Bettina Malnic

The mouse olfactory receptor gene family

In mammals, odor detection in the nose is mediated by a diverse family of olfactory receptors (ORs), which are used combinatorially to detect different odorants and encode their identities. The OR family can be divided into subfamilies whose members are highly related and are likely to recognize structurally related odorants. To gain further insight into the mechanisms underlying odor detection, we analyzed the mouse OR gene family. Exhaustive searches of a mouse genome database identified 913 intact OR genes and 296 OR pseudogenes. These genes were localized to 51 different loci on 17 chromosomes. Sequence comparisons showed that the mouse OR family contains 241 subfamilies. Subfamily sizes vary extensively, suggesting that some classes of odorants may be more easily detected or discriminated than others. Determination of subfamilies that contain ORs with identified ligands allowed tentative functional predictions for 19 subfamilies. Analysis of the chromosomal locations of members of each subfamily showed that many OR gene loci encode only one or a few subfamilies. Furthermore, most subfamilies are encoded by a single locus, suggesting that different loci may encode receptors for different types of odorant structural features. Comparison of human and mouse OR subfamilies showed that the two species have many, but not all, subfamilies in common. However, mouse subfamilies are usually larger than their human counterparts. This finding suggests that humans and mice recognize many of the same odorant structural motifs, but mice may be superior in odor sensitivity and discrimination.

Regulatory Properties of the NH2- and COOH-terminal Domains of Troponin T ATPase ACTIVATION AND BINDING TO TROPONIN I AND TROPONIN C

The contraction of skeletal muscle is regulated by Ca2+ binding to troponin C, which results in an internal reorganization of the interactions within the troponin-tropomyosin complex. Troponin T is necessary for Ca2+-dependent inhibition and activation of actomyosin. Troponin T consists of an extended NH2-terminal domain that interacts with tropomyosin and a globular COOH-terminal domain that interacts with tropomyosin, troponin I, and troponin C. In this study we used recombinant troponin T and troponin I fragments to delimit further the structural and regulatory interactions with the thin filament. Our results show the following: (i) the NH2-terminal region of troponin T activates the actomyosin ATPase in the presence of tropomyosin; (ii) the interaction of the globular domain of troponin T with the thin filament blocks ATPase activation in the absence of Ca2+; and (iii) the COOH-terminal region of the globular domain anchors the troponin C-troponin I binary complex to troponin T through a direct Ca2+-independent interaction with the NH2-terminal region of troponin I. This interaction is required for Ca2+-dependent activation of the actomyosin ATPase activity. Based on these results we propose a refined model for the troponin complex and its interaction with the thin filament.

Ric-8B, an Olfactory Putative GTP Exchange Factor, Amplifies Signal Transduction through the Olfactory-Specific G-Protein Gαolf

The olfactory system is able to detect a large number of chemical structures with a remarkable sensitivity and specificity. Odorants are first detected by odorant receptors present in the cilia of olfactory neurons. The activated receptors couple to an olfactory-specific G-protein (Golf), which activates adenylyl cyclase III to produce cAMP. Increased cAMP levels activate cyclic nucleotide-gated channels, causing cell membrane depolarization. Here we used yeast two-hybrid to search for potential regulators for Gαolf. We found that Ric-8B (for resistant to inhibitors of cholinesterase), a putative GTP exchange factor, is able to interact with Gαolf. Like Gαolf, Ric-8B is predominantly expressed in the mature olfactory sensory neurons and also in a few regions in the brain. The highly restricted and colocalized expression patterns of Ric-8B and Gαolf strongly indicate that Ric-8B is a functional partner for Gαolf. Finally, we show that Ric-8B is able to potentiate Gαolf-dependent cAMP accumulation in human embryonic kidney 293 cells and therefore may be an important component for odorant signal transduction.

Ric-8B interacts with Gαolf and Gγ13 and co-localizes with Gαolf, Gβ1 and Gγ13 in the cilia of olfactory sensory neurons

Olfactory sensory neurons are able to detect odorants with high sensitivity and specificity. We have demonstrated that Ric-8B, a guanine nucleotide exchange factor (GEF), interacts with Galphaolf and enhances odorant receptor signaling. Here we show that Ric-8B also interacts with Ggamma13, a divergent member of the Ggamma subunit family which has been implicated in taste signal transduction, and is abundantly expressed in the cilia of olfactory sensory neurons. We show that Gbeta1 is the predominant Gbeta subunit expressed in the olfactory sensory neurons. Ric-8B and Gbeta1, like Galphaolf and Ggamma13, are enriched in the cilia of olfactory sensory neurons. We also show that Ric-8B interacts with Galphaolf in a nucleotide dependent manner, consistent with the role as a GEF. Our results constitute the first example of a GEF protein that interacts with two different olfactory G protein subunits and further implicate Ric-8B as a regulator of odorant signal transduction.

Searching for the Ligands of Odorant Receptors

Through the sense of smell mammals can detect and discriminate between a large variety of odorants present in the surrounding environment. Odorants bind to a large repertoire of odorant receptors located in the cilia of olfactory sensory neurons of the nose. Each olfactory neuron expresses one single type of odorant receptor, and neurons expressing the same type of receptor project their axons to one or a few glomeruli in the olfactory bulb, creating a map of odorant receptor inputs. The information is then passed on to other regions of the brain, leading to odorant perception. To understand how the olfactory system discriminates between odorants, it is necessary to determine the odorant specificities of individual odorant receptors. These studies are complicated by the extremely large size of the odorant receptor family and by the poor functional expression of these receptors in heterologous cells. This article provides an overview of the methods that are currently being used to investigate odorant receptor–ligand interactions.

Genome-Wide Detection of Serpentine Receptor-Like Proteins in Malaria Parasites

Serpentine receptors comprise a large family of membrane receptors distributed over diverse organisms, such as bacteria, fungi, plants and all metazoans. However, the presence of serpentine receptors in protozoan parasites is largely unknown so far. In the present study we performed a genome-wide search for proteins containing seven transmembrane domains (7-TM) in the human malaria parasite Plasmodium falciparum and identified four serpentine receptor-like proteins. These proteins, denoted PfSR1, PfSR10, PfSR12 and PfSR25, show membrane topologies that resemble those exhibited by members belonging to different families of serpentine receptors. Expression of the pfsrs genes was detected by Real Time PCR in P. falciparum intraerythrocytic stages, indicating that they potentially code for functional proteins. We also found corresponding homologues for the PfSRs in five other Plasmodium species, two primate and three rodent parasites. PfSR10 and 25 are the most conserved receptors among the different species, while PfSR1 and 12 are more divergent. Interestingly, we found that PfSR10 and PfSR12 possess similarity to orphan serpentine receptors of other organisms. The identification of potential parasite membrane receptors raises a new perspective for essential aspects of malaria parasite host cell infection.

Ric-8A, a Gα Protein Guanine Nucleotide Exchange Factor Potentiates Taste Receptor Signaling

Taste receptors for sweet, bitter and umami tastants are G-protein-coupled receptors (GPCRs). While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS), RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs) are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

Nuclear compartmentalization of odorant receptor genes

Significance The mechanisms underlying odorant receptor (OR) monoallelic and monogenic expression are unclear. We show for the first time that the nuclei of olfactory neurons have a characteristic organization of facultative heterochromatin: it is highly concentrated around a large constitutive heterochromatin block located centrally in the nucleus. In addition, we show that the homologous alleles of a given OR gene are located in different chromatin compartments in the olfactory nucleus: while one of the alleles is associated with deeply repressed constitutive heterochromatin, the other one is located in or close to the more plastic facultative heterochromatin. This spatial organization is likely to be important for both the monoallelic and monogenic expression of these genes. Odorants are detected by odorant receptors, which are located on olfactory sensory neurons of the nose. Each olfactory sensory neuron expresses one single odorant receptor gene allele from a large family of odorant receptor genes. To gain insight into the mechanisms underlying this monogenic and monoallelic expression, we examined the 3D nuclear organization of olfactory sensory neurons and determined the positions of homologous odorant receptor gene alleles in relation to different nuclear compartments. Our results show that olfactory neurons exhibit a singular nuclear architecture that is characterized by a large centrally localized constitutive heterochromatin block and by the presence of prominent facultative heterochromatin domains that are localized around this constitutive heterochromatin block. We also found that the two homologous alleles of a given odorant receptor gene are frequently segregated to separate compartments in the nucleus, with one of the alleles localized to the constitutive heterochromatin block and the other one localized to the more plastic facultative heterochromatin, or next to it. Our findings suggest that this nuclear compartmentalization may play a critical role in the expression of odorant receptor genes.

CD36 is expressed in a defined subpopulation of neurons in the olfactory epithelium.

The sensory neurons in the olfactory epithelium (OSNs) are equipped with a large repertoire of olfactory receptors and the associated signal transduction machinery. In addition to the canonical OSNs, which express odorant receptors (ORs), the epithelium contains specialized subpopulations of sensory neurons that can detect specific information from environmental cues and relay it to relevant neuronal circuitries. Here we describe a subpopulation of mature OSNs in the main olfactory epithelium (MOE) which expresses CD36, a multifunctional receptor involved in a series of biological processes, including sensory perception of lipid ligands. The Cd36 expressing neurons coexpress markers of mature OSNs and are dispersed throughout the MOE. Unlike several ORs analyzed in our study, we found frequent coexpression of the OR Olfr287 in these neurons, suggesting that only a specific set of ORs may be coexpressed with CD36 in OSNs. We also show that CD36 is expressed in the cilia of OSNs, indicating a possible role in odorant detection. CD36-deficient mice display no signs of gross changes in the organization of the olfactory epithelium, but show impaired preference for a lipid mixture odor. Our results show that CD36-expressing neurons represent a distinct population of OSNs, which may have specific functions in olfaction.

Identification of agonists for a group of human odorant receptors

Olfaction plays a critical role in several aspects of the human life. Odorants are detected by hundreds of odorant receptors (ORs) which belong to the superfamily of G protein-coupled receptors. These receptors are expressed in the olfactory sensory neurons of the nose. The information provided by the activation of different combinations of ORs in the nose is transmitted to the brain, leading to odorant perception and emotional and behavioral responses. There are ~400 intact human ORs, and to date only a small percentage of these receptors (~10%) have known agonists. The determination of the specificity of the human ORs will contribute to a better understanding of how odorants are discriminated by the olfactory system. In this work, we aimed to identify human specific ORs, that is, ORs that are present in humans but absent from other species, and their corresponding agonists. To do this, we first selected 22 OR gene sequences from the human genome with no counterparts in the mouse, rat or dog genomes. Then we used a heterologous expression system to screen a subset of these human ORs against a panel of odorants of biological relevance, including foodborne aroma volatiles. We found that different types of odorants are able to activate some of these previously uncharacterized human ORs.