Mechanistic Dissection of Spatially Encoded GPCR Signaling Through High‐throughput CRISPR Screening

Our recent work has overturned the prevailing textbook model for GPCR signaling, which held that GPCR activation at the plasma membrane is responsible for all downstream consequences of signaling. We have demonstrated that receptor/second messenger signaling on internal compartments but not plasma membrane drives all receptor‐dependent transcriptional responses, and the site of receptor activation can dictate how cells “interpret” different ligands, including medically relevant drugs. This phenomenon of functional specialization of signaling based on receptor localization has been gaining significant support in the last few years from us and other groups, yet not much is known about its underlying mechanisms and functional consequences. To begin to address these open questions, we set up a functional genomic screen aimed at identifying factors regulating ß2‐adrenergic receptor (ß2‐AR)‐dependent transcription‐ a location‐biased GPCR signaling response. We combined pooled CRISPR interference libraries targeting all annotated transcripts in the human genome and a novel fluorescent transcriptional reporter for GPCR/cAMP signaling that we have developed with fluorescence‐activated cell sorting and deep sequencing. Through this comprehensive screen, we have identified both known and novel mediators of the GPRC pathway. We are currently defining the biochemical mechanisms and specificity of the new regulators and their contributions to location‐biased receptor signaling, and have intriguing data linking a highly‐penetrant genetic risk factor for psychosis to these pathways. Support or Funding Information National Institute of Mental Health [Grant MH10963301]