G protein-coupled receptors (GPCRs) represent the largest family of cell surface proteins in metazoa. GPCRs are expressed in diverse cell types, regulating a plethora of physiological and pathological processes. The human genome encodes approximately 800 GPCRs, which cluster in five major classes based on sequence similarity and evolutionary relationships. GPCRs are at the interface of environmental stimuli and intracellular responses, translating external signals into internal representations. They share a basic common structure of seven transmembrane domains, but display remarkable diversity in their ligand binding properties, intracellular signal transduction, and physiological functions. The expansion of the GPCR family, coupled with a high degree of functional redundancy, creates substantial complexities in systematically dissecting their functions. In particular, for many GPCRs, it has been challenging to identify their physiologically relevant ligands. Rapid advances in high-throughput screening techniques, computational modeling, and structural analyses have contributed significantly to the understanding of GPCR activation and signaling. However, these analyses have typically been performed in heterologous systems. A systematic in vivo approach to deorphanize the receptors and decipher their functions remains to be described.
The genome of the free-living nematode C. elegans encodes one of the largest GPCR repertoires among any sequenced organisms. In C. elegans, GPCRs mediate or regulate chemosensation, nociceptive responses, lipid homeostasis, social behavior, immunity, and mating, allowing animals to thrive in a complex ecological niche. A subset of GPCRs in C. elegans, mainly neurotransmitter receptors, is highly conserved. By contrast, predicted chemoreceptor GPCRs are highly divergent and dramatically expanded, numbering over 1300. The chemoreceptors are expressed in a small nervous system consisting of 302 neurons, of which 32 are chemosensory. Since each chemosensory neuron expresses multiple chemoreceptors, individual neurons likely detect and discriminate a variety of sensory stimuli to elicit the appropriate behavioral responses.
Many neuropeptide receptors in C. elegans have been deorphanized in vitro, facilitating the subsequent functional analyses. However, the majority of chemoreceptors, as well as a proportion of neuropeptide receptors, remain fully uncharacterized. In particular, only a few chemoreceptors have been paired with defined ligands since the description of the first olfactory receptor ODR-10. The large number of GPCRs in C. elegans, their functional redundancy, and the complexities associated with expressing these GPCRs in heterologous systems render existing approaches, such as forward and reverse genetics, inefficient in determining the functions of GPCRs in physiologically relevant contexts.
To this end, we sought to generate a comprehensive, versatile, and widely applicable resource that could overcome the current obstacles associated with the dissection of GPCR function in C. elegans. We used CRISPR/Cas9 genome editing to disrupt 1654 GPCR-encoding genes and 152 neuropeptide-encoding genes in 284 and 38 strains, respectively. To bypass possible functional redundancy, we specifically targeted multiple genes encoding closely related GPCRs in individual strains. We then systematically screened mutant strains for their responses to a range of environmental signals. Our analyses established a role for peptidergic signaling in acute response to hypoxia, identified a panel of GPCRs and neuropeptides involved in response to pathogens, and determined the putative receptors for the attractive volatile odorants pyrazine and 2,3-pentanedione (Fig. 1a). In particular, we identified GPCRs that exhibit partial redundancy in their functions, highlighting a distinctive advantage of our approach over existing methods. We expect that our resource will streamline the analyses of GPCR function in C. elegans, leading to new insights into how GPCRs translate external information into intracellular responses.
a Schematic drawing of our approach to phenotypic profiling of nearly all GPCR genes in C. elegans. b The gene expression pattern of unannotated GPCR genes, according to a previously published single-cell RNA-seq dataset of L4 worms. ‘Others’ indicates the non-neuronal tissues, such as intestine and muscle. c Phylogenetic tree analysis of 1675 GPCRs in C. elegans. The GPCR sub-families and relevant genes are highlighted. Six receptors of SRW subfamily, as indicated in blue, were clustered to the clade of neuropeptide receptors. 10 DUF621 domain-containing receptors (magenta) were closely related to chemoreceptors, and 5 DUF1182 domain-containing receptors (brown) were in the clade of neuropeptide receptors. 11 putative GPCRs (Teal), which were annotated as Chromadorea class in the Wormbase, were clustered closely to chemoreceptors. d Strategy for the generation and validation of GPCR mutant strains.
The number of GPCRs encoded by the C. elegans genome is predicted to be more than 1300. We reasoned that updated gene annotation might allow us to identify additional GPCR-encoding genes. From C. elegans genome release WS273 (and later versions), we obtained a list of 1442 putative chemoreceptor encoding genes, including 126 that had not been annotated (Supplementary Data 1a, b; See methods for details). Unannotated genes belong to multiple chemoreceptor subfamilies, and are predicted to be expressed predominantly in chemosensory neurons based on single-cell transcriptomics analysis (Fig. 1b and Supplementary Data 1b, c). Six unannotated members of the srw family are closely related to neuropeptide receptors (Fig. 1c.
