Trafficking prerogatives of olfactory receptors

McClintock, Timothy S.; Sammeta, Neeraja

doi:10.1097/00001756-200308260-00001

Cited by 75 publications

(52 citation statements)

References 79 publications

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“…It is believed that the major reason for the inefficient expression of ORs in HEK293 cells is the fact that they are retained in the endoplasmic reticulum and cannot reach the cell surface (10,11). Therefore, accessory proteins that help ORs to overcome this problem would be necessary to enable functional OR expression in heterologous cells.…”

Section: Discussionmentioning

confidence: 99%

“…However, so far only Ϸ30 ORs have been linked to odorants they recognized (6)(7)(8)(9). The major reason for this fact is that the functional expression of ORs in heterologous cell types has proven difficult, mostly because ORs cannot reach the plasma membrane (10)(11)(12). To circumvent this problem, we previously used a combination of Ca 2ϩ imaging and single-cell RT-PCR to identify the ORs expressed by olfactory neurons that responded to different aliphatic odorants (6).…”

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confidence: 99%

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Ric-8B promotes functional expression of odorant receptors

Dannecker

Mercadante

Malnic

2006

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

109

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Odorants are detected by a large family of odorant receptors (ORs) expressed in the nose. The information provided by the ORs is transmitted to specific regions of the brain, leading to odorant perception. The determination of the odorant specificities of the different ORs will contribute to the understanding of how odorants are discriminated by the olfactory system. However, to date only a few ORs have been linked to odorants they recognize, because ORs are poorly expressed on the cell surface of heterologous cells. Here we show that Ric-8B, a putative guanine nucleotide exchange factor for G␣olf, promotes efficient heterologous expression of ORs. Our results also show that Ric-8B enhances accumulation of G␣olf at the cell periphery, indicating that it promotes functional OR expression by improving the efficiency of OR coupling to G␣olf. Expression systems containing G␣olf and Ric-8B should contribute to the functional characterization of ORs.guanine nucleotide exchange factor ͉ olfactory receptor ͉ synembryn ͉ G protein ␣ olf ͉ heterologous expression M ammals can discriminate a vast number of odorants with remarkable sensitivity and accuracy. Odorants are first detected by a large family of odorant receptors (ORs) expressed in the cilia of the olfactory sensory neurons (OSNs) of the nose (1). The information provided by the different ORs is then transmitted to the olfactory bulb of the brain, which in turn relays information to the olfactory cortex. A series of experiments indicated that the information provided by the different ORs in the nose is organized into a sensory map in the olfactory bulb (2-4). In the olfactory cortex, which receives input from the bulb, there is another map of OR inputs, different from the map in the bulb (5).The discrimination of odorants presumably derives from the different ligand specificities of the ORs. Therefore, the determination of the odorant specificities of individual ORs should provide information regarding how odor identities are encoded. However, so far only Ϸ30 ORs have been linked to odorants they recognized (6-9). The major reason for this fact is that the functional expression of ORs in heterologous cell types has proven difficult, mostly because ORs cannot reach the plasma membrane (10-12). To circumvent this problem, we previously used a combination of Ca 2ϩ imaging and single-cell RT-PCR to identify the ORs expressed by olfactory neurons that responded to different aliphatic odorants (6). By using this approach we identified 13 different ORs that responded to various aliphatic alcohols and acids. Our results revealed that the olfactory system uses combinatorial receptor codes to encode odorant identities.The complete repertoires of mouse and human OR genes have recently been determined from the corresponding genome sequences (13-19). All together, 1,200 intact OR genes were identified in the mouse, and Ϸ388 intact genes were identified in humans (20). Gene expression studies confirmed that a large fraction of the mouse OR genes is expressed in the olfactory ep...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Ric-8B promotes functional expression of odorant receptors

Dannecker

Mercadante

Malnic

2006

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

109

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“…If one of the subunits is missing this specific interaction is no longer possible. Recently, a checkpoint protein (UNC-101) was identified that controls trafficking of odorant receptors into the olfactory cilia of Caenorhabditis elegans (47,48). Similar proteins may have evolved to control ciliary CNG channel targeting.…”

Section: Cngb1mentioning

confidence: 99%

Loss of CNGB1 Protein Leads to Olfactory Dysfunction and Subciliary Cyclic Nucleotide-gated Channel Trapping

Michalakis

Reisert

Geiger

et al. 2006

Journal of Biological Chemistry

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Olfactory receptor neurons (ORNs) employ a cyclic nucleotide-gated (CNG) channel to generate a receptor current in response to an odorant-induced rise in cAMP. This channel contains three types of subunits, the principal CNGA2 subunit and two modulatory subunits (CNGA4 and CNGB1b). Here, we have analyzed the functional relevance of CNGB1 for olfaction by gene targeting in mice. Electro-olfactogram responses of CNGB1-deficient (CNGB1 ؊/؊ ) mice displayed a reduced maximal amplitude and decelerated onset and recovery kinetics compared with wild-type mice. In a behavioral test, CNGB1؊/؊ mice exhibited a profoundly decreased olfactory performance. Electrophysiological recordings revealed that ORNs of CNGB1 ؊/؊ mice weakly expressed a CNG current with decreased cAMP sensitivity, very rapid flicker-gating behavior and no fast modulation by Ca 2؉ -calmodulin. Co-immunoprecipitation confirmed the presence of a CNGA2/CNGA4 channel in the olfactory epithelium of CNGB1 ؊/؊ mice. This CNGA2/CNGA4 channel was targeted to the plasma membrane of olfactory knobs, but failed to be trafficked into olfactory cilia. Interestingly, we observed a similar trafficking defect in mice deficient for the CNGA4 subunit. In conclusion, these results demonstrate that CNGB1 has a dual function in vivo. First, it endows the olfactory CNG channel with a variety of biophysical properties tailored to the specific requirements of olfactory transduction. Second, together with the CNGA4 subunit, CNGB1 is needed for ciliary targeting of the olfactory CNG channel. Cyclic nucleotide-gated (CNG)3 channels are molecular switches converting receptor-mediated increases in cytosolic concentrations of cAMP or cGMP into an influx of cations. The channels play a key role in visual and olfactory transductions. The CNG channel involved in olfaction is expressed at high density in the sensory cilia of olfactory receptor neurons (ORNs) where it is activated by an odorant-induced rise of cAMP. When open, the channel conducts mainly Ca 2ϩ and Na ϩ (1, 2). The subsequent rise in internal Ca 2ϩ

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“…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%

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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Trafficking prerogatives of olfactory receptors

Cited by 75 publications

References 79 publications

Ric-8B promotes functional expression of odorant receptors

Ric-8B promotes functional expression of odorant receptors

Loss of CNGB1 Protein Leads to Olfactory Dysfunction and Subciliary Cyclic Nucleotide-gated Channel Trapping

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

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