Membrane Trafficking and Signaling of the Delta Opioid Receptor Within the Biosynthetic Pathway

Abstract

G protein-coupled receptors (GPCRs) transduce diverse signals, including light, ions, hormones, and neurotransmitters, into equally diverse cellular responses. These cellular responses underlie complex physiological processes, including sensation, learning and memory, cardiac function, and immune function. Understanding the variables which contribute to GPCR signaling diversity at a cellular level is essential to understanding the role of GPCRs in physiology and disease. The subcellular location from which GPCR signaling occurs is an increasingly recognized variable which contributes to signaling diversity. I have used the delta opioid receptor (DOR) as a prototype GPCR to investigate mechanisms regulating GPCR localization and the effects of subcellular location on GPCR function. DOR is an ideal and therapeutically relevant prototype GPCR to study these questions. In neuronal cells, DOR localizes to multiple membrane compartments, including the plasma membrane and the Golgi apparatus. Relocation of DOR from intracellular sites to the plasma membrane is associated with enhanced pain-relieving effects of DOR agonists, which highlights the therapeutically relevant link between DOR localization and function. I first investigated the mechanisms which regulate DOR localization to the Golgi in a rat neuroendocrine cell line which shares common mechanisms with primary neurons in regulation of DOR trafficking. Through systematic mutagenesis of the DOR C-terminal primary amino acid sequence and high-resolution imaging, we identified conserved dual RXR amino acid motifs which are required for signal-regulated retention of DOR in the Golgi. Using biochemical approaches, we showed that these RXR motifs also mediate interaction with the coatomer protein I (COPI) complex. These data support a model in which DOR retention in the Golgi is mediated by active retrograde trafficking within the biosynthetic pathway. I next explored the effect of subcellular location on DOR activation. GPCR activation and coupling to effectors is driven by conformational changes in the receptor upon agonist binding. We used fluorescently tagged biosensors which recognize these conformational changes and high-resolution imaging to visualize DOR activation in different subcellular locations. We found that DOR in the plasma membrane and the Golgi differentially recruit two active conformation biosensors in response to the same agonist. These results indicate that subcellular location drives distinct engagement of effectors and suggest the exciting possibility that subcellular location may alter GPCR conformational landscapes upon ligand binding. I also determined the effect of subcellular location on DOR signaling using biosensors for second messenger signaling molecules cAMP and calcium. We found that DOR activation in both the plasma membrane and the Golgi inhibits cAMP production, suggesting that DOR couples to inhibitory G proteins regardless of compartment-specific effects on effector engagement or conformational landscapes. In a rat neuroendocrine cell line, DOR activation at the plasma membrane modulates calcium release from intracellular stores in a Gi/o, Gq/11, and phospholipase C- dependent manner. Modulation of calcium is specific to DOR signaling from the plasma membrane and is not observed upon DOR activation in the Golgi. These data suggest that DOR subcellular location influences the signaling profile of active receptors. Together this work adds to our understanding of how GPCR subcellular localization is regulated and how subcellular location can drive distinct GPCR activation and signaling. In the future, this mechanistic understanding could be applied to tune localization of therapeutically relevant GPCRs like DOR or to target GPCRs in specific subcellular compartments for desired therapeutic effects.