Báo cáo khoa học: Identification of a novel alternative splicing variant of RGS5 mRNA in human ocular tissues ppt

Identification of a novel alternative splicing variant
of RGS5 mRNA in human ocular tissues
Yanbin Liang, Chen Li, Victor M. Guzman, William W. Chang, Albert J. Evinger III, Dyna Sao
and David F. Woodward
Department of Biological Science, Allergan, Inc., Irvine, CA, USA
Heterotrimeric guanine nucleotide-binding proteins
(G proteins) comprise a superfamily that is involved in
the transmission of ligand binding to cell surface recep-
tor events into intracellular responses that regulate
various physiological processes [1–5]. G proteins play
important roles in determining the specificity and
temporal characteristics of cellular responses to signals.
They are composed of a, b and c subunits; each
subunit contains subtypes (20 a, six b and 12 c), allow-
ing many combinatorial possibilities [4]. The main
G proteins can be classified as follows: Gas, which
activates adenylyl cyclase; Gai ⁄ Gao, which inhibit
Keywords
alternative splicing; cannibinoid receptor;
G protein; prostaglandin FP receptor; RGS5
Correspondence
Y. Liang, Department of Biological Science,
Allergan, Inc., Irvine, CA 92612, USA
Tel: +1 714 2465966
Fax: +1 714 2465578
E-mail:
(Received 14 June 2004, revised 2 November
2004, accepted 6 December 2004)
doi:10.1111/j.1742-4658.2004.04516.x
Regulator of G protein signaling (RGS) proteins act as GTPase-activating
proteins (GAPs) for Ga subunits and negatively regulate G protein-coupled

receptor signaling. Using RGS5 gene-specific RT-PCR, we have identified
a novel alternative splicing variant of RGS5 mRNA in human ocular
tissues. The alternative splicing of RGS5 mRNA occurred at position +44
(GenBank NM_003617), spliced out 174 bp (+44 to +218 bp) of the cod-
ing region, and encoded an RGS5s protein with a 108 amino acid N-ter-
minal deletion. This study is the first to document alternative splicing of an
RGS5 gene. We therefore studied RGS5 and RGS5s mRNA distribution in
human tissues. In the eye, RGS5s was found to be highly expressed in the
ciliary body and trabecular meshwork. It was also expressed in the kidney,
brain, spleen, skeletal muscle and small intestine, but was not detectable in
the liver, lung, heart. RGS5s was not found in monkey and rat ocular tis-
sues, indicating species specificity for the eye. Comparing the recombinant
RGS5 and RGS5s expression in HEK293 ⁄ EBNA cells, RGS5s was present
almost exclusively in the cytosolic fraction, whereas RGS5 was present in
both membrane and cytosolic fractions. The data suggest that the
N-terminal of RGS5 may be important for protein translocation to the cell
membrane. Both RGS5 and RGS5s antagonized the rapid phosphorylation
of p44 ⁄ 42 MAP kinase induced by Gai coupled cannibinoid receptor-1 acti-
vation. RGS5, but not RGS5s, inhibited the Ca
2+
signaling initiated by
activation of Gaq coupled angiotensin II receptors (AT1) and prostaglandin
FP receptors. Cotransfection of RGS5s with RGS5 resulted in the blockade
of RGS5 actions with respect to inhibition of the signal transduction initi-
ated by activation of both AT1 and FP receptor, suggesting that RGS5s
may contain functional domains that compete with RGS5 in the regulation
of the Gaq coupled AT1 and FP receptors. The unique expression pattern,
cellular localization and functions of RGS5s suggest that RGS5s may play
a critical role in the regulation of intracellular signaling pathways.
Abbreviations

AT1, angiotensin II receptor; CB-1, cannibinoid receptor-1; FP, F prostanoid; GAP, GTPase-activating proteins; RGS, regulator of G protein
signaling; TM, trabecular meshwork.
FEBS Journal 272 (2005) 791–799 ª 2005 FEBS 791
adenylyl cyclase; Gaq, which activates phospholipase
C; and G12 and G13, which activate the Rho pathway.
Activation of G protein coupled receptors initiated by
agonist binding promotes GDP ⁄ GTP exchange, active
GTP binding to the Ga subunit, and Gbc dissociation
and interaction with target effector proteins. G protein
signaling is terminated by the hydrolysis of GTP to
GDP and subsequent reformation of the heterotrimer.
The strength and duration of a particular G protein-
directed signaling event is determined by the length of
time Ga remains in the active GTP-bound confor-
mation. The activities of G proteins can be regulated
by numerous extracellular and intracellular factors.
The regulator of G protein signaling (RGS) is one of
the factors that regulate G proteins functions.
The RGS proteins were first identified as signal
transduction molecules that have structural homology
to the Sst2 gene in Saccharomyces cereviseae [6] and
Caenorhabditis elegans (EGL10) [7]. Among all RGS
proteins, a conserved 120 amino acid domain has been
defined as the RGS domain that is both necessary and
sufficient for the stimulatory effects of RGS proteins on
Ga GTPase activity [8]. RGS proteins regulate G pro-
tein coupled receptors through three known mecha-
nisms: (a) RGS proteins are GTPase-activating proteins
(GAPs); (b) RGS proteins can act as effector antago-
nists that block GTP-bound Ga subunits from binding

to their effectors; and (c) RGS proteins can block Ga
subunit dissociation from Gbc subunits by enhancing
the affinity of Ga subunits for Gbc subunits [8].
RGS5 was first identified and isolated from neuro-
blastoma cells [9]. The messenger RNA for RGS5 was
abundantly expressed in aorta, skeletal muscle, small
intestine and brain, and at low levels in heart, placenta,
liver, colon, and leukocytes [9,10]. RGS5 acts as a
potent GTPase activating protein for Gaq and Gai,
but not Gas, and it attenuates angiotensin II-, endo-
thelin-1-, and PDGF-induced ERK-2 phosphorylation
[11,12]. In our previous study, we found that RGS5
mRNA was up-regulated in ocular hypertensive
monkey eyes [13]. In this study, we further explored
regulation of RGS5 mRNA in human normal and
glaucomatous ocular tissues. We first described identifi-
cation of a novel alternative splicing variance of RGS5
in human ocular tissues, and then studied tissue distri-
bution, cellular localization and, functional changes of
RGS5 alternative splicing variant (RGS5s). The infor-
mation gained from this study will help further under-
standing of the molecular mechanisms of RGS5 and its
alternative splicing variant (RGS5s) in the regulation of
G protein and G protein-coupled receptors.
Results
Identification of alternative splicing of RGS5
mRNA in human, monkey and rat ocular tissues
Identification of up-regulation of RGS5 mRNA in
monkey hypertensive eyes led us to further study the
regulation of RGS5 mRNA in human glaucoma eyes.

Using human RGS5 specific primers (RGS5 primers 1
and 2) corresponding to nucleotides 82–627 (GenBank
NM_003617), a 500 bp (RGS5) and a 300 bp (RGS5s)
PCR product was amplified from human normal and
glaucoma eyes (Fig. 1A). We screened three glaucoma
eyes and five normotensive eyes. Three glaucoma eyes
were obtained from National Disease Research
Interchange (NDRI, Philadelphia, PA, USA) at 48 h
postmortem. All of them were at age 70–79 years old
and male Caucasian with over 10 years glaucoma his-
tory. Five normotensive eyes were obtained from
NDRI at 48 h postmortem. All donors were at age
70–79 years old and male Caucasian. Using densitome-
try analysis, the ratios of RGS5s to RGS5 density in
RGS 5
RGS 5S
Glaucoma Eye
Human Eye
RGS5
RGS5s
CSM NoRT TM NoRT ODM NoRT
Human Ocular Tissue
Monkey
RGS 5
C
i
li
ary
B
o

d
y
R
etina
R
at E
ye
Rat RGS5
H
u
m
a
nRet
ina
RGS5
RGS5s
A
CD
B
Fig. 1. Identification of alternative splicing of
RGS5 mRNA in human, monkey and rat
ocular tissues. One hundred nanograms of
each total RNAs isolated from (A) human
eyes, (B) human ocular tissues, (C) monkey
eye and (D) rat eyes were used for RT-PCR
analysis. Arrows indicate a 550 bp PCR prod-
uct of RGS5 and 300 bp PCR product (alter-
native splicing of RGS5). CSM, clilary
smooth muscle; TM, trabecular meshwork;
ODM, a human NPE cell line; NoRT, no

reverse transcription control.
Alternative mRNA splicing variant of RGS5 Y. Liang et al.
792 FEBS Journal 272 (2005) 791–799 ª 2005 FEBS
three glaucoma eyes and five normotensive eyes are
measured. The ratios in three glaucoma eyes were 1.5,
1, 0.5, respectively, whereas the ratios in five normo-
tensive eyes were less than 0.5. RGS5s mRNA was
also identified in primary ciliary smooth muscle cells,
trabecular meshwork, and retina from normal human
eyes and in ODM-2 cells (Fig. 1B). Ciliary smooth
muscle, ODM-2 cells and retina showed ratio of
RGS5s to RGS5 is less than 1, the TM exhibited
almost an equal amount of RGS5s to RGS5, suggest-
ing that RGS5s expression is tissue-specific. Using
monkey and rat RGS5-specific primers (the same
primer sites as human), only a 500 bp PCR product
was amplified from monkey (Fig. 1C) and rat eyes
(Fig. 1D), suggesting that the alternative splicing of
RGS5 mRNA might be human specific.
Sequencing analysis showed that alternative splicing
occurred at +44 to +218 in the RGS5 coding region
(Figs 2 and 3A) and encoded a 73 amino acid RGS5s
protein (Fig. 3B).
Tissue distribution of RGS5s
To further identify RGS5s tissue expression, the RGS5
primer 1 and 2 set was used to detect RGS5s among
different human tissue RNAs (Fig. 2). RGS5s mRNA
was detected in the human kidney, brain, spleen, skel-
etal muscle and small intestine, but was not detectable
in liver, heart and lung (Fig. 4), suggesting that RGS5s

expression may be tissue-specific.
Cellular localization of RGS5 and RGS5s proteins
Hydropathic analysis (Kyte–Doolittle) showed that the
N-terminal (30 amino acid from ATG) of RGS5 is
highly hydrophobic, suggesting that this region may be
important for binding of RGS5 to the cell membrane.
RGS5s, by virtue of a deletion of 108 amino acids from
RGS5 N-termini, may be a membrane-unassociated
protein. To test this hypothesis, RGS5-V5-pcDNA3.1
plasmids or RGS5s-V5-pcDNA3.1 plasmids were trans-
aaaaaa
Splicing site
+1 +44 +45 +155 +156 +217 +218 +383 +384 +546
(34.4 kb) (6.3 kb) (9.2 kb) (5.04 kb)
RGS 5-s mRNA
RGS 5 Genomic Structure
Intron 1
Exon 1 Exon 2 Exon 3 Exon 4 Exon 5
Intron 2 Intron 3 Intron 4
Exon1
Exon1 Exon 4 Exon 5
Exon2 Exon3 Exon4 Exon5
aaaaaa
RGS 5 mRNA
Fig. 2. Alternative splicing of human RGS5
mRNA (RGS5s). Translation start site (ATG)
defined as +1. Blocks represent exons and
lines represent introns.
A
B

Fig. 3. Sequence analysis of alternative
splicing of RGS5 mRNA (RGS5-s). (A)
Blue color (ga) indicates the splicing site.
Red color ATG is the translation start
site. (B) Alternative splicing of RGS5
mRNA encodes a short amino acid sequ-
ence (73 amino acids).
Y. Liang et al. Alternative mRNA splicing variant of RGS5
FEBS Journal 272 (2005) 791–799 ª 2005 FEBS 793
fected in HEK293 ⁄ EBNA cells. Total protein, cytosolic
and membrane proteins were isolated from the trans-
fected cells and used for Western blotting analysis.
Western blotting analysis (Fig. 5) showed that RGS5 is
expressed in total, cytosolic and membrane fractions,
whereas RGS5s is not membrane associated.
RGS5s act as an endogenous negative regulator
of RGS5 in Gaq-coupled receptors
Human angiotensin II receptors (AT1) and prostaglan-
din F prostanoid (FP) receptor are Gaq protein-cou-
pled receptors. In previous reports, RGS5 was shown
to specifically interact with Gaq and Gai proteins
[14,15], and overexpression of RGS5 attenuated AT1
receptor associated Ca
2+
signaling [12]. The human
AT1 receptor was used in this study to see if RGS5s
alters AT1 receptor activity. In a parallel study, the
prostaglandin FP receptor was also tested to see if
RGS5 and RGS5s alter function. The results showed
that RGS5, but not RGS5s, inhibited 33% of the

maximum Ca
2+
signal response to angiotensin II- and
PGF
2a
(Fig. 6A,B), suggesting that RGS5 antagonized
both AT1 and FP receptor activities. Overexpression
of RGS5s along with RGS5 demonstrated that RGS5s
attenuated RGS5-antagonized AT1 and FP receptor-
associated Ca
2+
signaling, suggesting that RGS5s may
RGS5
RGS5s
Li
ve
r
Kidney
Brain
Smal
li
ntestine
Spleen
L
ung
Skeletal Muscle
Hear
t
Reti
na

Fig. 4. Tissue distribution of human RGS5 and RGS5s mRNA. One
hundred nanograms of each total RNA from liver, kidney, brain,
small intestine, spleen, lung, skeletal muscle, heart and retina was
used for RT-PCR analysis. The arrow indicates a 550 bp PCR prod-
uct of RGS5.
0
1000
2000
3000
4000
5000
-11 -10 -9 -8 -7 -6 -5 -4
FP+vector
FP+RGS5+RGS5s
FP+RGS5
FP+RGS5s
Concentration ( Log M)
*
**
*
*
** **
Fluorescence Units
Fluorescence Units
0
1000
2000
3000
4000
5000

6000
7000
8000
-11 -10 -9 -8 -7 -6 -5 -4
AT1+vector
AT1+RGS5+RGS5s
AT1 +R G S 5
AT1 +R G S 5s
Concentration ( Log M)
*
***
*
*
** **
***
A
B
Fig. 6. Effects of RGS5 and RGS5s on Ca
2+
signaling initiated by
Gaq-coupled AT1 and FP receptors. RGS5 and ⁄ or RGS5s cDNA
expression plasmids were cotransfected with AT1 or FP receptor
cDNA expression plasmid into HEK293 ⁄ EBNA cells. The trans-
fected cells were then treated with angiotensin II or PGF
2a
at con-
centrations ranging from 10
)12
M to 10
)5

M. FLIPR assay results
shown in the figures are representative of experiments independ-
ently repeated at least three times. *P<0.01 FP + vector or
FP + RGS5s vs FP + RGS5; **P<0.05 FP + RGS5 + RGS5s vs
FP + RGS5.
Western Blot Analysis
11 kDa
19 kDa
17 kDa
31 kDa
52 kDa
98 kDa
RGS5
RGS5s
TMCTMC
Fig. 5. Cellular localization of RGS5 and RGS5s. Twenty micro-
grams of each protein fraction was loaded into each lanes. Dilution
(1 : 1000) of rabbit V5 antibody was used to detect V5 antigen
fused with RGS5 and RGS5s proteins. T, total protein; M, mem-
brane protein; C, cytosolic protein. Arrows indicate a 26 kDa protein
of RGS5 and a 13 kDa protein of RGS5s.
Alternative mRNA splicing variant of RGS5 Y. Liang et al.
794 FEBS Journal 272 (2005) 791–799 ª 2005 FEBS
contain functional domains that compete with RGS5
protein in the regulation of the Gaq coupled receptors.
RGS5s antagonized Gai coupled CB-1 receptor
In addition to Gaq-coupled receptors, AT1 and FP,
the effects of RGS5 and RGS5s on Gai and Gas cou-
pled receptors (CB-1 or EP
2

receptor) were also tested.
Activation of CB-1 receptor by the agonist, WIN
55212–2, is coupled to Gai protein, which in turn acti-
vates signal transduction cascades, such as a rapid
phosphorylation of p44 ⁄ 42 MAP kinase [16]. Using
p44 ⁄ 42 MAP kinase assays, we further studied the
effects of RGS5 and RGS5s on the Gai-coupled
cannabinoid receptor-1 (CB-1) activities. The results
showed that both RGS5 and RGS5s attenuated CB-1
agonist (WIN 55212–2)-induced rapid phosphorylation
of p42 ⁄ 44 MAP kinase (Fig. 7). Cotransfection of
RGS5s with RGS5 did not result in more inhibition,
suggesting that RGS5s and RGS5 may interact with
the same inhibitory site in the Gai protein coupled to
the CB-1 receptor. Neither RGS5 nor RGS5s altered
the cAMP messenger initiated by the activation of the
Gas coupled EP
2
receptor (data not shown). In sum-
mary, RGS5 selectively inhibited Gaq-(AT1a and FP)
and Gai-(CB-1) coupled receptors, but RGS5s only
antagonized Gai-coupled CB-1 receptors.
Discussion
Using RGS5 gene specific RT-PCR, we have identified
a novel alternative splicing variant of RGS5 mRNA in
human ocular tissues. The alternatively spliced RGS5
mRNA encoded a 73 amino acid RGS5s protein with
an N-terminal 108 amino acid deletion. Functional
studies showed that RGS5s selectively regulated Gai-
coupled CB-1 receptors, and acted as an endogenous

negative modulator for RGS5 in Gaq coupled AT1
and FP receptor signal transduction. This is the first
study to document the existence of an alternative spli-
cing of the RGS5 gene.
It has been reported that over 20 different RGS pro-
teins have been identified and isolated, of which RGS3,
RGS8, RGS9 and RGS12 were found to present alter-
natively spliced RGS mRNA. RGS3T was the first iden-
tified RGS alternative splicing isoform, and it encoded
a C-terminal truncated form of RGS3 [17,18]. The
truncated form of RGS3 was found tissue-specifically
expressed in kidney, lung and brain. Functional studies
indicated that RGS3T not only modulated Gai and
Gaq proteins mediated signaling, but also modulated
Gas in intact cells. This provided the first evidence that
the C-terminal region of RGS3 comprised the functional
domain for negative regulation of Gas protein. Since
then, alternatively spliced RGS protein isoforms were
detected for many RGS proteins. RGS8 alternatively
spliced mRNA encoded an RGS8s protein with nine
amino acid deletion in the N-termini of RGS8 protein
[19,20]. The N-terminal deletion of RGS8 resulted in a
remarkable decrease in the inhibitory effects of RGS8
on Gaq-coupled responses. The nine amino acids in the
N-termini of RGS8 were determined to be important for
the inhibition of Gaq-coupled receptor specificity. This
is similar to our findings for RGS5 protein. The 108
amino acid N-terminal deletion of RGS5 caused RGS5
to completely lose its inhibitory effects on the Gaq cou-
pled AT1a and FP receptors. The N-terminal deletion

of RGS5 (RGS5s) also resulted in tissue-specific expres-
sion and changed its cellular localization. The expres-
sion pattern of RGS family members might contribute
to the physiological specificity of RGS proteins. Two
RGS9 proteins contained substantially different C-ter-
mini [21,22]. RGS9-1 is 191 amino acids shorter than
RGS9-2. RGS9-1 is exclusively expressed in the retina,
where it serves as a specific GAP for transducin, whereas
RGS9-2 is specifically expressed in the striatum, where it
Fig. 7. Effects of RGS5 and RGS5s on p44 ⁄ 42 phosphorylation
induced by activation of Gai-coupled cannibinoid receptor-1 (CB-1).
RGS5 and ⁄ or RGS5s cDNA expression plasmids were cotransfected
with CB-1 receptor cDNA expression plasmid into HEK293 ⁄ EBNA
cells. The transfected cells were treated with 10
)6
M of the CB-1
agonist WIN55212-2 for 10 min 20 lg of each protein fraction were
loaded into each lane. Top panels represent the phosphorylated form
of MAP kinases. Bottom panels represent the unphosphorylated
form of MAP kinases. The unphosphorylated form of MAP kinases
was used as a loading control. Densitomoter data (mean ± SD)
shown are representative of experiments independently repeated at
least three times. *P<0.01 vs. control; **P<0.01 vs. WIN55212-2
stimulation alone.
Y. Liang et al. Alternative mRNA splicing variant of RGS5
FEBS Journal 272 (2005) 791–799 ª 2005 FEBS 795
is involved in desensitization of Gi ⁄ o-coupled receptors.
RGS12 alternative splicing occurred at both 5¢ and 3¢
regions generating four alternative isoforms encoding
four distinct N-terminal domains and three distinct

C-terminal domains [23]. These intramolecular arrange-
ments created diverse regulatory mechanisms for
RGS12 proteins. Three different N-terminal RGS12
proteins were found to be exclusively localized in the cell
nucleus, suggesting that the N-termini of RGS12 pro-
teins are critical for the intranuclear distribution.
Besides RGS proteins, there are many alternative spli-
cing events that change gene expression pattern and
functionality [24]. Acetycholinesterase (ACHE) variants
encoded five different N-termini of ACHE isoforms.
Each of the ACHE isoforms showed tissue-specific
expression patterns and lost the ability to bind to cell
membranes [25,26]. In this study, we found that RGS5
was ubiquitously expressed in human tissues, and
RGS5s was tissue-specifically expressed in certain
human tissues. RGS5 was mainly expressed in the cell
surface membrane and cytoplasm, RGS5s was exclu-
sively present in the cytoplasm. The N-terminus of
RGS5 protein determined tissue-specific expression, the
ability of RGS5 to bind to the cell surface membrane,
and selectivity inhibiting G protein and G protein-
coupled receptors.
The GTPase-activating protein activity of RGS pro-
teins appears to be limited to the Gai and Gaq family
[6,8,15] and negatively regulates G protein-coupled
receptors in a receptor-specific manner. Endogenous
RGS3 and RGS5 in rat A-10 vascular smooth muscle
cells have differential effects on muscarinic and angio-
tensin receptors [23]. In an RGS3 and RGS5 knock-
down study, RGS3 selectively suppressed muscarinic m3

receptors but not angiotensin receptors (AT1a), whereas
RGS5 selectively modulated angiotensin receptors
(AT1a) but not muscarinic m3 receptors [23]. Overex-
pression of RGS5 did not alter platelet activating factor
(PAF) receptor signaling [27], but negatively regulated
angiotensin receptors (AT1a) [12,23]. In this study, we
confirmed that overexpression of RGS5 attenuated AT1
receptor-coupled Ca
2+
mechanisms, and showed for the
first time that overexpression of RGS5 selectively antag-
onized prostaglandin FP receptor mediated Ca
2+
signa-
ling. RGS5s did not interfere Gaq-coupled receptor
(AT1a and FP) activities, but impaired Gai-coupled
CB-1 receptor-activated p44 ⁄ 42 MAPK phosphoryla-
tion. Figure 8 exhibited a summary of RGS5s action in
Gaq and Gai coupled receptors. Up-regulation of
RGS5s was found in the glaucomatous eyes. It is, per-
haps, possible that RGS5s induced desensitization of
Gai-coupled receptors such as CB-1 which are known
to mediate decreased intraocular pressure [28], may
contribute to the development of ocular hypertension in
some glaucomatous patients.
Taken together, the identification of RGS5s provides
new clues for further understanding of the roles of RGS
proteins in the regulation of physiological processes. It
is possible that modulation of RGS5 and ⁄ or RGS5s
may provide a novel approach for glaucoma treatment.

Materials and methods
Cell cultures
HEK293 ⁄ EBNA cells were obtained from American Type
Culture Collection. HEK293 ⁄ EBNA cells were routinely
maintained in Dulbecco’s modified Eagle’s medium
(DMEM) with 10% fetal bovine serum, 1% glutamine,
0.5% penicillin ⁄ streptomycin. They were kept in humidified
5% CO
2
, 95% air at 37 °C.
Human ciliary smooth muscle (SM) cells were isolated
from a 69-year-old male donor eye. The donated human
eyes were collected by the National Disease Research Inter-
change (NDRI, Philadelphia, USA) under applicable regu-
lations and guidelines regarding consent issues, protection
of human subjects and donor confidentiality, and cultured
in DMEM with 10% fetal bovine serum and 0.5% penicil-
lin ⁄ streptomycin, according to the method previously repor-
ted by Woldemussie et al. [29].
Human trabecular meshwork (TM) cells were a gift from
J Polansky (University of California, San Francisco, CA,
USA). The human TM cells were derived from a 30-year-
old male donor eye and cultured in DMEM with 10% fetal
bovine serum and 0.5% penicillin ⁄ streptomycin in humid-
ified 8% CO
2
, 92% air at 37 °C. Both human primary TM
and SM cells were grown to confluence before addition of
the appropriate compounds.
Isolation of total RNA and reverse transcription-

polymerase chain reaction (RT-PCR)
Total RNA was isolated from the human eyes and human
ocular tissues (ciliary smooth muscles, trabecular mesh-
GTP GDP
Receptor
Pi
RGS5
βγ
Signal
Gα
GDP
(-) RGS5s
Gαq
GTP
βγ
Gαi
GTP
(-)
Fig. 8. Diagrammatic representation of RGS5 and RGS5s involved
in the mechanisms of G protein and G protein-coupled receptors.
Alternative mRNA splicing variant of RGS5 Y. Liang et al.
796 FEBS Journal 272 (2005) 791–799 ª 2005 FEBS
work, ODM-2) using a Qiagen total RNA isolation kit
according to manufacturer’s instructions. Human heart,
brain, lung, spleen, small intestine, skeletal muscle, kidney
and liver total RNA were purchased from Clontech (Palo
Alto, CA, USA). Using 5 lg of human total RNA, first
strand cDNA was synthesized by SuperScript II RNase H
reverse transcriptase (Life Technologies, Carlsbad, CA,
USA). Twenty-microliter reactions containing 5 lgof

RNA, 250 ng of oligo(dT) and 100 units of reverse tran-
scriptase were incubated at 42 °C for 1 h and terminated at
100 °C for 3 min.
The PCR buffer contained 10 mm Tris ⁄ HCl, pH 8.3,
50 mm KCl, 2 mm MgCl, 2.5 units Ampli Taq DNA
polymerase, 0.2 lm upstream and downstream primers, in a
final volume of 50 lL. After an initial incubation for 5 min
at 94 °C, samples were subjected to 30 cycles of 30 s at
95 °C, 30 s at 60 °C, and 30 s at 72 °C in a PerkinElmer
9700 thermal cycler. PCR products were sequenced by
Sequetech (Mountain View, CA, USA). The primers used
for the amplification of full length human RGS5, RGS5s
and angiotensin II receptor were as follows:
Primers (RGS5primer 1 and 2) corresponding to nucleo-
tides at 82–627 of human RGS5 sequence (GenBank,
NM_003617) were used for detection of alternative splicing:
5¢- ATGTGCAAAGGACTTGCAGC-3¢ (forward); 5¢-CAG
GAGTTAATCAAGTAG-3¢ (reverse).
Primers (RGS5 primer 3 and 4) corresponding to nucleo-
tides at 17–627 of human RGS5 sequence (GenBank,
NM_003617) were used for RGS5-pcDNA3.1-V5 plasmid:
5¢-TTCAAAGACTGGCTCTGCTGTTA-3¢ (forward); 5¢-
CTTGATTAACTCCTGATAAAACTCAGAGC-3¢ (reverse,
NON-STOP CODON).
Primers (RGS5s primer S1 and S2) corresponding to nu-
cleotides at 178 to 627 of human RGS5 sequence (GenBank,
NM_003617) were used for RGS5s-pcDNA3.1-V5 plasmid:
5¢-GTTGGTGACCTTGTCATTCCG-3¢ (forward); 5¢-CT
TGATTAACTCCTGATAAAACTCAGAGC-3¢ (reverse,
NON-STOP CODON).

Primers used for angiotensin II receptor (AT1a) cDNA
cloning: 5¢-CGCGGATGAAGAAAATGAAT-3¢ (forward);
5¢-CCCTTTGGAAACTGGACAGA-3¢ (reverse).
Primers used for cannabinoid receptor-1 (CB-1) cDNA
cloning: 5¢-GAGGACCAGGGGATGCGAAGG-3¢;5¢-TG
CCCCCTGTGGGTCACTTTCT-3¢.
Plasmids and transfection
Full-length RGS5 and RGS5s cDNA were subcloned into
TOPO pcDNA3.1 vector to create RGS5-pcDNA3.1 and
RGS5s-pcDNA3.1 plasmids. Full-length RGS5 and RGS5s
fused with V5 antigen were also subcloned into pcDNA3
vector and created RGS5-V5-pcDNA3.1 and RGS5s-
V5-pcDNA3.1 plasmids were created. Angiotensin II recep-
tor subtype 1 (AT1a) was subcloned into TOPO pcDNA3.1
vector to create AT1a-pcDNA3.1 plasmid. Human
prostaglandin FP receptor cDNA was subcloned into
pCEP4 vector and an hFP-pCEP4 plasmid was obtained.
Supercoiled plasmid DNA was transfected into 5 · 10
3
cells of HEK293 ⁄ EBNA by the FuGENE 6 method (Roche
Diagnostics Corp., Inc., Indianapolis, IN, USA), according
to manufacturer’s instructions. In brief, cells were washed
twice and resuspended in 1 mL of DMEM. One microgram
of plasmid DNA in 1 mL of DMEM containing 10 lL Fu-
GENE 6 solution was mixed with the cell suspension, and
the cells were cultured for 24 h at 37 °C.
Calcium signal studies on the FLIPR
TM
HEK293 ⁄ EBNA cells were seeded at a density of 5 · 10
3

cells per well in BiocoatÒ poly d-lysine-coated black-wall,
clear-bottom 96-well plates (Becton-Dickinson, Franklin
Lakes, NJ, USA) and allowed to attach overnight. Forty-
eight hours after transfection, the cells were washed two
times with HBSS ⁄ Hepes buffer (Hanks’ balanced salt solu-
tion without bicarbonate and phenol red, 20 mm Hepes,
pH 7.4) using a Laboratory Systems Cellwash plate washer.
After 45 min of dye-loading in the dark, using the calcium-
sensitive dye Fluo-4 AM at a final concentration of 2 lm,
the plates were washed four times with HBSS ⁄ Hepes buffer
to remove excess dye leaving 100 l L in each well. Plates
were re-equilibrated to 37 °C for a few minutes.
The cells were excited with an argon laser at 488 nm, and
emission was measured through a 510–570 nm bandwidth
emission filter (FLIPR
TM
, Molecular Devices, Sunnyvale,
CA, USA). Drug solution was added in a 50 lL volume to
each well to give the desired final concentration. The peak
increase in fluorescence intensity was recorded for each
well.
To generate concentration-response curves, angiotensin II
or PGF2a were tested in duplicate in a concentration range
between 10
)11
and 10
)5
m. The duplicate values were aver-
aged.
Western blotting analysis

RGS5-V5-pcDNA3.1 and RGS5s-V5-pcDNA3.1 were trans-
fected into HEK293 ⁄ EBNA cells. After 48 h, the transfected
cells were harvested and transferred to ice-cold lysis buffer
containing 30 mm Tris ⁄ Cl, 150 mm NaCl, 10% NP-40, 10%
glycerol, 0.5 mm EDTA, 0.5 mm phenylmethanesulfonyl
fluoride, 1 mm Na
3
VO
4
,40mm NaF, and incubated on ice
for 30 min. The cell lysate was then centrifuged at 13 000 g
for 10 min. The supernatant (total protein) was transferred
to new tubes, aliquoted and stored at )80 °C until the time
of electrophoresis. For membrane and cytosolic protein
isolation, the cells were homogenized in Tris ⁄ EDTA
(pH 7.4) buffer with Physcotron (Microtec Co., Funabashi,
Japan). The homogenates were then centrifuged at 36 000 g
for 30 min to obtain membrane and cytosolic fractions. Fif-
teen micrograms of each protein fraction (total, membrane,
Y. Liang et al. Alternative mRNA splicing variant of RGS5
FEBS Journal 272 (2005) 791–799 ª 2005 FEBS 797
cytosolic) were separated on 12% SDS ⁄ PAGE gels in Tris-
glycine, 0.1% SDS buffer, and transferred to poly(vinylidene
difluoride) membrane in NuPAGE transferring buffer at
130 V for 1 h. The blot was incubated for 2 h at room
temperature in 5% nonfat milk to block nonspecific binding.
The blot was then washed and incubated with anti-V5-HRP
IgG (Invitrogen, Inc., Carlsbad, CA, USA; 1 : 1000 dilution)
overnight at 4 °C, and then washed three times with NaCl ⁄ P
i

containing 0.1% Tween 20. Protein–antibody complexes
were visualized using ECL Western Blotting Detection Rea-
gents (Amersham, Inc., Piscataway, NJ, USA) following the
manufacturer’s protocol. The blot was exposed to Kodak
BioMax Light film (Kodak, Inc., Rochester, NY, USA) for
5 min.
Stimulation of MAP kinase phosphorylation
and immunoblots
HEK293 ⁄ EBNA cells were plated in six-well plates, and
transfected with human CB1, RGS5 and ⁄ or RGS5s expres-
sion plasmids. Forty-eight hours after transfection, the cells
were cultured in serum-free medium containing 0.1% bovine
serum albumin for 6 h, and then the cells were stimulated
with 10
)6
m WIN55212 for 10 min. The stimulation was ter-
minated by rapidly rinsing twice with ice-cold NaCl ⁄ P
i
.
Thereafter, the cells were lysed by adding RIPA buffer
(50 mm Tris ⁄ HCl pH 7.5, 1% Triton X-100, 0.1% deoxycho-
late, 150 mm NaCl, 1 mm sodium vanadate, 50 mm NaF,
2.5 mm sodium pyrophosphate, 1 m m b-glycerol phosphate
and protease inhibitors) and the cell lysates were immediately
scraped off the plates and transferred to a microfuge tube.
The cellular debris was removed by centrifugation at
10 000 g for 10 min, and the supernatant (total protein) was
transferred to new tubes, aliquoted and stored at )80 °C
until the time of electrophoresis. Fifteen micrograms of the
cell proteins were applied to SDS ⁄ PAGE, and the proteins

were transferred to nitrocellulose membranes. MAP kinase
activation was assayed by incubating nitrocellulose blots with
an antiserum that recognizes only the phosphorylated forms
of p42 and p44 MAP kinases. The control blots were also
probed with an antiserum recognizing only the unphosphor-
ylated forms of MAP kinases. The immunoreactive bands
were visualized by enhanced chemiluminescence using horse-
radish peroxidase-linked secondary antibodies. The blots
were exposed to Kodak BioMax Light film (Kodak, Inc.) for
5 min. The density of immunoreactive bands was determined
by Personal Densitometer SI (Molecular Devices).
References
1 Gilman AG (1987) G proteins: transducers of receptor-
generated signals. Annu Rev Biochem 56, 615–649.
2 Bourne HR, Sanders DA & McCormick F (1990) The
GTPase superfamily: a conserved switch for diverse cell
functions. Nature 348 , 125–132.
3 Simon MI, Strathmann MP & Gautam N (1991) Diver-
sity of G proteins in signal transduction. Science 252,
802–808.
4 Hamm HE (1998) The many faces of G protein signa-
ling. J Biol Chem 273, 669–672.
5 Neer EJ (1995) Heterotrimeric G proteins: organizers of
transmembrane signals. Cell 80, 249–257.
6 Dohlman HG & Thorner J (1997) RGS proteins and
signaling by heterotrimeric G proteins. J Biol Chem 272,
3871–3874.
7 Koelle MR & Horvitz HR (1996) EGL-10 regulates G
protein signaling in the C. elegans nervous system and
shares a conserved domain with many mammalian pro-

teins. Cell 84, 115–125.
8 Berman DM & Gilman AG (1998) Mammalian RGS
proteins: barbarians at the gate. J Biol Chem 273, 1269–
1272.
9 Seki N, Sugano S, Suzuki Y, Nakagawara A, Ohira M,
Muramatsu M, Saito T & Hori T (1998) Isolation, tis-
sue expression, and chromosomal assignment of human
RGS5, a novel G-protein signaling regulator gene.
J Hum Genet 43, 202–205.
10 Chen C, Zheng B, Han J & Lin S-C (1997) Characteri-
zation of a novel mammalian RGS protein that binds to
Galpha proteins and inhibits pheromone signaling in
yeast. J Biol Chem 272, 8679–8685.
11 Wang Q, Liu M, Mullah B, Siderovski DP & Neubig
RR (2002) Receptor-selective effects of endogenous
RGS3 and RGS5 to regulate mitogen-activated protein
kinase activation in rat vascular smooth muscle cells.
J Biol Chem 277, 24949–24958.
12 Zhou J, Moroi K, Nishiyama M, Usui H, Seki N, Ish-
ida J, Fukamizu A & Kimura S (2001) Characterization
of a novel C. elegans RGS protein with a C2 domain:
evidence for direct association between C2 domain and
Galphaq subunit. Life Sci 68, 1457–1469.
13 Liang Y, Li C, Burke J, Protzman C, Krauss A-H,
Nieves AL & Woodward DF (2002) Upregulation
of regulator of G protein signaling 5 (RGS5) in the
iris-ciliary body of chronic ocular hypertensive primates.
Invest Ophthalmol Vis Sci 43, 3387.
14 Watson N, Linder ME, Druey KM, Kehrl JH & Blumer
KJ (1996) RGS family members: GTPase-activating

proteins for heterotrimeric G-protein alpha-subunits.
Nature 383, 172–175.
15 Vries LD, Zheng B, Fisher T, Elenko E & Farquhar
MG (2000) The regulator of G protein signaling family.
Annu Rev Pharmacol Toxicol 40, 235–271.
16 Galve-Roperh I, Rueda D, Gomez del Pulgar T,
Velasco G & Guzman M (2002) Mechanism of extra-
cellular signal-regulated kinase activation by the
CB (1) cannabinoid receptor. Mol Pharmacol 62, 1385–
1392.
17 Chatterjee TK, Eapen AK & Fisher RA (1997) A trun-
cated form of RGS3 negatively regulates G protein-
Alternative mRNA splicing variant of RGS5 Y. Liang et al.
798 FEBS Journal 272 (2005) 791–799 ª 2005 FEBS
coupled receptor stimulation of adenylyl cyclase and
phosphoinositide phospholipase C. J Biol Chem 272,
15481–15487.
18 Mittmann C, Schuler C, Chung CH, Hoppner G, Nose
M, Kehrl JH & Wieland T (2001) Evidence for a short
form of RGS3 preferentially expressed in the human
heart. Naunyn-Schmiedeberg’s Arch Pharmacol 363,
456–463.
19 Saitoh O, Murata Y, Odagiri M, Itoh M, Itoh H, Mis-
aka T & Kubo Y (2002) Alternative splicing of RGS8
gene determines inhibitory function of receptor type-
specific Gq signaling. Proc Natl Acad Sci USA 99,
10138–10143.
20 Saitoh O, Masuho I, Terakawa I, Nomoto S, Asano T
& Kubo Y (2001) Regulator of G protein signaling 8
(RGS8) requires its NH2 terminus for subcellular locali-

zation and acute desensitization of G protein-gated K+
channels. J Biol Chem 276, 5052–5058.
21 Zhang K, Howes KA, He W, Bronson JD, Pettenati
MJ, Chen C, Palczewski K, Wensel TG & Baehr W
(1999) Structure, alternative splicing, and expression of
the human RGS9 gene. Gene 240, 23–34.
22 He W, Cowan CW & Wensel TG (1998) RGS9, a
GTPase accelerator for phototransduction. Neuron 20,
95–102.
23 Chatterjee TK & Fisher RA (2000) Novel alternative
splicing and nuclear localization of human RGS12 gene
products. J Biol Chem 275, 29660–29671.
24 Caceres JF & Kornblihtt AR (2002) Alternative spli-
cing: multiple control mechanisms and involvement in
human disease. Trends Genetic 18, 186–193.
25 Pick M, Flores-Flores C & Soreq H (2004) From brain
to blood: alternative splicing evidence for the cholingeric
basis of mammalian stress responses. Ann N Y Acad Sci
1018, 85–98.
26 Meshorer E, Toiber D, Zurel D, Sahly I, Dori A, Cag-
nano E, Schreiber I, Grisaru D, Tronche F & Soreq H
(YEAR?) Combinatorial complexity of 5’-alternative
acetycholinesterase transcripts and protein products.
J Biol Chem 279, 29740–29751.
27 Zhang Y, Neo SY, Han J, Yaw LP & Lin SC (1999)
RGS16 attenuates galphaq-dependent p38 mitogen-acti-
vated protein kinase activation by platelet-activating
factor. J Biol Chem 274 , 2851–2857.
28 Song Z-H & Slowey C-A (2000) Involvement of canna-
binoid receptors in the intraocular pressure-lowering

effects of WIN55212-2. J Pharmacol Exp Ther 292,
136–139.
29 Woldemussie E, Feldmann BJ & Chen J (1993) Charac-
terization of muscarinic receptors in cultured human iris
sphincter and ciliary smooth muscle cells. Exp Eye Res
56, 385–392.
Y. Liang et al. Alternative mRNA splicing variant of RGS5
FEBS Journal 272 (2005) 791–799 ª 2005 FEBS 799