Structural and functional characterization of G protein-coupled receptors with deep mutational scanning

Abstract
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Abstract

In humans, the >800 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.

Data availability

Sequencing data have been submitted to GEO and the accession code is GSE144819.

The following data sets were generated

1. Jones EM
2. Lubock NB
3. Venkatakrishnan A
4. Wang J
5. Tseng AM
6. Paggi JM
7. Latorraca NR
8. Cancilla D
9. Satyadi M
10. Davis JE
11. Babu MM
12. Dror RO
13. Kosuri S
(2020) Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning
NCBI Gene Expression Omnibus, GSE144819.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144819

Article and author information

Author details

Eric M Jones

Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, Quantitative and Computational Biology Institute, Eli and Edythe Broad Center o, University of California, Los Angeles, Los Angeles, United States

Competing interests
Eric M Jones, holds equity and is employed by Octant, Inc., a company to which patent rights based on this work have been licensed (Application No. 62/528,833).
Nathan B Lubock

Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, United States

Competing interests
Nathan B Lubock, Employed by and holds equity in Octant Inc. to which patent rights based on this work have been licensed (Application No. 62/528,833).
A J Venkatakrishnan

Department of Computer Science, Institute for Computational and Mathematical Engineering, Department of Molecular and Cellular Physiology, Department of Structural Biology, Stanford University, Stanford, United States

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0003-2819-3214
Jeffrey Wang

Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, Quantitative and Computational Biology Institute, Eli and Edythe Broad Center o, University of California, Los Angeles, Los Angeles, United States

Competing interests
No competing interests declared.
Alex M Tseng

Department of Computer Science, Institute for Computational and Mathematical Engineering, Department of Molecular and Cellular Physiology, Department of Structural Biology, Stanford University, Stanford, United States

Competing interests
No competing interests declared.
Joseph M Paggi

Department of Computer Science, Institute for Computational and Mathematical Engineering, Department of Molecular and Cellular Physiology, Department of Structural Biology, Stanford University, Stanford, United States

Competing interests
No competing interests declared.
Naomi R Latorraca

Biophysics Program, Stanford University, Stanford, United States

Competing interests
No competing interests declared.
Daniel Cancilla

Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, Quantitative and Computational Biology Institute, Eli and Edythe Broad Center o, University of California, Los Angeles, Los Angeles, United States

Competing interests
No competing interests declared.
Megan Satyadi

Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, Molecular Biology Institute, Quantitative and Computational Biology Institute, Eli and Edythe Broad Center o, University of California, Los Angeles, Los Angeles, United States

Competing interests
No competing interests declared.
Jessica E Davis

Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, United States

Competing interests
No competing interests declared.
M Madan Babu

MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

Competing interests
No competing interests declared.
Ron O Dror

Biophysics Program, Stanford University, Stanford, United States

For correspondence
ron.dror@stanford.edu

Competing interests
No competing interests declared.
Sriram Kosuri

Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, United States

For correspondence
sri@ucla.edu

Competing interests
Sriram Kosuri, holds equity and is employed by Octant, Inc., a company to which patent rights based on this work have been licensed to (Application No. 62/528,833).

"This ORCID iD identifies the author of this article:" 0000-0002-4661-0600

Funding

National Science Foundation (1556207)

Sriram Kosuri

National Institutes of Health (GM007185)

Sriram Kosuri

National Institutes of Health (5T32GM008496)

Sriram Kosuri

National Institutes of Health (DP2GM114829)

Sriram Kosuri

Medical Research Council (MC_U105185859)

Sriram Kosuri

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.