• The most prevalent signaling pathway in the body and largest superfamily in the human genome

• Examples include:VisionSmellNeurotransmissionEndocrineParacrineOrphan

• 50% of all prescribed drugs act at GPCRs:Histamine type 1 receptor antagonistsHistamine type 2 receptor antagonistsβ1-adrenergic receptor antagonistsβ1-adrenergic receptor agonistsβ2-adrenergic receptor agonistsCysteinyl leukotriene antagonistsM3-muscarinic antagonistsα2-adrenergic receptor agonistsAngiotensin II receptor antagonists

G-Protein Coupled Receptors

TM1TM7

amino-terminus

carboxy-terminus

ECL-1ECL-2

ECL-3

ICL-1ICL-2

ICL-3“ICL-4”

Topography of GPCRs

extracellular

intracellular

transmembrane

GPCR Signaling

αs βγG-protein

αs stimulates adenylyl cyclase

αi inhibits adenylyl cyclase

αq/11 stimulates phospholipase C

Variability in GPCR Function

• Diseases known to involve GPCR signaling (heart failure, hypertension, asthma, depression) show heterogeneity in risk, severity, and progression

• Response to administered agonists and antagonists in treatment of such diseases shows marked interindividual variability

• Atypical or paradoxical responses to GPCR agonists and antagonists are common in drug development and early clinical trials

• Potential basis of GPCR-response variabilityPolymorphisms of the genesAlternative splicing which results in expression of multiple receptor “isoforms” with different properties

RNA Splicing Mediates Gene Expression Diversity

A B

Isoform AFunctionA

Isoform BFunction B

~25,000 genes

>100,000 unique mRNAs

conditionalspatialtemporaldisease

Why are Microarrays that Detect Splicing Events Necessary?

RNA Splicing is critical for generating a diverse proteome from a limited set of genes– <30K genes leading to >100K transcripts!– 60-90% of all genes estimated to exhibit alternative splicing– 70% of the isoforms from genes on chromosomes 19 and 22

have altered protein coding sequence, which could lead to altered function

RNA Splicing is altered in human diseases– The Human Gene Mutation Database currently lists 4182

mutations that disrupt consensus splice sites [Stenson et al (2003), The Human Gene Mutation Database (HGMD®): 2003 Update. Hum Mutat (2003) 21:577-581]

Human Airway Smooth Muscle (HASM) GPCRs

• We had previously estimated that ~25 GPCRs are expressed on human airway smooth muscle

• Examples include:M3-muscarinic – constricts airwayβ2AR – bronchodilates airwayProstaglandin receptorsLeukotriene receptors

• HypothesisHuman airway smooth muscle express many more GPCRs than predicted based on classic pharmacologyThese undergo frequent alternative splicing events leading to a highly diversified receptor milieu

highly variable

Primary HASM Cells in Culture from 5 Individuals

RNA

ExonHit Human GPCR SpliceArrays• Designed by ExonHit using eArray (Agilent Technologies)

• Slides printed by Agilent Technologies

1. What is the complement of GPCRs expressed in HASM?

2. What are the relative (hierarchical) expression levels of these GPCRs?

3. How many isoforms from alternative splicing are expressed?

4. What are the structural consequences of alternative splicing in selected GPCRs?

ExonHit Human GPCR SpliceArray

• 441 GPCR genes

• Bioinformatics (ESTs, mRNAs, RefSeqs) indicates the potential for 256 genes to be alternatively spliced

• Probes designed to detect each potential splice variantAlternative splice donorsAlternative splice acceptorsExon(s) skipsNovel exonsIntron retentionsNovel introns

Results – GPCRs expressed on HASM

• Transcripts representing 353 different GPCRs detected (many more receptors than predicted)

• Expression varied by ~900-fold

• The “benchmark” GPCRs β2AR and M3R were not among the higher expressing GPCRs (lower 50th percentile)

• 111 orphan GPCRs

Distribution of GPCRs Expressed in HASM by Class and G-protein Pathway

Reference Gene

Accession Gene AnnotationMean Signal

Mean BG P-value

NM_001295 chemokine (C-C motif) receptor 1* 198216 423 1.21E-21NM_000676 adenosine A2b receptor* 44122 418 1.61E-08NM_002030 formyl peptide receptor-like 2* 13310 415 1.01E-16NM_002511 neuromedin B receptor* 11395 410 3.35E-18NM_001504 chemokine (C-X-C motif) receptor 3 8877 415 4.11E-18NM_006564 chemokine (C-X-C motif) receptor 6* 7400 414 1.24E-17NM_000025 adrenergic, beta-3-, receptor* 6124 410 5.66E-13NM_000738 cholinergic receptor, muscarinic 1 5324 411 2.10E-14NM_000955 prostaglandin E receptor 1 (subtype EP1), 42kDa 5052 416 4.71E-16NM_000956 prostaglandin E receptor 2 (subtype EP2), 53kDa* 4444 424 2.25E-09NM_000677 adenosine A3 receptor 4182 414 2.75E-11NM_005508 chemokine (C-C motif) receptor 4* 3676 421 5.23E-16NM_175057 trace amine receptor 3 (TAR3), mRNA* 3671 421 3.35E-16NM_000710 bradykinin receptor B1* 3023 413 3.27E-12NM_001059 tachykinin receptor 3 2966 420 4.28E-16NM_000675 adenosine A2a receptor 2288 417 4.24E-14NM_000797 dopamine receptor D4 2111 422 4.14E-13NM_000798 dopamine receptor D5 1955 413 1.96E-13NM_006639 cysteinyl leukotriene receptor 1 1631 430 5.56E-10NM_000959 prostaglandin F receptor (FP) 1483 419 2.99E-08NM_003382 vasoactive intestinal peptide receptor 2 1452 415 2.46E-11NM_001841 cannabinoid receptor 2 (macrophage)* 1421 417 1.71E-12NM_000960 prostaglandin I2 (prostacyclin) receptor (IP) 1105 411 1.12E-11NM_181657 leukotriene B4 receptor 1079 417 2.07E-11NM_000739 cholinergic receptor, muscarinic 2 1064 417 5.26E-07NM_012125 cholinergic receptor, muscarinic 5 969 413 2.24E-10NM_000674 adenosine A1 receptor 965 411 4.28E-08NM_000794 dopamine receptor D1* 927 423 1.01E-10NM_019839 leukotriene B4 receptor 2 919 412 4.01E-10NM_020377 cysteinyl leukotriene receptor 2* 913 413 2.17E-10

Expression levels (non-spliced variants) of selected GPCR transcripts in human airway smooth muscle

Some Intriguing Receptors are Highly ExpressedCompared to Benchmark GPCRs

Results – Splice Variants of GPCRs in HASM

• Of the 353 GPCRs detected, 192 had at least 1 alternatively spliced form (54%)

• A total of 967 events detected; thus on average each of the spliced GPCR had ~5 splicing events

• Novel exons and exon skip events were the most common. Note: the designation of a novel or deleted exon depends on which sequence is the reference

Distribution of Alternatively Spliced Forms of GPCRS Expressed on HASM

Splice Variants of the High Affinity Leukotriene B4 Receptor (LTB4R or BLT1)

• Expression of the unspliced form was moderate in HASM

• Several potential splicing events identified

• RT-PCR was performed with specific primers utilized to amplify two alternatively spliced LTB4R transcripts (alternative donor and alternative acceptor splice sites)

• Products purified and sequenced to identify the precise sequence

ALL= full length sequence of LTB4RLong Red = deleted from LTB4R-AS2Bracketed Red = deleted from LTB4R-AS1

Sequencing Results

Alternative Splicing Results in Expression of Three Forms of the LTB4R1 in HASM

Wild-typeTMD

1 2 3 4 5 6 7

LTB4R-AS1

LTB4R-AS2

TMD

4 5 6 7

1 3 4 5 6 7

AS2AS1WT

P= probability from transmembrane spanning prediction modeling

Western blot

Changes in LTB4R Structure due to Alternative Splicing

AS1 AS2

XX X

XXXX

X

Conclusions

• Human airway smooth muscle (HASM) expresses many more GPCRs than expected

• Many transcripts were expressed at higher levels than those of GPCRs currently utilized for treatment of asthma and chronic obstructive lung disease

• However, a large proportion of HASM GPCR transcripts are alternatively spliced, with an average 5 splicing events/splicedreceptor

Conclusions (cont.)

• Paradoxic airway responses to activation of the LTB4R are well known and remain unexplained

• LTB4R had 2 confirmed alternative splicing events which were further investigated

• At the transcript level, these were identified as deletions of portions of the 5′ region of the receptor

• Protein predictions revealed a loss of – The amino terminus, 1st, 2nd and part of 3rd TMDs and 2 loops– The 2nd TMD and part of the 1st ECL

• At the protein level, all three isoforms were expressed, with the full-length and lowest molecular size isoforms expressed at similar levels

Conclusions (cont.)

• Both alternatively spliced LTB4Rs could act as dominant-negative proteins against the full-length receptor, thus LTB4R antagonists would be unlikely to have therapeutic efficacy in asthma

• These results reveal the complexity of GPCR signaling in the lung, and show that substantial diversity is present due to alternative splicing.

• Such information is critical in developing a better understanding of GPCR function, and drug development, for obstructive lung diseases.