G protein-coupled receptors (GPCRs) constitute a large family of seven-transmembrane cell surface receptors that play important roles in signal transduction by mediating cellular responses to a wide array of extracellular stimuli. While classically GPCR signaling was thought to occur solely at the plasma membrane, research in recent years has shown that GPCRs also signal from intracellular organelles, such as endosomes and the Golgi apparatus, broadening our fundamental understanding of GPCR function and pharmacology. The subcellular localization of GPCRs is tightly controlled by receptor passage through the secretory pathway. Nascent GPCRs are synthesized at the endoplasmic reticulum (ER), where they undergo folding, post-translational modifications, and quality control checks important for trafficking and function. Properly folded GPCRs are then transported to the Golgi apparatus for further processing and sorting, before transport to their final destinations at the plasma membrane or intracellular sites. Despite a deep mechanistic understanding of the secretory pathway in eukaryotic cells, the cellular factors that govern intracellular retention or forward trafficking of GPCRs in ER and Golgi organelles have not been comprehensively identified.

Here we adapted and validated a synchronized secretory protein trafficking assay, known as the retention using selective hooks (RUSH) method, to study GPCR trafficking through the secretory pathway. Our work focused on the delta-opioid receptor (DOR), a physiologically and clinically relevant GPCR targeted by opioid drugs and a prototypical member of the rhodopsin-like class A GPCR family. Using the synchronized transport of DOR from the ER to the plasma membrane in HeLa cells as a read-out, we performed an unbiased genome-wide CRISPR/Cas9 knockout screen to identify the cellular machinery that determines DOR anterograde transport. Our screen identified 89 hit genes that promoted DOR export and 115 hit genes that reduced DOR export, based on decreased or increased DOR cell surface levels measured in a flow-cytometry based RUSH assay. We orthogonally validated several individual hits by showing that their siRNA-mediated knockdown in cells significantly decreased DOR cell surface levels. One of our top hits, the cornichon protein family member 1 (CNIH1), was mechanistically characterized. We found that CNIH1 localized to ER and Golgi organelles and regulated the efficient ER exit of DOR. In the absence of CNIH1, DOR was intracellularly retained leading to reduced plasma membrane signaling. Extending our analyses beyond DOR, we tested the susceptibility of 13 other class A GPCRs and a non-GPCR secretory cargo to regulation by CNIH1. Strikingly, only the transport of a subset of GPCRs was affected by decreased CNIH1 levels. CNIH4, a distantly related homolog of CNIH1 that we also analyzed in this study, showed a broader effect on the transport of the diverse secretory cargos.

Our study provides a comprehensive analysis of GPCR anterograde transport and identifies novel cellular factors that control GPCR export. Overall, the exploration of regulated GPCR transport through secretory organelles and the link to signaling has the potential to shape drug responses including of opioid receptors, key therapeutic targets for pain relief.