolina Autism Project
and Autism Genetic Resource Exchange (AGRE) Consortium and the
participating families. This study was supported in part by research Grant
#2487 from the Autism Speaks Foundation and Grant R01HD052680 from
the National Institute of Child Health and Human Development (to T.W.);
a postdoctoral fellowship from the Ministry of Education and Science of
Spain (to R.M.); and fellowships from the International Human Frontiers
Science Program (LT00399/2008-L) and Australian National Health and Med-
ical Research Council (ID477108) (to V.A.). R.L.H. is an investigator with the
Howard Hughes Medical Institute. AGRE is a program of Autism Speaks and
is supported, in part, by Grant 1U24MH081810 from the National Institute of
Mental Health to Clara M. Lajonchere (PI).
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| March 22, 2011
| vol. 108
| no. 12