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: 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 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. 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