Dual RXR motifs regulate nerve growth factor–mediated intracellular retention of the delta opioid receptor

Abstract

The delta opioid receptor (DOR), a physiologically relevant prototype for G protein–coupled receptors, is retained in intracellular compartments in neuronal cells. This retention is mediated by a nerve growth factor (NGF)-regulated checkpoint that delays the export of DOR from the trans-Golgi network. How DOR is selectively retained in the Golgi, in the midst of dynamic membrane transport and cargo export, is a fundamental unanswered question. Here we address this by investigating sequence elements on DOR that regulate DOR surface delivery, focusing on the C-terminal tail of DOR that is sufficient for NGF-mediated regulation. By systematic mutational analysis, we define conserved dual bi-arginine (RXR) motifs that are required for NGF- and phosphoinositide-regulated DOR export from intracellular compartments in neuroendocrine cells. These motifs were required to bind the coatomer protein I (COPI) complex, a vesicle coat complex that mediates primarily retrograde cargo traffic in the Golgi. Our results suggest that interactions of DOR with COPI, via atypical COPI motifs on the C-terminal tail, retain DOR in the Golgi. These interactions could provide a point of regulation of DOR export and delivery by extracellular signaling pathways.

In vivo mapping of a GPCR interactome using knockin mice

“…analyses with other studies that used MS analyses, coimmunoprecipitation (coIP), and bioluminescence resonance energy transfer (BRET) to identify proteins interacting with DOPr. We confirmed 24 proteins that were previously reported by us, and others, to interact with DOPr, including β-arrestin-1 (18,19), the cannabinoid receptor 1 (20,21), heterotrimeric G proteins (22), the cyclin-dependent-like kinase 5 (23), calnexin (24), as well as subunits of the coatomer (25,26) and the V-type proton ATPase complexes (27) ( Table 1).…”

β-arrestin mediates communication between plasma membrane and intracellular GPCRs to regulate signaling

“…In accordance with previous literature, K3R mutant receptors partially colocalized with a Golgi compartment marker (Fig. 1d ) 38 , 39 . We also noticed a steady-state population of WT CXCR4 retained at the Golgi (Fig.…”

“…While the CXCR4 K3R mutant has been previously used to study CXCR4 degradation 36,37 , we found that these mutations caused a drastic change in the spatial distribution of CXCR4. This was due to the unintended creation of an R-X-R motif, which has been shown to increase GPCR retention in the Golgi [38][39][40] . Indeed, mutating a single residue in the R-X-R motif (i.e., K3R/Q) restored receptor plasma membrane localization to near WT levels ( Fig.…”

Rab43 GTPase directs postsynaptic trafficking and neuron-specific sorting of G protein–coupled receptors

“…The players involved in GPCR targeting in neurons have just begun to be revealed. Recent studies have identified several highly conserved motifs embedded within neuronal GPCRs, which dictate receptor delivery to the surface through regulating receptor correct folding and exit from the ER or the Golgi ( 6 , 7 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ). A number of receptor-interacting proteins have also been identified to control GPCR delivery in neurons, such as Yif1B, in the dendritic targeting of serotonin-1A receptor, prenylated Rab acceptor 1 domain family member 2 in γ-aminobutyric acid B receptor export from the ER, and GGAs (Golgi-associated, γ-adaptin homologous, ADP-ribosylation factor-interacting proteins) in α 2 -AR transport from the Golgi ( 8 , 9 , 10 , 11 , 46 , 47 , 48 ).…”