Structural and Functional Characterization of G Protein-Coupled Receptors with Deep Mutational Scanning

AbstractIn 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.

Download Full-text

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

eLife ◽

10.7554/elife.54895 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Eric M Jones ◽

Nathan B Lubock ◽

AJ Venkatakrishnan ◽

Jeffrey Wang ◽

Alex M Tseng ◽

...

Keyword(s):

G Protein ◽

Drug Targets ◽

Functional Characterization ◽

Structure And Function ◽

G Protein Coupled Receptors ◽

Single Amino Acid ◽

Loss Of Function ◽

And Function ◽

Drug Receptors ◽

G Protein Coupled

The >800 human 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 7800 of 7828 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 identify 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.

Download Full-text

GPCRdb in 2021: integrating GPCR sequence, structure and function

Nucleic Acids Research ◽

10.1093/nar/gkaa1080 ◽

2020 ◽

Vol 49 (D1) ◽

pp. D335-D343 ◽

Cited By ~ 1

Author(s):

Albert J Kooistra ◽

Stefan Mordalski ◽

Gáspár Pándy-Szekeres ◽

Mauricio Esguerra ◽

Alibek Mamyrbekov ◽

...

Keyword(s):

G Protein ◽

Drug Targets ◽

Olfactory Receptors ◽

Structure And Function ◽

G Protein Coupled Receptors ◽

Sequence Structure ◽

Protein Interfaces ◽

And Function ◽

G Protein Coupled ◽

Refined Version

Abstract G protein-coupled receptors (GPCRs) form both the largest family of membrane proteins and drug targets, mediating the action of one-third of medicines. The GPCR database, GPCRdb serves >4 000 researchers every month and offers reference data, analysis of own or literature data, experiment design and dissemination of published datasets. Here, we describe new and updated GPCRdb resources with a particular focus on integration of sequence, structure and function. GPCRdb contains all human non-olfactory GPCRs (and >27 000 orthologs), G-proteins and arrestins. It includes over 2 000 drug and in-trial agents and nearly 200 000 ligands with activity and availability data. GPCRdb annotates all published GPCR structures (updated monthly), which are also offered in a refined version (with re-modeled missing/distorted regions and reverted mutations) and provides structure models of all human non-olfactory receptors in inactive, intermediate and active states. Mutagenesis data in the GPCRdb spans natural genetic variants, GPCR-G protein interfaces, ligand sites and thermostabilising mutations. A new sequence signature tool for identification of functional residue determinants has been added and two data driven tools to design ligand site mutations and constructs for structure determination have been updated extending their coverage of receptors and modifications. The GPCRdb is available at https://gpcrdb.org.

Download Full-text