Insulation of Signaling Pathways
Authors: John R. Carlson
Journal: Neuron (2000)

An intriguing problem in neurobiology concerns the regulation of signal integration versus signal insulation within an individual neuron. In some cases signaling pathways within a cell converge and integrate to generate a common unified output. In other cases, where a single neuron must be able to keep track of multiple independent signaling cascades, insulation to prevent “cross-talk” between the pathways becomes critically important. The mechanisms of pathway insulation are particularly interesting in cases in which the insulated pathways share many common molecular components. An excellent system in which to address the issue of signal pathway insulation is the olfactory system of the nematode Caenorhabditis elegans. The worm is faced with a daunting signal-to-noise problem: it must be able to chemotax toward an odor source—a bacterial meal—through the rich odorous background of the soil in which it lives. Moreover, it must accomplish this feat with a vanishingly small number of olfactory receptor neurons. In particular, the worm uses two pairs of receptor neurons to sense a wide diversity of attractive odors. One of these pairs, AWC, expresses multiple odorant receptor genes, and all the odors it senses, including benzaldehyde, butanone, isoamyl alcohol, 2,3-pentanedione, and 2,4,5-trimethylthiazole, appear to share components of a single transduction pathway. Despite this lean economy at both cellular and molecular levels, the worm is able to distinguish among odors, as shown in two distinct paradigms. First, prolonged exposure to one odorant decreases response to that odorant, termed odorant adaptation, but not to another odorant. Second, in the presence of a saturating background of one AWC-sensed odorant, the worm is unable to chemotax toward a point source of the same odorant but is able to migrate toward a point source of a different AWC-sensed odorant, an ability referred to as odorant discrimination. In each of these paradigms, then, two different AWC-sensed odors evoke different responses. How are two response pathways, which share many components, regulated independently within the same cell? A fresh perspective on this problem is reported by 3L'Etoile N. Bargmann C Neuron. 2000; 25 (this issue,): 575-586Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar in this issue of Neuron. The study concerns the odr-1 gene, whose mutations affect chemotaxis to all AWC-sensed odors. odr-1 is shown to encode a transmembrane guanylyl cyclase (tGC) containing a signal sequence, a large extracellular domain, a kinase-like region, and a predicted cyclase domain near the carboxyl terminus. The identification of ODR-1 as a tGC is of special interest in that it belongs to a large family of more than 25 predicted GCs described previously (11Yu S. Avery L. Baude E. Garbers D Proc. Natl. Acad. Sci. USA. 1997; 94: 3384-3387Crossref PubMed Scopus (338) Google Scholar). The expression of these receptors in chemosensory neurons, along with the large size of the family, prompted the proposal that they might represent a new family of odorant receptors. Isolation of mutants defective in one of the C. elegans GCs allows functional testing of this proposal. In this regard, L'Etoile and Bargmann show that a truncated version of ODR-1 lacking the extracellular domain of ODR-1 functions almost as well as the wild-type product, which supports a role for ODR-1 downstream in olfactory signaling, rather than a role as an odorant receptor. Further experiments showed that the cyclase domain is required for olfactory signaling. This result is consistent with a requirement for cGMP production and is of interest because all AWC-sensed odorants are believed to act through a cGMP-sensitive cation channel. ODR-1 was also shown, using GFP fusion constructs, to be expressed in AWC and a small number of other chemosensory neurons (although different constructs containing different extents of flanking DNA showed somewhat different expression patterns, providing a healthy reminder that such experiments require caution in interpretation). L'Etoile and Bargmann then show a link between ODR-1 and adaptation. When ODR-1 was overexpressed by introducing high-copy transgenes of the wild-type genomic clone into odr-1 mutant animals, a provocative result was obtained: these ODR-1(OE) animals no longer adapted normally to butanone, although adaptation to benzaldehyde or isoamyl alcohol was normal. The same results were obtained when a cyclase-defective gene was overexpressed, showing that this odor-specific effect on adaptation does not depend on excess cGMP production. A link was also shown between ODR-1 and odor discrimination in the saturation paradigm. While wild-type animals are able to chemotax toward benzaldehyde or isoamyl alcohol in a saturating background of butanone, ODR-1(OE) animals were defective in chemotaxis toward both odorants in the presence of butanone. By contrast, ODR-1(OE) animals responded normally to butanone in a background of benzaldehyde; thus, the effect exhibits an interesting asymmetry. Interpretations of this effect are further constrained by another degree of specificity: ODR-1(OE) animals in saturating levels of butanone retain normal responses to a point source of 2,3-pentanedione, which is also sensed by AWC. The discrimination defect caused by overexpression of ODR-1 is different from the adaptation defect in that the discrimination effect requires a functional cyclase domain, as if the effect depended upon excess cGMP production. Why should butanone saturation evoke an effect not elicited by other odorants? Butanone signaling, unlike signaling by other AWC-sensed odorants, is mediated in part by the Gα protein GPA-2 (5Roayaie K. Crump J. Sagasti A. Bargmann C Neuron. 1998; 20: 55-67Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Mutation of this Gα subunit suppressed the discrimination defects of ODR-1(OE) animals, as if the effect of overexpression on odor discrimination depended on hyperactivation of the normal butanone signaling pathway. What does this elaborate series of observations tell us about the mechanism of odor discrimination? One interpretation is that in wild type, saturating levels of one odorant cause a specific downregulation of one odorant response, such that a gradient of a second odorant can be detected. In the mutant that overexpresses ODR-1, however, high levels of butanone signaling downregulate not one but several odor responses. In other words, the normal insulation between butanone signaling and other signaling pathways is compromised. In one version of this model, the downregulation occurs locally in wild-type animals and is effected by localized production of cGMP. In mutants overexpressing ODR-1, high levels of cGMP would result in a more global effect, spreading to downregulate additional pathways. Such a model would fit especially well if olfactory signaling complexes were spatially segregated from one another, as is found, for example, in Drosophila phototransduction (2Huber A. Sander P. Gobert A. Bahner M. Hermann R. 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One possible model is that ODR-1 binds to a protein required for butanone adaptation, titrates it, and thereby blocks adaptation. Perhaps signaling complexes are heterogeneous, such that complexes responding to other odors lack the protein bound by ODR-1 and are not affected by ODR-1 overexpression. These results invite further experimentation. It will be interesting to determine whether olfactory signaling components in various organisms do in fact cluster in discrete, spatially segregated complexes. As more is learned about the binding specificity of odorant receptors (12Zhao H. Ivic L. Otaki J.M. Hashimoto M. Mikoshiba K. Firestein S Science. 1998; 279: 237-242Crossref PubMed Scopus (504) Google Scholar, 4Malnic B. Hirono J. Sato T. Buck L.B Cell. 1999; 96: 713-723Abstract Full Text Full Text PDF PubMed Scopus (1594) Google Scholar, 8Speca D.J. Lin D.M. Sorensen P.W. Isacoff E.Y. Ngai J. Dittman A.H Neuron. 1999; 23: 487-498Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 9Touhara K. Sengoku S. Inaki K. Tsuboi A. Hirono J. Sato T. Sakano H. Haga T Proc. Natl. Acad. Sci. USA. 1999; 96: 4040-4045Crossref PubMed Scopus (249) Google Scholar), it will become more apparent whether competition for receptor binding sites plays any role in odor discrimination in various species, as has been proposed previously (7Siddiqi O Trends Genet. 1987; 3: 137-142Abstract Full Text PDF Scopus (73) Google Scholar). It should also be noted that many of the most interesting results from this work come from overexpression studies. Such studies can be enormously illuminating and incisive, but additional insight can often be gained by complementing them with studies of loss-of-function mutations. Insulation of signaling pathways is likely to be critical not only in C. elegans olfactory neurons but also in a wide variety of mammalian neurons, many of which express multiple receptors that converge on common signaling pathways. Thus, our understanding of signaling in many neuronal types may benefit from further consideration of olfactosomes.