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Alternative splicing of the G -coupled superfamily in human airway smooth muscle diversifies the complement of receptors

Richard Einstein*, Heather Jordan*, Weiyin Zhou*, Michael Brenner*, Esther G. Moses†, and Stephen B. Liggett†‡

*ExonHit Therapeutics, Inc., 217 Perry Parkway, Building 5, Gaithersburg, MD 20877; and †Department of Medicine, Cardiopulmonary Genomics Program, 20 Penn Street, HSF-2, Room S-112, University of Maryland, Baltimore, MD 21201

Communicated by Rita R. Colwell, University of Maryland, College Park, MD, February 8, 2008 (received for review October 27, 2007) G protein-coupled receptors (GPCRs) are the largest signaling heterogeneity in GPCR signaling has also been observed be- family in the genome, serve an expansive array of functions, and tween individuals that is not readily resolved by physiologic are targets for Ϸ50% of current therapeutics. In many tissues, such setting or disease state. This interindividual variability in GPCR as airway smooth muscle (ASM), complex, unexpected, or para- function, which may be responsible for disease risk or modifi- doxical responses to /antagonists occur without known cation and altered response to therapy, has led to the notion of mechanisms. We hypothesized that ASM express many more heterogeneity of either structure or expression of GPCRs within GPCRs than predicted, and that these undergo substantial alter- the human population. And indeed, quantitative measurements native splicing, creating a highly diversified receptor milieu. Tran- of GPCR expression and/or function in otherwise matched script arrays were designed detecting 434 GPCRs and their pre- individuals typically reveal as much as 10-fold variability (8). dicted splice variants. In this cell type, 353 GPCRs were detected Similarly, common polymorphisms of GPCRs with functional (including 111 orphans), with expression levels varying by Ϸ900- implications have been identified, which appear to associate with fold. Receptors used for treating airway disease were expressed some, but not all, of this variability (9). Another mechanism that lower than others with similar signaling properties, indicating introduces diversity of expression or function is alternative potentially more effective targets. A disproportionate number of mRNA splicing, giving rise to several ‘‘isoforms’’ of a receptor. Class-A -group receptors, and those coupling to Gq/11 or Gs The identification and characterization of alternatively spliced (vs. Gi), was found. Importantly, 192 GPCRs had, on average, five within the group of known and orphan GPCRs have not different expressed receptor isoforms because of splicing events, been delineated, so the extent of diversity imposed by this including alternative splice donors and acceptors, novel introns, mechanism within this important superfamily of signaling mod- intron retentions, exon(s) skips, and novel exons, with the latter ules is unknown. two events being most prevalent. The consequences of splicing In the airways, GPCRs expressed on smooth muscle regulate were further investigated with the , known airway contraction and relaxation and have been extensively for its aberrant responsiveness in lung. We found transcript ex- studied in regard to asthma pathogenesis and treatment (10). pression of three variants because of alternative donor and accep- Local increases in endogenous agonists acting at histamine, tor splice sites, representing in-frame deletions of 38 and 100 aa, leukotriene, prostaglandin, muscarinic, thromboxane, and other with protein expression of all three isoforms. Thus, alternative GPCRs result in markedly enhanced bronchial hyperresponsive- splicing, subject to conditional, temporal, and cell-type regulation, ness to contraction, leading to airway constriction. Other recep- is a major mechanism that diversifies the GPCR superfamily, cre- tors, such as the ␤2- (␤2AR), appear to ating local recepteromes with specialized environments. oppose constriction or evoke relaxation (10), and thus airway ͉ ͉ smooth muscle (ASM) GPCRs have represented important pharmacogenetics signal transduction splice variants targets for therapeutic agents, both agonists and antagonists, in asthma treatment. Nevertheless, a large body of pharmacologic protein-coupled receptors (GPCRs) represent the largest data reveals complex, contradictory, or paradoxical responses of Gsuperfamily of receptors in the . They are ASM GPCRs, bringing into question the role of certain receptors expressed on every cell and carry out an extensive array of or pathways in smooth muscle pathophysiology and the potential biologic signaling. These include receptors for vision and smell, utility of novel drugs targeting members of this superfamily. We neurotransmission, and thousands of endocrine, autocrine and hypothesized that ASM expresses many more GPCRs than paracrine functions throughout the body. Critical GPCR circuits predicted, and that these undergo frequent alternative splicing, have been identified in , cardiovascular, resulting in unexpected receptor isoforms and thus creating a gastrointestinal, pulmonary, endocrine, and renal disease (re- highly diversified receptor milieu. viewed in ref. 1), resulting in enhanced understanding of the pathologic basis of disease, new diagnostics, and new drug Results Ϸ targets. Indeed, it has been estimated that 50% of pharma- Complement of Expressed GPCRs from Human ASM. To identify ceuticals are directed at GPCRs or their immediate downstream expression of the GPCR superfamily in ASM cells, RNA was signaling events (2, 3). There are several well recognized phe- nomena in GPCR signaling events, at both the in vitro and clinical levels, which have revealed a complexity beyond that Author contributions: R.E. and S.B.L. designed research; H.J., M.B., E.G.M., and S.B.L. initially expected. One issue has been the recognition that one performed research; R.E. and S.B.L. contributed new reagents/analytic tools; R.E., W.Z., receptor can regulate another through any number of heterol- M.B., E.G.M., and S.B.L. analyzed data; and R.E., H.J., and S.B.L. wrote the paper. ogous mechanisms (4, 5). This ‘‘cross-talk’’ appears to be nec- The authors declare no conflict of interest. essary for the cell to integrate the large number of GPCR Freely available online through the PNAS open access option. signaling events underway at any given time (6, 7). Thus, the ‡To whom correspondence should be addressed. E-mail: [email protected]. complement of receptors expressed on a cell, even those that do This article contains supporting information online at www.pnas.org/cgi/content/full/ not appear to be directly involved with a pathway, can be a 0801319105/DCSupplemental. determinant of the cellular response to a given GPCR. Extensive © 2008 by The National Academy of Sciences of the USA

5230–5235 ͉ PNAS ͉ April 1, 2008 ͉ vol. 105 ͉ no. 13 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801319105 Downloaded by guest on September 27, 2021 Fig. 2. Distribution of GPCR splice variants detected in human ASM cells. The number of splice events detected in human ASM cells is shown by event type. The six types of alternative splicing events considered are defined in Methods Fig. 1. Distribution of Gs-, Gq/11-, and Gi/Go-coupled GPCRs stratified by and summarized in Fig. S1. See Dataset S2 for results from specific genes and relative expression in human ASM cells. Data represent mean expression of the Dataset S3 for detailed splice-event annotation. unspliced (‘‘wild-type’’) transcripts from cells derived from five individuals stratified by quartiles. coupling was predicted by an algo- rithm as described in Methods. The diversity of GPCR expression is further illustrated by representative genes expressed in each quartile assembled by Fig. 1. Gq/11- and Gs-coupled receptors were the most prevalent GPCR Class (A, -like; B, -like) and group (amine, in human ASM (Fig. 1). This appears to be independent of the peptide, or other). names in black indicate disputed or dual G protein level of receptor expression, because the number of detected coupling. See Dataset S1 for expression results for specific GPCR genes. receptors which couple to Gq/11 and Gs is similar in all expression quartiles, and greater than the number of detected Gi/o-coupled receptors. isolated and analyzed by using expression microarrays that provide for resolution of transcripts produced from the same Diversity of GPCR Expression Due to Alternative Splicing in Human by alternative splicing. The overall expression level per ASM. The number of GPCRs with potential splice events that GPCR can be determined by using probes monitoring the classic were identified in silico amounted to 252 of the 434 GPCR genes, ‘‘nonspliced’’ form. The arrays used here contain probes that representing a total of 1,500 events. Probe sets on the array were monitor a total of 434 GPCR genes. The expression results from designed to detect each specific splicing event using the strategy cultured ASM cells derived from five individuals are provided in depicted in Fig. S1. Alternative splicing events of GPCR genes supporting information (SI) Dataset S1. A total of 353 GPCR detected in human ASM are shown in Fig. 2 classified by event genes had statistically significant expression levels, defined as a type. The data from individual GPCR splicing events from these 2-fold signal over background, and all such signals had Bonfer- Ϫ cells are provided by sample in Dataset S2. A specific variant can roni-corrected P values of Ͻ10 4. Of interest, because they are be further assessed as to event-type, location within the gene of targets for current pharmacologic therapy in obstructive lung the splice event(s), and splice-site scores from the frequency disease, are the M3-muscarinic receptor (CHRM3) and ␤2- ␤ matrix (12) in Dataset S3. Of the 252 potentially alternatively adrenergic receptor ( 2AR, ADRB2). Knowledge of expression MEDICAL SCIENCES of these ‘‘benchmark’’ therapeutic targets places into perspective spliced GPCR genes, 192 had one (or more) alternative isoforms, expression results from other GPCRs, because they may relate with a total of 967 events detected. Exons and exon(s) skip events to function or suitability as new drug targets. Antagonists to the were the most common in ASM (Fig. 2). Of note, these two former receptor are potent blockers of bronchoconstriction, categories should be considered similar, because the difference whereas agonists acting at the ␤ AR are the most efficacious lies in which sequence has been selected as the reference (Fig. 2 Ϸ Ϸ bronchodilators known. Yet, both of these were among the S1). Of the 600 potential novel exons, 50% were expressed lower-expressing GPCRs in ASM (Ϸ100-fold lower than the in ASM, which represented the smallest percentage of detected highest expressors), suggesting an unrecognized complement of vs. probed events. A somewhat higher percentage was found for receptors in this cell type that may be involved in pathogenesis exon skip, alternative splice donor, and alternative splice accep- of airway obstruction or may be new therapeutic targets. Among tor events, and particularly so for the less-common novel intron all significantly expressed GPCRs, expression varied by as much and intron retention events. There was no apparent relationship as Ϸ900-fold. Of the 353 receptors that were expressed, 111 were between ‘‘wild-type’’ expression levels and the occurrence of a orphan GPCRs. Receptors were stratified by class (11) and by particular splicing event. That is, these events occurred regard- the major G to which they couple based on published less of the degree of overall expression of a given GPCR gene. studies or by modeling techniques. As expected, the expressed GPCRs were dominated by members of Class A, which ac- Three Expressed Forms of the Leukotriene B4 Receptor Due to Alter- counted for 72% of the nonorphan receptors. Within this class, native Splicing. A select group of 30 GPCRs was chosen for the group of peptide receptors was most prevalent (60%), further study based on high, intermediate, and low levels of followed by the amine receptors (22%). The remainder of the expression, and their known relevance to ASM signal transduc- Class A receptors were primarily of the hormone, prostanoid, tion (Table 1). Overall expression levels within this group of and nucleotide families. The majority of non-Class A receptors genes (taking into account all variants) differed by up to were Class B secretin-like receptors. Examples of the heteroge- Ϸ400-fold. Of the 30 genes selected for further analysis, 13 had neity of GPCRs expressed in human ASM and the relationship no evidence of alternative splicing, whereas the remaining 17 between G protein-coupling and expression level are shown in genes contained 70 splicing events. Of these, 39 events were

Einstein et al. PNAS ͉ April 1, 2008 ͉ vol. 105 ͉ no. 13 ͉ 5231 Downloaded by guest on September 27, 2021 Table 1. Expression levels of selected GPCR transcripts from airway smooth muscle GenBank accession no. Gene annotation Mean signal Mean BG P value

NM࿝001295 Chemokine (C-C motif) receptor 1* 198216 423 1.21E-21 NM࿝000676 * 44122 418 1.61E-08 NM࿝002030 -like 2* 13310 415 1.01E-16 NM࿝002511 receptor* 11395 410 3.35E-18 NM࿝001504 Chemokine (C-X-C motif) receptor 3 8877 415 4.11E-18 NM࿝006564 Chemokine (C-X-C motif) receptor 6* 7400 414 1.24E-17 NM࿝000025 Adrenergic, beta-3-, receptor* 6124 410 5.66E-13 NM࿝000738 receptor, muscarinic 1 5324 411 2.10E-14 NM࿝000955 Prostaglandin E receptor 1 (subtype EP1), 42kDa 5052 416 4.71E-16 NM࿝000956 Prostaglandin E receptor 2 (subtype EP2), 53kDa* 4444 424 2.25E-09 NM࿝000677 4182 414 2.75E-11 NM࿝005508 Chemokine (C-C motif) receptor 4* 3676 421 5.23E-16 NM࿝175057 Trace amine receptor 3 (TAR3), mRNA* 3671 421 3.35E-16 NM࿝000710 B1* 3023 413 3.27E-12 NM࿝001059 3 2966 420 4.28E-16 NM࿝000675 2288 417 4.24E-14 NM࿝000797 receptor D4 2111 422 4.14E-13 NM࿝000798 D5 1955 413 1.96E-13 NM࿝006639 Cysteinyl 1 1631 430 5.56E-10 NM࿝000959 (FP) 1483 419 2.99E-08 NM࿝003382 Vasoactive intestinal peptide receptor 2 1452 415 2.46E-11 NM࿝001841 2 (macrophage)* 1421 417 1.71E-12 NM࿝000960 Prostaglandin I2 (prostacyclin) receptor (IP) 1105 411 1.12E-11 NM࿝181657 Leukotriene B4 receptor 1079 417 2.07E-11 NM࿝000739 Cholinergic receptor, muscarinic 2 1064 417 5.26E-07 NM࿝012125 Cholinergic receptor, muscarinic 5 969 413 2.24E-10 NM࿝000674 965 411 4.28E-08 NM࿝000794 * 927 423 1.01E-10 NM࿝019839 Leukotriene B4 receptor 2 919 412 4.01E-10 NM࿝020377 Cysteinyl leukotriene receptor 2* 913 413 2.17E-10

Genes marked with an asterisk showed no evidence of splicing; mean signal and mean BG (background) represent intensity dye-normalized values, which were calculated as described in Methods. P values are Bonferroni corrected for the number of comparisons.

identified where novel exons or exon extension events were were pooled, and bands were purified, cloned, and sequenced to present and led to additional sequence in the alternatively verify the sequence structure for each variant. Slightly different spliced transcripts. Thirty-eight of these events were found to be variants were found in the 5ЈUTR but generated sequences that significantly expressed above background, whereas one event had exactly the same coding region as the reference sequence. was not statistically significant. In contrast, 31 events were found Two additional variants contained a deletion of part of the to consist of exon skip events or exon truncations, where splicing 5ЈUTR and 5Ј-coding sequence (Fig. 3); the junction sequences activity led to a loss of sequence. Twenty-four of these events were found to be significantly above background, whereas seven were not found to be expressed. From this set of expressed events, 17 were selected for validation by RT-PCR. The final list represented a variety of splice events (alternative donor and acceptor sites, novel introns, and novel and skipped exons), with multiple categories of supporting evidence for the event (se- quences from mRNAs, ESTs, and RefSeqs). Primers were specifically designed for capturing spliced isoforms or were specifically targeted to amplify only the longer isoform (Fig. S2 and Table S1). Data from the high-affinity leukotriene B4 receptor (13) (LTB4R, also denoted LTB4R1 and BLT1, Gen- Bank accession no. NM࿝181657) showed several potential splic- ing events (Table 2, Fig. 3) and was further explored to elucidate the ramifications of this diversification mechanism with a rele- vant signaling pathway in ASM. LTB4R has four splice events that were monitored on the array, and two were unequivocal, as determined from RT-PCR followed by sequencing. The two Fig. 3. Genomic organization and identification of potential splice variants validated events, the alternative donor and acceptor sites, are in the LTB4R gene. A schematic diagram for LTB4R is illustrated approximately illustrated in Fig. S2. Both events were identified as part of the to scale to indicate the exons and introns for the reference LTB4R gene and three splice variants that were detected. The location of RT-PCR primers is same transcript, so primers were designed to amplify the variant indicated and was used to isolate amplicons that verified the sequences for and the reference form of the transcript (primers are indicated each variant. The sequences were aligned to the genome and UTR and coding in Fig. 3; F19 and R19 amplify both the reference and the variant, regions are indicated. LTB4R-AS1 has lost 39 aa internal of the coding region, whereas F20 and R20 amplify only the reference). Amplicons whereas LTB4R-AS2 lost the initial 100 N-terminal aa.

5232 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801319105 Einstein et al. Downloaded by guest on September 27, 2021 Fig. 4. Nucleotide and translated sequence of LTB4R and the two splice variants. In A, sequence electropherograms from the RT-PCR products representing the indicated receptors are shown, with the point of deviation from wild-type LTB4R indicated by the arrows. In B, translated sequences are shown. The full-length sequence represents wild-type LTB4R, the bracketed red sequence represents amino acids absent in the LTB4R-AS1, and the entire red sequences represent the amino acids absent in LTB4R-AS2. *, ϩ, and # indicate the specific splice sites in the DNA (A) or (B) sequence.

for both of these variants were identical (Fig. 4A). This resulted (LTB4R-AS2). The wild-type and -AS2 forms were expressed at in a translated protein lacking the first 100 aa (LTB4R-AS2 in approximately equivalent levels, whereas the -AS1 form was Fig. 4B) compared with the reference protein. Another variant Ϸ10-fold lower in expression compared with either of the other was found (Fig. 3, clones A03 and A06), with alterations in the two. coding region distinct from the previous clones. The distinct junction for this clone was identified and revealed the loss of a Discussion portion of the second exon. This loss from alternative splicing is In the human genome, typical pre-mRNAs consist (on average) in-frame and results in the exclusion of amino acids 43–81 of the of 8 introns and 9 exons, spanning Ϸ27,000 nt (14). RNA splicing protein (LTB4R-AS1, Fig. 4B). at intron–exon boundaries provides for deletion of introns, Transmembrane modeling (Fig. 5 A–C) reveals that LTB4R- resulting in mature exon sequence. Such splicing, performed by AS1 lacks the second transmembrane domain and part of the 5Ј ribonucleoprotein complexes (spliceosomes), is subject to con- portion of the first extracellular loop. LTB4R-AS2 lacks the N ditional, temporal, and cell-type-specific regulation, resulting in terminus, first transmembrane domain, first intracellular loop, alternatively spliced transcripts and thus various isoforms of the second transmembrane domain, first extracellular loop, and Ϸ8 expressed protein (reviewed in refs. 15–17). Alternative splicing aa from the 5Ј portion of the third transmembrane domain. To is one mechanism by which the number of expressed proteins is ascertain whether the LTB4R-AS1 and -AS2 receptor proteins, thought to exceed the number of genes in the human genome. which lack 39 and 100 aa, are expressed on human ASM cells, Thus, considerable structural diversity of expressed proteins can Western blots were carried out with a polyclonal antibody be realized from alternative splicing of a single gene. In the directed to a peptide representing the third intracellular loop of current work, we identified and characterized the splice variants the human LTB4R. As shown in Fig. 5D, the full-length receptor of the GPCR superfamily because (i) it represents the largest was identified at its predicted molecular mass of Ϸ38 kDa. Two family of signaling proteins in the genome, (ii) these receptors other forms were also identified at the predicted molecular mass play a significant role in normal homeostasis and pathophysiol- Ϸ Ϸ of the two shorter forms at 33 kDa (LTB4R-AS1) and 27 kDa ogy, and (iii) extensive efforts are underway to design agonists MEDICAL SCIENCES

Fig. 5. Predicted topology of LTB4R and the two splice variants. Transmembrane-spanning domains and the extracellular and intracellular loops are depicted, with the x axis representing the amino acid number (A, LTB4R; B, LTB4R-AS1; C, LTB4R-AS2). The probabilities (P) were calculated by using the program as described in Methods. Representative Western blot (D) shows the three isoforms expressed in ASM at the predicted molecular mass of the receptors at 37.5 (full length), 33.2 (LTB4R-AS1), and 26.9 kDa (LTB4R-AS2). Results are from cells derived from two individuals.

Einstein et al. PNAS ͉ April 1, 2008 ͉ vol. 105 ͉ no. 13 ͉ 5233 Downloaded by guest on September 27, 2021 and antagonists for pharmacologic therapy. Genes encoding the contraction mediated by the Gq-coupled M3-muscarinic receptor majority of known and orphan GPCRs (not including those for (23). Other reports, though, have shown significant contraction smell or sight) were considered, and the cell-type interrogated of lung parenchymal strips by nanomolar concentrations of LTB4 was human ASM cells. This cell type was chosen because of its (24). Some studies have concluded that LTB4 antagonists have central role in the pathogenesis of airway obstruction in asthma minor effects on leukocyte accumulation but decrease bronchial and the known actions of GPCRs in ASM contraction, relax- hyperreactivity (25, 26), another showed decreased leukocyte ation, and proliferation. recruitment but no effect on pulmonary function (27), and Of the 434 GPCRs monitored on the splice variant-specific another found that antigen-induced bronchoconstriction was array, 353 were detected over a wide range of expression levels decreased but eosinophil accumulation and acute bronchial in this cell type. This number of GPCRs is much greater than the hyperreactivity were unaffected (28). Ϸ50 that were estimated to be expressed on human ASM based The discrepant bronchoconstriction effects from LTB4R an- on evidence from pharmacologic and limited expression studies tagonists may be due to the presence of splice variants of the (10). The several hundred GPCRs with relatively low expression receptor in ASM. We show here three forms of the transcribed levels should not be considered biologically insignificant, be- LTB4R and two additional variations in the 5ЈUTR, which may cause this group includes the M3-muscarinic receptor, the ␤2AR, alter the rate of transcription. The two splice variants involving and receptors for leukotrienes and prostanoids, all of which are the coding region lack domains that are likely necessary for LTB4 important in the pathogenesis of obstructive lung disease or binding to the receptor. However, both have intact G protein receptor-targeted therapy. Surveying the wild-type isoform ex- coupling domains (second and third intracellular loops) and thus pression levels of the Gq-coupled receptors, which mediate may act as dominant-negative proteins during wild-type LTB4R bronchoconstriction, we noted expression of Ϸ150 receptors, signaling. Of note, traditional transcript-detection methods extensive variation in levels of expression, and diversification via would have failed to identify the two alternatively spliced LTB4R splice variants. These findings indicate potential new targets for transcripts, giving a false impression of the level of expression of antagonists and potential complexities because of multiple iso- the full-length receptor. Given that LTB4R-AS2 protein is forms. For example, the CysLT1 receptor is currently a target for expressed at approximately the same level as wild-type LTB4R, antagonists such as montelukast in the treatment of asthma. functional LTB4R expression is at least Ϸ1/2 that expected. And, However, the orphan receptor GPCR 18, also predicted to be if the splice variants act as dominant negatives, functional Gq-coupled, was shown here to be expressed Ϸ4-fold higher than expression could be substantially lower or perhaps trivial. De- CysLT1 in ASM (Dataset S1) and may be an efficacious target velopment of LTB4R antagonists directed at the smooth muscle for therapy, assuming that local levels of its endogenous receptor would thus appear to have a low probability of thera- [N-arachidonylglycine (18)] are increased in asthma. Similarly, peutic efficacy. ␤ ␤ the 2AR,aGs-coupled receptor which is the target for -ago- Taken together, the current results reveal an unexpectedly nists used to dilate the airways during bronchospasm, is ex- large number of receptors in the GPCR superfamily expressed pressed Ϸ25-fold lower than the orphan receptor GPCR 101, in human ASM and extensive variability in structure because of which is also predicted to be Gs coupled. Thus, agonists to GPCR alternative splicing leading to many receptor isoforms. The 101 could be more effective bronchodilators than ␤-agonists, or utility of these findings was explored in the LTB4R gene, where these agents could be coadministered to achieve maximal bron- splicing events resulted in two previously unrecognized isoforms, chodilation. Interestingly, GPCR 101 does not have the two which may explain discrepant results with antagonists for treat- consensus sequences for protein kinase A ing asthma. In regard to the superfamily, alternative splice ␤ found in the 2AR, which contributes to -promoted donors and acceptors, skipped exons, novel exons, and novel and desensitization of this receptor (19). Thus, agonists for GPCR retained introns were all observed, leading to marked expansion 101 may show less tachyphylaxis than ␤-agonists. Of the 353 of this signaling modality. Given that GPCRs are expressed on GPCRs expressed on ASM, nearly two-thirds had in silico every cell type in the body and play prominent roles in signaling evidence for the potential to be alternatively spliced. Of these, events in many diseases, consideration of this previously unrec- 192 were found to express more than one isoform in this cell type, ognized diversity is necessary for understanding GPCR-related with a total of 967 splicing events detected in the superfamily. Of pathophysiology and therapeutic targets. the spliced GPCRs, then, an average of approximately five different isoforms per receptor were expressed in ASM. This Methods represents substantial diversity in a cell type where new phar- Bioinformatic Identification of Potential GPCR Splice Variants. An initial list of macologic agents are needed to control bronchospasm in asthma 434 GPCR gene representatives was determined by processing a set of nonviral and chronic obstructive lung disease. and nonolfactory GPCR sequences obtained from public databases to a non- We chose the LTB4R for additional investigation because of redundant set through genomic position (by alignment) clustering. These 434 its role in asthma and to emphasize the potential impact of splice representatives were compared with the exon structure of overlapping align- ments at the same locus of the human genome in silico. variants on pathophysiology and treatment strategies. LTB4, like a number of other arachidonic acid metabolites, is increased in Sequences were aligned to the human genome (build 33) using BLAT (29). RefSeq sequences met the criteria of 97% alignment identity and a minimum inflammatory states such as asthma, with elevated local levels in BLAT score of 200; in addition, EST alignments also had to include at least one the airway (20). The high-affinity receptor for LTB4, LTB4R, is splice junction, thereby having more than one exon, and Ͼ70% of the total well recognized as being expressed on neutrophils and other sequence had to be represented. Orientation of the EST alignment and leukocytes (eosinophils, T-lymphocytes) and, when activated by sequence was determined by comparison of consensus splice site scores of all its endogenous agonist, promotes leukocyte migration to the splice junctions for the sequence performed in both sequence orientations. lung and participates in activation and degranulation (21). To Overlapping sequences were compared by using genomic mapping coordi- our knowledge, LTB4R expression on human ASM cells has not nates, and differences in sequence content were determined through math- been previously identified. In recombinant expression systems, ematical differences in the derived coordinates. Each event was given a unique identification number. For example, 106.008.001 indicates an event the receptor has been shown to couple to both G␣ and G␣ (22). i q found in the 106th locus as found in the original gene list; the alignment was ␣ Given that G q-coupled receptors act to constrict ASM, this the eighth variant sequence checked in the locus, and this event was the first suggests that LTB4 could evoke bronchoconstriction directly, (001) event found in that variant sequence. Probe names consist of the event rather than via leukocyte recruitment. In guinea pig trachea, number with an underscore followed by a letter to indicate whether the probe LTB4 alone has been reported to have no effect on isometric is a common probe (A5 or A3), a long-form exon probe (B), a long-form contraction, but significantly enhances acetylcholine promoted junction probe (C or D), or a short-form junction probe (E) (Fig. S1). Splice-site

5234 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801319105 Einstein et al. Downloaded by guest on September 27, 2021 scores were calculated by using a frequency matrix from the AltExtron Data- using the default settings. Dataset S2 contains probe level information from base (www.ebi.ac.uk/asd/altextron/data/splice࿝site࿝dists.html) (12). all 10 hybridizations (five samples processed in duplicate). Intensity dye- normalized values and processed background values are given for each probe Probe Design and Microarrays for Splice Variant Identification. A set of probes (see Data Analysis and Modeling for details). was synthesized for each event identified bioinformatically and was designed to obtain a common thermodynamic profile as described (30). Probes directed Data Analysis and Modeling. The mean signal and background values from the against exon–exon junctions were limited to 24 bases to limit cross-reactivity output files were used to produce datasets that were compared to determine from hybridization of half the junction probe to a single exon (30). The probes significantly expressed over background. The dye normalization factor microarrays were manufactured by Agilent Technologies with each probe was calculated from values in the file for each probe/sample combination and printed in duplicate and randomly distributed on the microarray slide. Primary multiplied by the background and mean signals to produce dye normalized human ASM (bronchial) cells derived from five individuals were obtained from values. To calculate gene level expression values, the median value from all Clonetics and were maintained in monolayers in SMBM media (Clonetics). ‘‘A’’ probes for each gene or locus was calculated (A1 and A2 were used for Cells at passages 3 or 4 were detached and total RNA prepared. (We are genes without splice events, A3 and A5 probes for genes with splice events, unaware of data suggesting that short-term culture alters the fundamental Dataset S1). The median data values were log2 transformed, and a two-way processes of splicing, but overall expression levels of genes, including GPCRs, ANOVA model was performed by using signal type (signal vs. background) and could be affected by culture conditions.) Five micrograms of total RNA from dye as a factor to generate Bonferroni-corrected P values for all probes to each sample was labeled by using a random primed labeling protocol de- determine probes that were expressed significantly above background. Re- scribed by Xiang (31) with the following modifications: the final concentra- ceptors were classified by their signaling to the three major classes of G tions of dTTP and aadUTP were 250 ␮M, instead of 300 and 200 ␮M, respec- protein: Gi/o,Gs, and Gq/11, using the International Union of Pharmacology tively; the first-strand synthesis was not stopped with EDTA but directly database (32) and literature searches. Predicted G protein-coupling specificity hydrolyzed for 10 min at 65°C; after neutralization, the labeled material was for unclassified orphan receptors was determined by a support vector machine purified by using the Rapid PCR purification system (Marligen) and eluted and hidden Markov model using the program GRIFFIN (33). Transmembrane twice with 50 ␮l of each of RNase-free water preheated to 65°C. For hybrid- spanning predictions were made by the TMHMM Server v2.0 (www.cbs. ization, sets of dye-swap experiments were performed with each sample using dtu.dk). 2.5 ␮g each of Cy3- and Cy5-labeled cDNA along with 40 ␮g of herring sperm DNA (Invitrogen). The mixture was heated at 98°C for 3 min and then brought Western Blots. To detect expression of the LTB4R isoforms, a polyclonal to room temperature for 5 min in the presence of 25 ␮lof10ϫ Control Targets antibody (Sigma) directed against a peptide representing the third intracel- from an in situ hybridization kit (Agilent Technologies). After addition of the lular loop of LTB4R was used in Western blots from whole-cell ASM lysates. 2ϫ hybridization buffer, the mixture was hybridized to the GPCR arrays at Studies were performed with 40 ␮g of protein and a primary antibody titer of 65°C for 17 h. The arrays were washed by using a 60-mer oligo microarray 1:1,000 using methods described (4). processing protocol (Agilent), with washes of 5 min each. The arrays were scanned by using an Agilent Microarray Scanner. The images were analyzed ACKNOWLEDGMENTS. This work was supported by National Institutes of with the Feature Extraction software, version 8.1 (Agilent Technologies), Health Grants HL071609 and HL065899 (to S.B.L.).

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