Human serotonin transporter variants display
altered sensitivity to protein kinase G and
p38 mitogen-activated protein kinase
Harish C. Prasad*, Chong-Bin Zhu*, Jacob L. McCauley†, Devadoss J. Samuvel‡, Sammanda Ramamoorthy‡,
Richard C. Shelton*§¶, William A. Hewlett*§, James S. Sutcliffe†¶?, and Randy D. Blakely*§¶**
Departments of *Pharmacology,†Molecular Physiology and Biophysics, and§Psychiatry, and Centers for?Human Genetics Research and¶Molecular
Neuroscience, Vanderbilt University School of Medicine, Nashville, TN 37232-8548; and‡Department of Neurosciences, Medical University
of South Carolina, Charleston, SC 29425
Edited by Susan G. Amara, University of Pittsburgh School of Medicine, Pittsburgh, PA, and approved June 8, 2005 (received for review February 19, 2005)
(hSERT, 5HTT, and SLC6A4) inactivate 5-HT after release and are
prominent targets for therapeutic intervention in mood, anxiety,
and obsessive-compulsive disorders. Multiple hSERT coding vari-
ants have been identified, although to date no comprehensive
functional analysis of these variants has been reported. We trans-
fected hSERT or 10 hSERT coding variants and examined total and
surface protein expression, antagonist recognition, and trans-
porter modulation by posttranslational, regulatory pathways. Two
variants, Pro339Leu and Ile425Val, demonstrated significant
changes in surface expression supporting alterations in 5-HT trans-
port capacity (Vmax). Regardless of basal transport activity, all SERT
variants displayed a capacity for rapid, phorbol ester-triggered
down-regulation. Remarkably, five variants (Thr4Ala, Gly56Ala,
Glu215Lys, Lys605Asn, and Pro612Ser) demonstrated no capacity
for 5-HT uptake stimulation after acute protein kinase G (PKG)?p38
mitogen-activated protein kinase (MAPK) activation. Epstein–Barr
virus (EBV)-transformed lymphocytes natively expressing the most
common of these variants (Gly56Ala) exhibited a similar loss of
5-HT uptake stimulation by PKG?p38 MAPK activators. HeLa cells
transfected with the Gly56Ala variant demonstrated elevated
basal phosphorylation and, unlike hSERT, could not be further
phosphorylated after 8-bromo cGMP (8BrcGMP) treatments. These
studies reveal cellular phenotypes associated with naturally occur-
ring human SERT coding variants and suggest that altered trans-
porter regulation by means of PKG?p38 MAPK-linked pathways
may influence risk for disorders attributed to compromised 5-HT
serotonin [5-hydroxytryptamine(5-HT)] transporters
transport ? antidepressant ? polymorphism ? regulation ? autism
physiological processes, including vasoconstriction, gastrointestinal
motility and secretion, respiration, sleep, appetite, aggression, and
mood (1, 2). Disrupted 5-HT signaling has been implicated in a
similarly wide spectrum of disorders, including primary pulmonary
hypertension, irritable bowel syndrome, sudden infant death syn-
drome (SIDS), anorexia, obsessive-compulsive disorder (OCD),
autism, depression, and suicide (3–6). A major determinant of
5-HT signaling is the antidepressant-sensitive 5-HT transporter
(SERT, 5HTT). Human SERT (hSERT) protein is encoded by a
single locus mapping to chromosome 17q11.2 (7). Although evi-
dence of alternative splicing of 5? noncoding exons exists (8, 9), the
same ORF is translated in brain, platelets, lymphocytes, and
placenta, producing a protein of 630 aa with closest identify to
norepinephrine and dopamine transporters (NET and DAT re-
spectively). Initial hydropathy-based predictions of SERT second-
ary structure proposed 12 transmembrane domains (TMs) with
intracellular NH2and COOH termini (10), a model supported by
biochemical and immunocytochemical studies (11, 12). SERT
proteins can be rapidly regulated by multiple G protein-coupled
the brain and periphery, and modulates a wide variety of
receptors and protein kinase-linked pathways, including those
triggered by activation of PKC, protein kinase G (PKG), and p38
mitogen-activated protein kinase (MAPK) (13–17). Phosphoryla-
tion and down-regulation of SERT through the PKC-linked path-
way is sensitive to extracellular 5-HT (14), revealing an intrinsic
capacity for temporal integration of ongoing 5-HT clearance de-
mand with modulatory inputs.
The importance of SERT in presynaptic 5-HT homeostasis,
synaptic 5-HT clearance, and psychoactive drug action has raised
questions as to whether the hSERT gene exhibits functional
polymorphisms that impact expression and activity in vivo (18).
A common promoter variant (5HTTLPR) was found to support
altered hSERT mRNA and protein expression (19) and has been
associated with anxiety traits as well as multiple psychiatric
syndromes, including autism, OCD, and depression (18). A
variable nucleotide tandem repeat sequence (VNTR) in the
intron following the first coding exon has also been described
and seems to have enhancer-like properties (20). Ten nonsyn-
onymous SNPs have been identified in hSERT (21, 22), although
few have been explored for their functional impact. Recently,
Kilic et al. (23) established a gain-of-function phenotype asso-
ciated with the hSERT Ile425Val variant, attributing alterations
to constitutive elaboration of regulation normally supported by
PKG stimulation. Ozaki et al. (24) found the variant in two
families, tracking the allele (as well as the 5HTTLPR ‘‘L’’ allele)
with subjects exhibiting a complex psychiatric phenotype, in-
cluding, among other things, OCD and Asperger’s syndrome.
The increasing awareness that rare, functional alleles can define
disrupted pathways bearing other disease susceptibility genes
(25, 26) encouraged us to achieve a comprehensive, functional
observed is a striking pattern of regulatory disruption, wherein
half of the hSERT variants, including all four that are present on
cytoplasmic domains, seem specifically refractory to PKG and
p38 MAPK-linked signaling pathways. We discuss our findings
with respect to a possible role for compromised hSERT regu-
lation in disorders linked to 5-HT dysfunction.
Materials and Methods
DNA Constructs. The full-length cDNA encoding hSERT in the
mammalian expression vector pcDNA3.1(Invitrogen) has been
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: 5-HT, 5-hydroxytryptamine; SERT, serotonin transporter; hSERT, human
SERT; PKG, protein kinase G; MAPK, mitogen-activated protein kinase; OCD, obsessive-
compulsive disorder; TM, transmembrane domain; 8BrcGMP, 8-bromo cGMP; ?-PMA,
?-phorbol 12-myristate 13-acetate; RTI-55, (3?-(4-iodophenyl)tropan-2?-carboxylic acid
**To whom correspondence should be addressed at: Vanderbilt Center for Molecular
Neuroscience, Suite 7140, MRBIII, Vanderbilt School of Medicine, Nashville, TN 37232-
8548. E-mail: firstname.lastname@example.org.
© 2005 by The National Academy of Sciences of the USA
August 9, 2005 ?
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described (27). Mutations in hSERT were produced by using the
QuikChange mutagenesis kit (Stratagene). All mutations were
confirmed by fluorescent dideoxynucleotide sequencing (Center
for Molecular Neuroscience Neurogenomics Core).
Transfection and Transport Studies. HeLa cells, maintained at 37°C
in a 5% CO2 humidified incubator, were grown in complete
medium [DMEM, (Invitrogen)?10% FBS?2 mM L-glutamine?100
units/ml penicillin?100 ?g/ml streptomycin]. Transfections (1 ?g of
DNA per 500,000 cells per 6-well-plate or 0.05 ?g per 10,000 cells
per 24-well-plate) were performed by using FuGENE 6 reagent
(Roche, Indianapolis) in Opti-MEM I (Invitrogen) as suggested by
the manufacturer. Transfected cells were cultured as above for 36 h
before 5-HT transport and biochemical assays.
Transport, Binding, Biotinylation, and Phosphorylation Studies.
CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 1.8 g?liter glucose, and
10 mM Hepes (pH 7.4), as described (15), defining specific 5-HT
uptake by using 10 ?M paroxetine. For quantitative assessment of
SERT total and surface density, we measured [125I]RTI-55 (3?-(4-
to intact cells on ice, using either paroxetine (10 ?M) or 5-HT (100
?M) as displacer (15). To establish levels and biosynthetic progres-
sion of hSERT protein produced from mutant cDNAs, HeLa cells
were plated in six-well dishes at 500,000 cells per well and trans-
fected 12 h later. Twenty four hours after transfection, whole-cell
detergent extracts were blotted for hSERT (mAb Technologies,
Stone Mountain, GA, 1:1000) by using enhanced chemilumines-
cence (ECL, Amersham Pharmacia) detection. Altered density of
SERT surface proteins was validated by using immunoblotting of
biotinylated whole-cell extracts produced by using the lysine-
directed, membrane-impermeant biotinylating reagent sulfo-NHS-
SS-biotin (Pierce) (27). Phosphorylation of hSERT and Gly56Ala
variants in transfected HeLa cells was examined as described (13)
by using 100 ?M 8-bromo cGMP (8BrcGMP) (60 min) as stimulus.
Specificity of labeling was verified by using parallel cultures trans-
fected with pcDNA3.
from autistic pedigrees recruited by S. E. Folstein (The Johns
Hopkins University, Baltimore), from the Autism Genetics Re-
National Institute of Mental Health Human Genetics Initiative
Repository (www.nimhgenetics.org) at Rutgers University. DNA
based allelic discrimination, by using an Applied Biosystems Assay-
by-Design and independently confirmed by PCR and direct se-
quence analysis. Genotyped lymphocytes were cultured in
suspension in RPMI medium 1640, supplemented with 15% FBS,
2 mM L-glutamine, 100 units?ml penicillin, and 100 ?g?ml strep-
tomycin at 37°C in a humidified incubator at 5% CO2and main-
tained in uniform growth conditions. Lymphocyte were pelleted at
200 ? g for 5 min and washed with Krebs–Ringer–Hepes (KRH)
assay buffer. A total of 1 ? 106cells in triplicate were prewarmed
(37°C) in a shaking water bath (10 min) in 12 ? 75 polypropylene
tubes in KRH buffer containing 100 ?M pargyline and 100 ?M
ascorbic acid ? modifiers. After a 5-min incubation with [3H]5-HT
(20 nM) ? 10 ?M paroxetine at 37°C, uptake assays were termi-
nated by immersion on ice, and uptake in pelleted, SDS (1%)-
extracted cells was quantitated by scintillation spectrometry.
The location of 10 identified hSERT coding variants is described in
Fig. 1A (see also Tables 1 and 2, which are published as supporting
information on the PNAS web site). The reference hSERT cDNA
and each hSERT variant were separately transfected into HeLa
cells, and 5-HT transport activity was assessed as described in
Materials and Methods. As shown in Fig. 1B, five variants (Thr4Ala,
Gly56Ala, Ser293Phe, Leu362Met, and Ile425Val) displayed en-
hanced 5-HT transport activity relative to hSERT and one variant
(Pro339Leu) displayed markedly reduced uptake activity. These
variations persisted across multiple plasmid preparations and thus
seem to arise from intrinsic differences in protein abundance,
Ile425Val and Pro339Leu. Kinetic analysis of Ile425Val revealed
significant changes in 5-HT Vmax(190 ? 28% of hSERT, P ? 0.05)
also Fig. 6, which is published as supporting information on the
PNAS web site). With Pro339Leu, 5-HT Vmax was significantly
reduced (3.0 ? 1.2% of hSERT, P ? 0.05). The activity of
Pro339Leu was too low to allow the 5-HT Km to be reliably
To establish a physical basis for the altered transport activities,
immunoblots of transfected HeLa cell extracts were obtained. As
shown in Fig. 2A, hSERT and almost all variants produced com-
parable levels of the 80-kDa protein that is characteristic of mature
variants bearing the largest changes in 5HT uptake, Pro339Val and
Ile425Val, revealed a significant reduction and elevation, respec-
(Fig. 2 B and C). Total [125I]RTI-55 binding, defined with the
hydrophobic displacer paroxetine, did not demonstrate differences
between hSERT and hSERT variants (Fig. 7A, which is published
as supporting information on the PNAS web site). Surface SERT
density, as defined by 5-HT displacement, was significantly de-
pressed for Pro339Val and elevated for Ile425Val (Fig. 7B), but
unchanged for the other variants. In contrast, Thr4Ala, Gly56Ala,
Ser293Phe, and Leu362Met variants also display enhanced basal
transport activity (30–50%) that could not be explained by en-
hanced surface density. We also evaluated whether variant SERTs
COOH termini oriented inside the cell. Variants in extramembrane domains are
shaded black whereas those in membrane domains are shaded white. (B) 5-HT
transport activity of SERT-coding variants in transfected HeLa cells. Data reflect
mean values ? SEM of three separate experiments. Means were compared with
means against hSERT values (*, P ? 0.05 taken as significant).
www.pnas.org?cgi?doi?10.1073?pnas.0501432102Prasad et al.
retained normal antagonist sensitivities. Several variants demon-
cocaine (Table 3, which is published as supporting information on
the PNAS web site). Most prominently, we observed a 10-fold shift
in cocaine potency with Pro339Leu, accompanied by a significant
although less substantial loss of potency for citalopram and
cGMP-linked pathways enhance SERT activity in native (15, 28,
29) and transfected (15, 23, 30) cells. Similarly, when we treated
hSERT-expressing HeLa cells with 8BrcGMP (10–100 ?M, 10
min), we achieved a dose-dependent stimulation of 5-HT transport
which could be completely antagonized by coincubation with the
PKG antagonist H8 (10 ?M) (Fig. 3A). Parallel changes in
[125I]RTI-55 surface-binding support increased surface trafficking
triggered by the PKG pathway (Fig. 4). For variants Leu255Met,
Ser293Phe, Pro339Leu, Leu362Met, and Ile425Met, 8BrcGMP
triggered a dose-dependent, H8-sensitive stimulation of SERT
activity comparable with hSERT. In contrast, Thr4Ala, Gly56Ala,
Glu215Lys, Lys605Asn, and Pro621Ser were completely insensitive
to 8BrcGMP application. The hSERT variants that responded with
uptake increases also demonstrated elevated [125I]RTI-55 surface
binding. Remarkably, the five hSERT variants that failed to elicit
uptake increases after 8BrcGMP treatments actually demonstrated
a reduction in [125I]RTI-55 surface binding (Fig. 4). These reduc-
tions in surface density were still specific because they could be
completely blocked by H8. 8BrcGMP and H8 had no effects on
total binding as assessed in parallel assays using paroxetine as the
displacer (data not shown).
SERTs are known to be rapidly internalized by phorbol ester
treatments, effects that are blocked by PKC antagonists (14). Thus,
we treated transfected cells with the phorbol ester ?-phorbol
12-myristate 13-acetate (?-PMA) and monitored changes in 5-HT
transport activity. As expected, hSERT expressed transiently in
HeLa cells displays an ?40% down-regulation after a 15-min
treatment with 10 ?M ?-PMA, down-regulation blocked by the
PKC antagonist bisindolylmaleimide (BIM, 1 ?M) (Fig. 8, which is
published as supporting information on the PNAS web site). In
each of the hSERT variants (Thr4Ala, Gly56Ala, Lys605Asn, and
Pro621Ser) displayed down-regulation after ?-PMA treatments
equal to or slightly greater than that seen for hSERT.
In studies of adenosine receptor and PKG-linked up-regulation
of SERT, we discovered that enhanced SERT activity requires
activated p38 MAPK (15). More recent studies reveal that direct
p38 MAPK activators such as anisomycin trigger a rapid, traffick-
transfected cells with 1 ?M anisomycin for 10 min before 5-HT
transport assays and, as previously found, achieved a 40–50%
noblots of total cell extracts prepared from HeLa cells transfected with hSERT or
one of the variants described in the study. (B) Cell surface expression alterations
in hSERT Pro339Leu and Ile425Val. Variants were transfected in parallel with
hSERT into HeLa cells, and cell surface transporters were identified by immuno-
blotting of biotinylated samples, captured as described in Materials and Meth-
mean values of three separate experiments ? SEM. Means were compared with
a one-way ANOVA followed by Dunnett’s test to compare variant surface ex-
pression to that achieved with hSERT (*, P ? 0.05 taken as significant).
Analysis of protein expression of hSERT and coding variants. (A) Immu-
examined for 5-HT transport activities as described in Materials and Methods
after pretreatments of cells with either 100 ?M 8BrcGMP ? H8 or vehicle for
cells. Cells transfected with hSERT or hSERT-coding variants were examined
after pretreatments of cells with either 1 ?M anisomycin ? SB203580 or
vehicle for 10 min. Results reflect mean values ? SEM of three separate
experiments normalized to each mutant’s control measured under vehicle-
treated conditions (100%). Results in A and B reflect mean values ? SEM of
three separate experiments normalized to each mutant’s level under vehicle-
treated conditions (100%). Data were analyzed by a one-way ANOVA with
post hoc Bonferonni tests comparing variant to hSERT 8BrcGMP?anisomycin
responses with P ? 0.05 taken as significant.
Impact of 8BrcGMP and p38 MAPK on hSERT activity. (A) Activity
Prasad et al. PNAS ?
August 9, 2005 ?
vol. 102 ?
no. 32 ?
stimulation of uptake activity (Fig. 3B). Just as with 8BrcGMP
treatments, variants Leu255Met, Ser293Phe, Pro339Leu,
Leu362Met, and Ile425Val each responded to anisomycin treat-
ment comparable with hSERT, with increased activity blocked by
cotreatments with SB203580. Neither the uptake stimulation of
hSERT nor the up-regulation achieved with these five variants was
accompanied by changes in total or surface [125I]RTI-55 binding
(data not shown), consistent with a trafficking-independent mode
five hSERT variants lacking 8BrcGMP sensitivity (Thr4Ala,
anisomycin, no uptake stimulation was observed.
Of the variants studied, only one, Gly56Ala, is found at frequen-
cies sufficient to permit identification of subjects carrying modified
alleles. We genotyped a large collection of 340 autism families
possessing, in many cases, banked, Epstein–Barr virus (EBV)-
transformed lymphocytes, because SERT is natively expressed in
lymphocytes (19) and because the 17q11.2 region harboring the
the 56Ala allele at a frequency of 1.1% in all families, but this
frequency increased to 2.3% in 120 families most contributing to
linkage. This frequency represents a significant difference (?2 ?
9.94, df ? 1, P ? 0.0016) between our sample and a separately
collected, nonclinical sample (21). Importantly, we identified two
probands bearing a homozygous Ala-56 genotype as well as mul-
tiple subjects carrying heterozygous genotypes.
As seen with transfected cells, lymphocytes homozygous for the
Gly variant (identical to reference hSERT) provided robust
8BrcGMP stimulation of 5-HT transport, stimulation that is sen-
sitive to H8 (Fig. 5A). Additionally, anisomycin stimulated uptake
activity, and this stimulation was sensitive to SB203580. In contrast,
the Ala-56 homozygous lines lacked sensitivity to either 8BrcGMP
or anisomycin. The Gly56Ala heterozygous cells displayed inter-
mediate sensitivity to these agents, consistent with a gene dosage-
dependent impact on regulation. As detected in transfected cells,
we found that all three genotypes displayed a similar degree of
down-regulation after ?-PMA treatments (data not shown). SERT
is phosphorylated under basal conditions, and phosphorylation can
be significantly elevated after PKG activation (13). To examine
whether the loss of regulation exhibited by the Gly56Ala variant
might derive from changes in its ability to receive regulatory
phosphorylation, we performed in situ phosphorylation studies,
immunoprecipitating SERT proteins after stimulation of
prelabeled cells with 8BrcGMP. In hSERT-transfected HeLa cells,
8BrcGMP (100 ?M, 60 min) triggered an ?80% elevation of basal
phosphorylation (Fig. 5B). In contrast, Gly56Ala-transfected cells
exhibited significantly elevated basal phosphorylation levels and
could not be further phosphorylated by 8BrcGMP treatments.
Western blotting of cell extracts revealed no differences in total
hSERT protein levels.
A number of disorders including anxiety, major depression, OCD,
autism, and irritable bowel syndrome have been associated with
the a common, functional variant in the hSERT promoter termed
the 5HTTLPR (18, 32, 33). Less attention has been given to the
functional status of hSERT-coding variants. In an early report,
with hSERT- or hSERT-coding variants were treated with either 100 ?M
(5 nM) binding with 5-HT (100 ?M) as displacer. Data were analyzed by a
one-way ANOVA with post hoc Bonferonni tests comparing variant to hSERT
anisomycin responses, with P ? 0.05 taken as significant.
Impact of 8BrcGMP on hSERT surface binding. HeLa cells transfected
lymphocytes and may involve altered transporter phosphorylation. (A) Lym-
and assessed for 5-HT uptake regulation as described for transfected HeLa
cells. Data presented derive from individual lymphocyte lines of determined
genotype. Findings were replicated in a separate set of genotyped lines with
equivalent results. Uptake levels for each genotype with vehicle-treated
conditions were taken as 100%. Transport activities were analyzed by a
two-way ANOVA with post hoc Bonferroni tests, with P ? 0.05 taken as
significant. (B) hSERT Gly56Ala variant displays altered basal phosphorylation
and sensitivity to 8BrcGMP. SERTs expressed in transfected HeLa cells were
examined 36 h after transfection. (Upper) Representative total extract immu-
noblot and autoradiogram from SERT immunoprecipitations. (Lower) Quan-
titation of SERT labeling from phosphorylation studies (n ? 3). Values are
expressed as mean ? SEM.*, P ? 0.01 versus WT-vehicle; ##, P ? 0.05 versus
WT-vehicle by one-way ANOVA with Bonferroni post hoc analysis.
Altered PKG?p38 MAPK sensitivity of 56Ala is evident in native
www.pnas.org?cgi?doi?10.1073?pnas.0501432102Prasad et al.
Lesch and coworkers (22) identified a single Leu255Met allele.
of 450 nonclinical subjects, revealing nine new coding variants, but
only one, Gly56Ala, was found more than once and still at a
frequency of ?0.5% (4?900 chromosomes; Table 1). Gly56Ala was
also identified at low frequency by Cargill et al. (34), along with
Lys605Asn, also found by Glatt et al. (21). None of these initial
studies characterized the function of hSERT-coding variants. Pos-
sibly, they could impact hSERT function and provide clues to
pathways contributing risk for 5-HT-linked clinical phenotypes. By
analogy, we identified Ala457Pro in human norepinephrine
(hNET) in a single family with orthostatic intolerance (OI) (35).
Despite the rarity of this variant, its segregation with tachycardia
and plasma catecholamines provides important evidence that id-
iopathic OI likely involves a hypernoradrenergic state. Each of the
major SERT alleles studied is highly conserved across currently
sequenced mammalian SERTs (Table 2). Conservation has been
demonstrated as one predictor of functional perturbations (36).
Overall, we found that 7 of the 10 variants bore functional pertur-
bations, including altered protein expression and basal 5-HT up-
take, cocaine and antidepressant recognition, or loss of regulation.
We found two variants whose changes in 5-HT uptake capacity
were accompanied by parallel changes in total and?or cell surface
protein expression. Pro339Leu exhibits a major loss of mature,
N-glycosylated protein consistent with improper folding leading to
inefficient biosynthetic progression or rerouting to degradative
functional loss is observed, suggesting further disruption of the
5-HT translocation mechanism. Pro339Leu lies in TM6, a domain
suggested to participate in transporter oligomerization (37).
Whereas Pro-339 is conserved down to Caenorhabditis elegans, the
residue is not conserved in norepinephrine and dopamine, consis-
tent with a more specific role in 5-HT translocation or unique
aspects of the transporter’s biosynthesis not tested in our studies.
transport activity coupled to increased cell surface density. Re-
cently, Ozaki et al. (24) identified this variant in two families with
a complex psychiatric phenotype including OCD and Asperger’s
carried a single copy of the allele in the background of a homozy-
gous 5HTTLPR L?L genotype. We were unable to reproduce the
loss of regulatory sensitivity for Ile425Val reported by Kilic et al.
(23), although we did observe the reported increase in basal 5-HT
transport capacity. We have found, using hSERT-inducible cell
the PKG?p38 MAPK pathways become less evident or is nonde-
tectible with higher level expression of hSERT, and, thus, a variant
such as Ile425Val, bearing constitutively elevated surface density
and 5-HT uptake, may more readily saturate the regulatory ma-
chinery upon heterologous expression. Regardless, both studies
agree that Ile425Val represents a hypermorphic mutation whose
inappropriately elevate synaptic 5-HT clearance.
The most striking finding in the current article is the complete
lack of sensitivity of 5 of the 10 hSERT variants to acute actrivators
of PKG or p38 MAPK. This loss of sensitivity does not correlate
with changes in basal 5-HT transport activity: both Pro339Leu and
Ile425Val, which show hypomorphic and hypermorphic pheno-
types, respectively, demonstrated stimulation by 8BrcGMP and
anisomycin. Additionally, all of the variants displayed a relatively
robust sensitivity to phorbol ester-triggered down-regulation. Fi-
nally, although there were changes noted for cocaine and antide-
pressant recognition, these changes were not highly correlated with
loss of transporter stimulation. In fact, the greatest number of
changes in antagonist recognition occurred within TMs 4–8
it is reasonable to speculate that variants may disrupt interactions
with accessory proteins that, in past years, have grown to include
syntaxin 1A (38), protein phosphatase 2A catalytic subunit
(PP2Ac) (39), Hic-5 (40), and MacMARCKS (41). The loss of
regulation by Glu215Lys is more difficult to explain but could
suggest a conformational linkage of EL2 to regions of TM3 linked
to 5-HT recognition (42). EL2 movement, when limited by zinc
coordination, blocks substrate transport in homologous dopamine
proteins (43), and thus sequence variation in this loop may perturb
regulatory conformational changes propagated from intracellular
pathway triggers an increase in 5-HT affinity, as assessed in
antagonist binding assays (17).
Measurement of Gly-56 and Ala-56 hSERT lymphocyte mRNA
by real-time PCR revealed no differences in SERT mRNA levels
defects in transporter regulation observed. hSERT proteins exhibit
basal phosphorylation and become further phosphorylated in re-
sponse to activators of PKA, PKC, and PKG (13). To consider the
integrity of hSERT PKG phosphorylation, we explored the extent
of phosphorylation of the Gly56Ala variant in transiently trans-
fected HeLa cells and obtained evidence that the variant exhibits
elevated basal phosphorylation and cannot be further phosphory-
lated in response to 8BrcGMP treatments. The Gly56Ala variant,
possibly through disrupted phosphatase interactions (39), may lack
normal inhibitory mechanisms restricting basal phosphorylation.
Alternatively, the Gly56Ala variant may impart a gain-of-function
phenotype that leads to elevated basal phosphorylation. For exam-
ple, 5-HT-gated channel activity is unmasked in SERT proteins by
elimination of regulatory syntaxin 1A interactions (38), and, pos-
that directly or indirectly enhance basal phosphorylation of SERT.
Additional studies are needed to expand this effort to p38 MAPK
stimulation, to clarify which of the several kinases targeting SERT
ylation studies to the other affected variants. Loss of regulation
through the PKG pathway may leave the endocytic mechanisms
five PKG?p38 MAPK nonresponsive alleles (Thr4Ala, Gly56Ala,
Lys605Asn, and Pro621Ser) actually demonstrated significantly
enhanced phorbol ester-mediated down-regulation, further en-
hancing this possibility. Enhanced sensitivity to phorbol ester-
mediated down-regulation may also be a clue as to why the variants
actually show decreased [125I]RTI-55 surface binding. Signals that
trigger the PKG-dependent shuttling of new transporters to the
surface may also enhance endocytic recycling rates (44) possibly
through crosstalk with PKC-linked pathways. As such, diminished
[125I]RTI-55 binding to the variants may report a stabilized, partial
conformational transition on the endocytic limb. We have recently
reported (45) that platelet SERT exhibits surface-resident inactive
states and that it is conceivable that [125I]RTI-55 binding might
report a state occupied before uptake inactivation. Although these
ideas remain speculative at best, the five variants lacking uptake
in probing different steps in the complex regulatory pathways that
ultimately establish 5-HT uptake capacity.
Because the changes we report are genetically encoded and
because SERT expression occurs early in development, the non-
responsive alleles could compromise the ability of SERT to mod-
ulate in response to environmental demands and elevate risk for
developmental disorders linked to altered 5-HT signaling (46–48).
on emotional behavior in adults, and genetic variation at the
hSERT promoter has been reported to interact with early child-
hood stressors to influence risk for depression and suicide in later
life (33). Possibly, carriers of regulatory nonresponsive hSERT
alleles may be at greater risk for adult onset disorders arising from
inappropriate hSERT activity at critical periods in development.
Prasad et al. PNAS ?
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TheGly56Alaallele,althoughuncommon,isstillcarriedby?1:200 Download full-text
Caucasian subjects (21), representing more than a million Ameri-
cans. Disrupted 5-HT signaling has long been discussed as a
potential underlying determinant of altered development and be-
havior in autism (6, 49–51). Because of our prior study noting
linkage of autism to the SERT locus at 17q11.2 (31) and our access
to a large collection of autism family DNA samples with matching
accessed banked cell lymphocyte lines to determine the functional
impact of the variant allele within native hSERT expressing cells.
Although details of allelic segregation with the autism phenotype
allele at a frequency of 2.3% in 120 families selected on the basis
of linkage to autism at 17q11.2, a ?5-fold increase in allele
frequency over the Gly56Ala frequency published by Glatt et al.
(21) in a study of 450 nonclinical subjects. The homozygous
Gly56Ala lines we identified derive from two male subjects with
(31, 52, 53) and male-specific autism risk in particular (54) argue
that further evaluation of the phenotype of hSERT Gly56Ala
carriers, as well as a directed search for additional hSERT alleles
that can similarly impact transporter regulation through PKG?p38
MAPK pathways, is warranted.
We gratefully acknowledge technical assistance from Qiao Han in cell
culture support, Dr. Keith Henry for guidance on transport and biotinyla-
tion studies, and Dr. Louis DeFelice for critical review of the manuscript.
MH55135 (to Dr. Susan E. Folstein), and funds from Vanderbilt Kennedy
Center Hobbs Research Awards (to R.D.B. and J.S.S.). H.C.P. and R.C.S.
by a predoctoral training grant from the National Alliance for Autism
Research. We gratefully acknowledge assistance from the Vanderbilt
Neurogenomics Core, the Center for Human Genetics Research DNA
Autism Genetic Resource Exchange Consortium, a program of Cure
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www.pnas.org?cgi?doi?10.1073?pnas.0501432102Prasad et al.