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G- Coupled Receptors

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

• Examples include: ¾ Vision ¾ Smell ¾ Neurotransmission ¾ Endocrine ¾ Paracrine ¾ Orphan

• 50% of all prescribed drugs act at GPCRs: ¾ Histamine type 1 antagonists ¾ Histamine type 2 receptor antagonists

¾ β1- antagonists

¾ β1-adrenergic receptor

¾ β2-adrenergic receptor agonists ¾ Cysteinyl leukotriene antagonists

¾ M3-muscarinic antagonists

¾ α2-adrenergic receptor agonists ¾ Angiotensin II receptor antagonists Topography of GPCRs

amino-terminus

ECL-2 ECL-1 extracellular ECL-3

TM7 transmembrane TM1

ICL-3 ICL-1 “ICL-4” ICL-2 intracellular

carboxy-terminus GPCR Signaling

α G-protein s βγ

αs stimulates

αi inhibits adenylyl cyclase

αq/11 stimulates Variability in GPCR Function

• Diseases known to involve GPCR signaling (heart failure, , 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 variability ¾ Polymorphisms of the ¾ Alternative splicing which results in expression of multiple receptor “isoforms” with different properties RNA Splicing Mediates Expression Diversity

~25,000 genes

conditional spatial A B temporal disease

>100,000 unique mRNAs

Isoform A Isoform B FunctionA Function B 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 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:

¾ M3-muscarinic – constricts airway

¾ β2AR – bronchodilates airway highly variable ¾ Prostaglandin receptors ¾ Leukotriene receptors

• Hypothesis ¾ Human airway smooth muscle express many more GPCRs than predicted based on classic pharmacology ¾ These undergo frequent alternative splicing events leading to a highly diversified receptor milieu 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 variant ¾ Alternative splice donors ¾ Alternative splice acceptors ¾ Exon(s) skips ¾ Novel exons ¾ Intron retentions ¾ Novel 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 Expression levels (non-spliced variants) of selected GPCR transcripts in human airway smooth muscle

Reference Gene Mean Mean Accession Gene Annotation Signal 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 formyl receptor-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 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 Some Intriguing Receptors are Highly Expressed Compared 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 Sequencing Results

ALL= full length sequence of LTB4R Long Red = deleted from LTB4R-AS2 Bracketed Red = deleted from LTB4R-AS1 Alternative Splicing Results in Expression of Three Forms of the LTB4R1 in HASM

TMD Wild-type LTB4R-AS1

1 2 3 4 5 6 7 1 3 4 5 6 7

TMD

WT 4 5 6 7 AS1 AS2

LTB4R-AS2 Western blot

P= probability from transmembrane spanning prediction modeling Changes in LTB4R Structure due to Alternative Splicing

X X X X XXX X

AS1 AS2 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/spliced receptor 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 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.