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

Using RGS5 gene-specific RT-PCR, we have identified a novel alternative splicing variant of RGS5 mRNA in human ocular tissues.. Cotransfection of RGS5s with RGS5 resulted in the blockade o

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: Liang_Yanbin@Allergan.com

(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 RGS5gene 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 Ca2+ 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.

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

Glau coma

E ye

Huma

n

ye

RGS5 RGS5s

CSM NoRT TM NoRT ODM NoRT Human Ocular Tissue

Monkey

RGS 5

C

ar

y B

o d

y

R

in a

R

E ye

Rat RGS5

H u

m an R

in a

RGS5 RGS5s A

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 prodprod-uct (alter-native splicing of RGS5) CSM, clilary smooth muscle; TM, trabecular meshwork; ODM, a human NPE cell line; NoRT, no reverse transcription control.

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 Ussuggest-ing

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

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).

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 Ca2+ 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 Ca2+ signal response to angiotensin II- and PGF2a (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 Ca2+signaling, suggesting that RGS5s may

RGS5

RGS5s

Li

r

K idney Br

ai n

Smal

l intestine

Splee

n

L ung Skele

ta l

M uscl e

Hear t

Re

ti 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

FP+vector FP+RGS5+RGS5s FP+RGS5

FP+RGS5s

Concentration ( Log M)

* * * *

* ** **

0 1000 2000 3000 4000 5000 6000 7000 8000

AT1+vector AT1+RGS5+RGS5s AT1+RGS5

AT1+RGS5s

Concentration ( Log M)

*

A

B

Fig 6 Effects of RGS5 and RGS5s on Ca2+ 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 PGF2aat con-centrations ranging from 10)12M to 10)5M 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

31 kDa

52 kDa

98 kDa

RGS5

RGS5s

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.

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 EP2receptor) 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 EP2 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 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)6M 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.

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 Ca2+mechanisms, and showed for the

first time that overexpression of RGS5 selectively

antag-onized prostaglandin FP receptor mediated Ca2+

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% CO2, 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% CO2, 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 Receptor GDP

Pi

RGS5

βγ

Signal

Gα

GDP

(-) RGS5s

G αq

GTP

βγ G αi

GTP

(-)

Fig 8 Diagrammatic representation of RGS5 and RGS5s involved

in the mechanisms of G protein and G protein-coupled receptors.

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 lg of

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:

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· 103

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 FLIPRTM

HEK293⁄ EBNA cells were seeded at a density of 5 · 103 cells per well in Biocoat 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 lL 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 (FLIPRTM, 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)11and 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 Na3VO4, 40 mm 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,

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 ⁄ Pi

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)6mWIN55212 for 10 min The stimulation was

ter-minated by rapidly rinsing twice with ice-cold NaCl⁄ Pi

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 mm 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-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 desensitilocali-zation 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