Olfactory Receptor Trafficking Involves Conserved Regulatory Steps

Gimelbrant, Alexander A.; Haley, Shannon; McClintock, Timothy S.

doi:10.1074/jbc.m005433200

Cited by 86 publications

(60 citation statements)

References 37 publications

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“…2E). The punctuated distribution of OR17-40 in the plasma membrane is similar to what has been reported for other ORs (8,14) and suggests receptor clustering. At Ϸ30 min after ACP labeling, the punctuated pattern on the cell surface gave rise to cytosolic bright spots (Fig.…”

supporting

confidence: 67%

“…In highly expressing cells, ORs did not exit the ER, possibly because of receptor self-aggregation. Previous studies on OR expression in the odora olfactory sensory neuron cell line proposed a model in which receptor trafficking is controlled at two regulatory checkpoints: first, at the exit from the ER, which is crucial in heterologous cells; and second, in a post-Golgi compartment where ORs are blocked in undifferentiated odora cells (8). Our results indicate that, in heterologous cells, the first checkpoint is critical but OR17-40 does not remain blocked at the second, because the receptor was visible at the cell surface only a few hours after being in the Golgi apparatus.…”

Section: Discussionmentioning

confidence: 99%

“…Functional expression of ORs in heterologous cells is, therefore, an important approach for elucidating the molecular mechanism of olfaction and helps to identify specific ligands for particular receptors. Unfortunately, ORs express very inefficiently at the plasma membrane of heterologous cells, apparently because of OR retention in intracellular compartments and degradation via the ubiquitin-proteasome pathway (8)(9)(10). However, direct monitoring of these processes in vivo is still immature.…”

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confidence: 99%

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Visualizing odorant receptor trafficking in living cells down to the single-molecule level

Jacquier

Prummer

Segura

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Despite the importance of trafficking for regulating G proteincoupled receptor signaling, for many members of the seven transmembrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellular trafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulselabeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Singlemolecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of Ϸ190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception.cell signaling ͉ G protein-coupled receptors ͉ single-particle tracking ͉ in vivo protein labeling ͉ fluorescence imaging T he sensation of smell is mediated by a specific family of olfactory G protein-coupled receptors (GPCRs), which recognize small volatile molecules (1). Although odorant receptors (ORs) account for the largest mammalian gene family, comprising up to 1,000 members, the mechanism of signal recognition and amplification in olfactory transduction remains elusive (2). This is partly because classical methods for OR detection, based on immunocytochemistry (3, 4) or genetic fusion to GFP (5), have not permitted the simultaneous live-cell imaging of olfactory processes at the cell membrane and in cytoplasmic compartments.Comprehensive functional studies on ORs in a native cellular environment, such as isolated olfactory sensory neurons, adenovirus-infected olfactory epithelia, or genetically engineered animals, are often hampered by practical limitations: (i) olfactory sensory neurons are difficult to maintain in primary culture (6), (ii) virus-mediated gene transfer does not consistently yield functional OR expression (7), and (iii) creating transgenic animals for each OR would be quite expensive. Functional expression of ORs in heterologous cells is, therefore, an important approach for elucidating the molecular mechanism of olfaction and helps to identify specific ligands ...

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supporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Visualizing odorant receptor trafficking in living cells down to the single-molecule level

Jacquier

Prummer

Segura

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Several studies have also demonstrated that postGolgi transport of GPCRs is a regulated process. For example, G protein-coupled olfactory and chemokine receptors are released from the ER, but accumulated in the Golgi (16,17). The opsin mutant E150K is also accumulated in the cis and medial Golgi (18).…”

mentioning

confidence: 99%

Rab8 Interacts with Distinct Motifs in α2B- and β2-Adrenergic Receptors and Differentially Modulates Their Transport

Dong

Yang

Zhang

et al. 2010

Journal of Biological Chemistry

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The molecular mechanism underlying the post-Golgi transport of G protein-coupled receptors (GPCRs) remains poorly understood. Here we determine the role of Rab8 GTPase, which modulates vesicular protein transport between the trans-Golgi network (TGN) and the plasma membrane, in the cell surface targeting of ␣ 2B -and ␤ 2 -adrenergic receptors (AR). Transient expression of GDP-and GTP-bound Rab8 mutants and short hairpin RNA-mediated knockdown of Rab8 more potently inhibited the cell surface expression of ␣ 2B -AR than ␤ 2 -AR. The GDP-bound Rab8(T22N) mutant attenuated ERK1/2 activation by ␣ 2B -AR, but not ␤ 2 -AR, and arrested ␣ 2B -AR in the TGN compartment. Co-immunoprecipitation revealed that both ␣ 2B -AR and ␤ 2 -AR physically interacted with Rab8 and glutathione S-transferase fusion protein pulldown assays demonstrated that Rab8 interacted with the C termini of both receptors. Interestingly, mutation of the highly conserved membrane-proximal C terminus dileucine motif selectively blocked ␤ 2 -AR interaction with Rab8, whereas mutation of residues (T22N) of a chimeric ␤ 2 -AR carrying the ␣ 2B -AR C terminus was similar to ␣ 2B -AR. These data provide strong evidence indicating that Rab8 GTPase interacts with distinct motifs in the C termini of ␣ 2B -AR and ␤ 2 -AR and differentially modulates their traffic from the TGN to the cell surface.

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“…Receptors involved in defensive signaling may be built as ion channels, G protein-coupled receptors (GPCRs), or accessory coreceptors that work in combination with another receptor. A few coreceptors that facilitate cell surface expression (10)(11)(12) and, in some cases, affect the pharmacology of associated receptors (13,14) have been identified. For example, receptor activity-modifying proteins (RAMPs) affect the pharmacology of their GPCR partners (15).…”

mentioning

confidence: 99%

Identification of RL-TGR, a coreceptor involved in aversive chemical signaling

Cohen

Haack

Halstead-Nussloch

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Chemical signaling plays an important role in predator–prey interactions and feeding dynamics. Like other organisms that are sessile or slow moving, some marine sponges contain aversive compounds that defend these organisms from predation. We sought to identify and characterize a fish chemoreceptor that detects one of these compounds. Using expression cloning in Xenopus oocytes coexpressing the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, the beta-2 adrenergic receptor (β 2 AR), and fractions of a zebrafish cDNA library, we isolated a cDNA clone encoding receptor activity–modifying protein (RAMP)-like triterpene glycoside receptor (RL-TGR), a novel coreceptor involved in signaling in response to triterpene glycosides. This coreceptor appears to be structurally and functionally related to RAMPs, a family of coreceptors that physically associate with and modify the activity of G protein–coupled receptors (GPCRs). In membranes from formoside-responsive oocytes, RL-TGR was immunoprecipitated in an apparent complex with β 2 AR. In HEK293 cells, coexpression of β 2 AR induced the trafficking of RL-TGR from the cytoplasm to the plasma membrane. These results suggest that RL-TGR in the predatory fish physically associates with the β 2 AR or another, more physiologically relevant GPCR and modifies its pharmacology to respond to triterpene glycosides found in sponges that serve as a potential food source for the fish. RL-TGR forms a coreceptor that responds to a chemical defense compound in the marine environment, and its discovery might lead the way to the identification of other receptors that mediate chemical defense signaling.

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Olfactory Receptor Trafficking Involves Conserved Regulatory Steps

Cited by 86 publications

References 37 publications

Visualizing odorant receptor trafficking in living cells down to the single-molecule level

Visualizing odorant receptor trafficking in living cells down to the single-molecule level

Rab8 Interacts with Distinct Motifs in α2B- and β2-Adrenergic Receptors and Differentially Modulates Their Transport

Identification of RL-TGR, a coreceptor involved in aversive chemical signaling

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