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| Status |
Public on Feb 06, 2020 |
| Title |
Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning |
| Organisms |
Escherichia coli; Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
In humans, the 813 G protein-coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state, and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G-protein signal transduction. We tested 7,800 of 7,828 possible single amino acid substitutions to the beta-2 adrenergic receptor (β2AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for β2AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we resolve residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.
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| Overall design |
The overall design of this experiment is to test the functional consequences of every amino acid substitution to the beta-2 adrenergic receptor (B2AR). This occurs in two stages that occurs roughly as follows (for more details, see Figure 1 of our bioRxiv preprint <https://www.biorxiv.org/content/10.1101/623108v3.full>). First, we use DNA microarray synthesis to generate a library of mutants at every position in the protein (think alanine scan, cysteine scan, aspartic acid scan, ...). We then clone these mutants into a plasmid backbone that's tagged with a DNA barcode. We use NGS to associate each variant with its corresponding barcode (this corresponds to the the Map_* and Eric_* fastq's that are eventually processed into the NextSeq_MiSeq.map.csv.gz).
Second, the mapped library is integrated into human cell lines such that each cell only receives a single B2AR mutant - barcode pair. Activation of the receptor by agonist incubation results in transcription of the mutant's barcode, which we can detect with RNA-seq (the second set of fastq's drug-*).
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| Contributor(s) |
Jones EM, Lubock NB, Venkatakrishnan A, Wang J, Tseng AM, Paggi JM, Latorraca NR, Cancilla D, Satyadi M, Davis JE, Babu MM, Dror RO, Kosuri S |
| Citation(s) |
33084570 |
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| Submission date |
Feb 05, 2020 |
| Last update date |
Dec 08, 2020 |
| Contact name |
Nathan Lubock |
| E-mail(s) |
[email protected], [email protected]
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| Organization name |
UCLA
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| Street address |
607 Charles E Young Drive
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90024 |
| Country |
USA |
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| Platforms (4)
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| GPL16085 |
Illumina MiSeq (Escherichia coli) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
| GPL21697 |
NextSeq 550 (Homo sapiens) |
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| Samples (27)
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| Relations |
| BioProject |
PRJNA604999 |
| SRA |
SRP247450 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE144819_NextSeq_MiSeq.map.csv.gz |
14.6 Mb |
(ftp)(http) |
CSV |
| GSE144819_drug-2_cond-bcs-counts.txt.gz |
26.1 Mb |
(ftp)(http) |
TXT |
| GSE144819_drug-3_cond-bcs-counts.txt.gz |
33.8 Mb |
(ftp)(http) |
TXT |
| GSE144819_drug-6_cond-bcs-counts.txt.gz |
78.6 Mb |
(ftp)(http) |
TXT |
| GSE144819_drug-7_cond-bcs-counts.txt.gz |
73.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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