Cross-linking evidence for motional constraints within chemoreceptor trimers of dimers.

Chemotactic behavior in bacteria relies on the sensing ability of large chemoreceptor clusters that are usually located at the cell pole. In Escherichia coli, chemoreceptors exhibit higher-order interactions within those clusters based on a trimer-of-dimers organization. This architecture is conserved in a variety of other bacteria and archaea, implying that receptors in many microorganisms form trimer-of-dimer signaling teams. To gain further insight into the assembly and dynamic behavior of receptor trimers of dimers, we used in vivo cross-linking targeted to cysteine residues at various positions that define six different levels along the cytoplasmic signaling domains of the aspartate and serine chemoreceptors, Tar and Tsr, respectively. We found that the cytoplasmic domains of these receptors are close to each other near the trimer contact region at the cytoplasmic tip and lie farther apart as the receptor dimers approach the cytoplasmic membrane. Tar and Tsr reporter sites within the same or closely adjacent levels readily formed mixed cross-links, whereas reporters located different distances from the tip did not. These findings indicate that there are no significant vertical displacements of one dimer with respect to the others within the trimer unit. Attractant stimuli had no discernible effect on the cross-linking efficiency of any of the reporters tested, but a strong osmotic stimulus reproducibly enhanced cross-linking at most of the reporter sites, indicating that individual dimers may move closer together under this condition.

Spatial choice is biased by chemical cues from conspecifics in the speckeled worm eel Myrophis punctatus

The speckeld worm eel Myrophis punctatus lives in high-densities assemblages, and usually digs through, or lies on the substrate. These behaviours could lead to chemical marks on the substrate and could modulate the spatial distribution in this species. We tested the hypothesis that the spatial choice of the speckled worm eel is modulated by the presence of conspecific odour on the substrate. Here, we showed that the speckled worm eel avoids the substrate area containing the conspecific odour, indicating that this chemical cue modulates the eel's spatial decision. The eels clearly detected the conspecific's odour. This perception might indicate the presence of conspecifics into the substrate. Since the eels avoided an area containing conspecific odour, we suggest this may be a response that avoids the consequences of invading a resident-animal's territory.

A enguia mirongo-mirim Myrophis punctatus vive em agrupamentos de alta densidade populacional e comumente se enterra ou permanece sob o substrato. Esses comportamentos podem levar a marcas químicas no subtrato e podem, portanto, modular o uso do espaço nessa espécie. Neste estudo, testamos a hipótese de que a preferência espacial da enguia mirongo-mirim é influenciada pela presença de odor do animal coespecífico no subtrato. Mostramos que as enguias evitam a área que contém tal odor, indicando que as decisões de ocupação espacial podem ser influenciadas por pistas químicas de coespecíficos. As enguias claramente detectaram o odor de um animal coespecífico e essa percepção poderia ser um indicativo da presença de um coespecífico enterrado no substrato. Visto que elas evitam uma área contendo tal odor, sugerimos que isso poderia ser uma resposta para evitar invadir o território de um animal residente.

Reconstitution of a chemical defense signaling pathway in a heterologous system.

Chemical signaling plays an important role in ecological interactions, such as communication and predator-prey dynamics. Since sessile species cannot physically escape predators, many contain compounds that deter predation; however, it is largely unknown how predators physiologically detect deterrent chemicals. Few studies have investigated ecologically relevant aversive taste responses in any predator. Our objective was to determine if a signaling pathway for detecting marine sponge-derived deterrent compounds could be reconstituted in a heterologous expression system to ultimately facilitate investigation of the molecular mechanism of such an aversive behavioral response. Zebrafish (Danio rerio) rejected artificial diets laced with sponge chemical defense compounds that were previously shown to deter a generalist marine predator, Thalassoma bifasciatum, suggesting that zebrafish can recognize deterrent compounds relevant to coral reef systems. Transcripts made from a zebrafish cDNA library were expressed in a heterologous system, Xenopus laevis oocytes, and tested for chemoreceptor activation via electrophysiology, using the cystic fibrosis transmembrane conductance regulator (CFTR) as a reporter. Oocytes expressing gene sequences from the library and CFTR exhibited a CFTR-like electrophysiological response to formoside and ectyoplasides A and B, sponge defense compounds. Therefore, the chemical defense-activated signaling pathway can be reconstituted in Xenopus oocytes. Kinetics of the responses suggested that the responses to formoside and ectyoplasides A and B were receptor-mediated and capable of using the G(alphas) signaling pathway in this system. This bioassay has the potential to lead to the identification of genes that encode receptors capable of interacting with deterrent chemicals, which would enable understanding of predator detection of chemical defenses.

Structure of the conserved HAMP domain in an intact, membrane-bound chemoreceptor: a disulfide mapping study.

The HAMP domain is a conserved motif widely distributed in prokaryotic and lower eukaryotic organisms, where it is often found in transmembrane receptors that regulate two-component signaling pathways. The motif links receptor input and output modules and is essential to receptor structure and signal transduction. Recently, a structure was determined for a HAMP domain isolated from an unusual archeal membrane protein of unknown function [Hulko, M., et al. (2006) Cell 126, 929-940]. This study uses cysteine and disulfide chemistry to test this archeal HAMP model in the full-length, membrane-bound aspartate receptor of bacterial chemotaxis. The chemical reactivities of engineered Cys residues scanned throughout the aspartate receptor HAMP region are highly correlated with the degrees of solvent exposure of corresponding positions in the archeal HAMP structure. Both domains are homodimeric, and the individual subunits of both domains share the same helix-connector-helix organization with the same helical packing faces. Moreover, disulfide mapping reveals that the four helices of the aspartate receptor HAMP domain are arranged in the same parallel, four-helix bundle architecture observed in the archeal HAMP structure. One detectable difference is the packing of the extended connector between helices, which is not conserved. Finally, activity studies of the aspartate receptor indicate that contacts between HAMP helices 1 and 2' at the subunit interface play a critical role in modulating receptor on-off switching. Disulfide bonds linking this interface trap the receptor in its kinase-activating on-state, or its kinase inactivating off-state, depending on their location. Overall, the evidence suggests that the archeal HAMP structure accurately depicts the architecture of the conserved HAMP motif in transmembrane chemoreceptors. Both the on- and off-states of the aspartate receptor HAMP domain closely resemble the archeal HAMP structure, and only a small structural rearrangement occurs upon on-off switching. A model incorporating HAMP into the full receptor structure is proposed.

Analyzing transmembrane chemoreceptors using in vivo disulfide formation between introduced cysteines.

The sulfhydryl chemistry possible at the thiol group of cysteine provides a very useful tool for probing protein structure and function. The power of site-specific mutagenesis makes it possible to use this tool at essentially any position in a polypeptide sequence. The reactivity of introduced cysteines is often assessed in vitro, using purified proteins or cell extracts. However, it can be particularly informative to probe the protein of interest in vivo, in its native cellular environment. Our laboratory has used in vivo approaches extensively in studies of bacterial transmembrane chemoreceptors, particularly by utilizing disulfide formation between pairs of introduced cysteines to learn about structural organization and mechanisms of function. We have concentrated on experimental conditions in which the cellular system of interest remained functional and thus the protein we were characterizing maintained not only its native structure but also its natural interactions. For this reason, our studies of bacterial transmembrane chemoreceptors using disulfide formation in vivo have focused in large part on cysteines separated from the reducing environment of the cell interior, in transmembrane or periplasmic domains. In this chapter, we discuss the applications and limitation of these approaches as well as the details of experimental manipulations and data analysis.

In vivo crosslinking methods for analyzing the assembly and architecture of chemoreceptor arrays.

The chemoreceptor molecules that mediate chemotactic responses in bacteria and archaea are physically clustered and operate as highly cooperative arrays. Few experimental approaches are able to investigate the structure-function organization of these chemoreceptor networks in living cells. This chapter describes chemical crosslinking methods that can be applied under normal physiological conditions to explore physical interactions between chemoreceptors and their underlying genetic and structural basis. Most of these crosslinking approaches are based on available atomic structures for chemoreceptor homodimers, the fundamental building block for higher-order networks. However, the general logic of our in vivo crosslinking approaches is readily applicable to other protein-protein interactions and other organisms, even when high-resolution structural information is not available.

Secretory cells of the airway express molecules of the chemoreceptive cascade.

Airway secretion is maintained by specialized non-ciliated epithelial cells whose phenotype varies with their topographical location. In addition, specialized epithelial cells located in the airway contain the molecular machinery of chemoreceptive elements. Our aim has been to evaluate whether the secretory cells themselves possess a chemoreceptive capability, which requires the simultaneous presence of chemosensory and secretory mechanisms. We performed immunohistochemical analysis with antibodies against the Clara-cell-specific secretory proteins, CC10 and CC26, as secretory markers. As chemoreceptive markers, we employed antibodies against alpha-gustducin and phospholipase C beta 2 (PLCbeta2), two components of the taste transduction pathway. We also attempted to characterize further the secretory cell type by using a marker of chloride secretion, cystic fibrosis transmembrane regulator (CFTR). We found alpha-gustducin localized in non-ciliated cells of the epithelium lining the trachea and bronchioles of adult rats, where it was also co-expressed with CC10 and CC26. Ultrastructural immunohistochemistry revealed alpha-gustducin in the apical cytoplasm of secretory cells, concentrated around and inside the granules. CFTR was also observed in a subpopulation of non-ciliated epithelial cells, co-localized with some alpha-gustducin- and PLCbeta2-immunoreactive cells, at all levels of the airway epithelium. We conclude that non-ciliated epithelial cells of the rat airway express components of distinct signaling mechanisms and suggest that secretory events are driven by a molecular mechanism activated by the binding of luminal substances to G-protein-coupled receptors.

Balls and chains--a mesoscopic approach to tethered protein domains.

Many proteins contain regions of unstructured polypeptide chain that appear to be flexible and to undergo random thermal motion. In some cases the unfolded sequence acts as a flexible tether that restricts the diffusion of a globular protein domain for the purpose of catalysis or self-assembly. In this article, we present a stochastic model for tethered protein domains under various conditions and solve it numerically to deduce the general and dynamic properties of these systems. A critical domain size dependent on the length of the tether is presented, above which a spherical domain tethered to an impenetrable wall by a flexible chain displays a restricted localization between two concentric half-shells. Results suggest that the diffusion of such a spherical domain is effectively reduced in its dimensionality and able to explore the available space with high efficiency. It also becomes clear that the orientation of the ball is not independent of the distance from the tethering point but becomes more constrained as the linking tether is extended. The possible biological significance of these and other results is discussed.

Soluble proteins in insect chemical communication.

Our understanding of the biochemical mechanisms that mediate chemoreception in insects has been greatly improved after the discovery of olfactory and taste receptor proteins. However, the presence of soluble polypeptides in high concentration around the dendrites of sensory neurons still poses unanswered questions. More than 2 decades after their discovery and despite the wealth of structural information available, the physiological function of odorant-binding proteins is not well understood. More recently, members of a second family of soluble polypeptides, the chemosensory proteins, were also discovered in the lymph of chemosensilla. Here we review the structural properties of both classes of soluble proteins, their affinity to small ligands, and their expression in the different parts of the insect body and subcellular localisation. Finally, we discuss current ideas and models of the role of such proteins in insect chemoreception.

Control of chemotactic signal gain via modulation of a pre-formed receptor array.

The remarkably wide dynamic range of the chemotactic pathway of Escherichia coli, a model signal transduction system, is achieved by methylation/amidation of the transmembrane chemoreceptors that regulate the histidine kinase CheA in response to extracellular stimuli. The chemoreceptors cluster at a cell pole together with CheA and the adaptor CheW. Several lines of evidence have led to models that assume high cooperativity and sensitivity via collaboration of receptor dimers within a cluster. Here, using in vivo disulfide cross-linking assays, we have demonstrated a well defined arrangement of the aspartate chemoreceptor (Tar). The differential effects of amidation on cross-linking at different positions indicate that amidation alters the relative orientation of Tar dimers to each other (presumably inducing rotational displacements) without much affecting the conformation of the periplasmic domains. Interestingly, the effect of aspartate on cross-linking at any position tested was roughly opposite to that of receptor amidation. Furthermore, amidation attenuated the effects of aspartate by several orders of magnitude. These results suggest that receptor covalent modification controls signal gain by altering the arrangement or packing of receptor dimers in a pre-formed cluster.

Diagnostic cross-linking of paired cysteine pairs demonstrates homologous structures for two chemoreceptor domains with low sequence identity.

Hundreds of bacterial chemoreceptors from many species have periplasmic, ligand-recognition domains of approximately the same size, but little or no sequence identity. The only structure determined is for the periplasmic domain of chemoreceptor Tar from Salmonella and Escherichia coli. Do sequence-divergent but similarly sized chemoreceptor periplasmic domains have related structures? We addressed this issue for the periplasmic domain of chemoreceptor Trg(E) from E. coli, which has a low level of sequence similarity to Tar, by combining homology modeling and diagnostic cross-linking between pairs of introduced cysteines. A homology model of the Trg(E) domain was created using the homodimeric, four-helix bundle structure of the Tar(S) domain from Salmonella. In this model, we chose four pairs of positions at which introduced cysteines would be sufficiently close to form disulfides across each of four different helical interfaces. For each pair we chose a second pair, in which one cysteine of the original pair was shifted by one position around the helix and thus would be less favorably placed for disulfide formation. We created genes coding for proteins containing four such pairs of cysteine pairs and investigated disulfide formation in vivo as well as functional consequences of the substitutions and disulfides between neighboring helices. Results of the experimental tests provided strong support for the accuracy of the model, indicating that the Trg(E) periplasmic domain is very similar to the Tar(S) domain. Diagnostic cross-linking of paired pairs of introduced cysteines could be applied generally as a stringent test of homology models.

Ligand-induced conformational changes in the Bacillus subtilis chemoreceptor McpB determined by disulfide crosslinking in vivo.

Previously, we characterized the organization of the transmembrane (TM) domain of the Bacillus subtilis chemoreceptor McpB using disulfide crosslinking. Cysteine residues were engineered into serial positions along the two helices through the membrane, TM1 and TM2, as well as double mutants in TM1 and TM2, and the extent of crosslinking determined to characterize the organization of the TM domain. In this study, the organization of the TM domain was studied in the presence and absence of ligand to address what ligand-induced structural changes occur. We found that asparagine caused changes in crosslinking rate on all residues along the TM1-TM1' helical interface, whereas the crosslinking rate for almost all residues along the TM2-TM2' interface did not change. These results indicated that helix TM1 rotated counterclockwise and that TM2 did not move in respect to TM2' in the dimer on binding asparagine. Interestingly, intramolecular crosslinking of paired substitutions in 34/280 and 38/273 were unaffected by asparagine, demonstrating that attractant binding to McpB did not induce a "piston-like" vertical displacement of TM2 as seen for Trg and Tar in Escherichia coli. However, these paired substitutions produced oligomeric forms of receptor in response to ligand. This must be due to a shift of the interface between different receptor dimers, within previously suggested trimers of dimers, or even higher order complexes. Furthermore, the extent of disulfide bond formation in the presence of asparagine was unaffected by the presence of the methyl-modification enzymes, CheB and CheR, or the coupling proteins, CheW and CheV, demonstrating that these proteins must have local structural effects on the cytoplasmic domain that is not translated to the entire receptor. Finally, disulfide bond formation was also unaffected by binding proline to McpC. We conclude that ligand-binding induced a conformational change in the TM domain of McpB dimers as an excitation signal that is likely propagated within the cytoplasmic region of receptors and that subsequent adaptational events do not affect this new TM domain conformation.

Espins are multifunctional actin cytoskeletal regulatory proteins in the microvilli of chemosensory and mechanosensory cells.

Espins are associated with the parallel actin bundles of hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction in mice and humans. Here, we report that espins are also concentrated in the microvilli of a number of other sensory cells: vomeronasal organ sensory neurons, solitary chemoreceptor cells, taste cells, and Merkel cells. Moreover, we show that hair cells and these other sensory cells contain novel espin isoforms that arise from a different transcriptional start site and differ significantly from other espin isoforms in their complement of ligand-binding activities and their effects on actin polymerization. The novel espin isoforms of sensory cells bundled actin filaments with high affinity in a Ca(2+)-resistant manner, bound actin monomer via a WASP (Wiskott-Aldrich syndrome protein) homology 2 domain, bound profilin via a single proline-rich peptide, and caused a dramatic elongation of microvillus-type parallel actin bundles in transfected epithelial cells. In addition, the novel espin isoforms of sensory cells differed from other espin isoforms in that they potently inhibited actin polymerization in vitro, did not bind the Src homology 3 domain of the adapter protein insulin receptor substrate p53, and did not bind the acidic, signaling phospholipid phosphatidylinositol 4,5-bisphosphate. Thus, the espins constitute a family of multifunctional actin cytoskeletal regulatory proteins with the potential to differentially influence the organization, dimensions, dynamics, and signaling capabilities of the actin filament-rich, microvillus-type specializations that mediate sensory transduction in various mechanosensory and chemosensory cells.

A highly conserved candidate chemoreceptor expressed in both olfactory and gustatory tissues in the malaria vector Anopheles gambiae.

Anopheles gambiae is a highly anthropophilic mosquito responsible for the majority of malaria transmission in Africa. The biting and host preference behavior of this disease vector is largely influenced by its sense of smell, which is presumably facilitated by G protein-coupled receptor signaling [Takken, W. & Knols, B. (1999) Annu. Rev. Entomol. 44, 131-157]. Because of the importance of host preference to the mosquitoes' ability to transmit disease, we have initiated studies intended to elucidate the molecular mechanisms underlying olfaction in An. gambiae. In the course of these studies, we have identified a number of genes potentially involved in signal transduction, including a family of candidate odorant receptors. One of these receptors, encoded by GPRor7 (hereafter referred to as AgOr7), is remarkably similar to an odorant receptor that is expressed broadly in olfactory tissues and has been identified in Drosophila melanogaster and other insects [Krieger, J., Klink, O., Mohl, C., Raming, K. & Breer, H. (2003) J. Comp. Physiol. A 189, 519-526; Vosshall, L. B., Amrein, H., Morozov, P. S., Rzhetsky, A. & Axel, R. (1999) Cell 96, 725-736]. We have observed AgOr7 expression in olfactory and gustatory tissues in adult An. gambiae and during several stages of the mosquitoes' development. Within the female adult peripheral chemosensory system, antiserum against the AgOR7 polypeptide labels most sensilla of the antenna and maxillary palp as well as a subset of proboscis sensilla. Furthermore, AgOR7 antiserum labeling is observed within the larval antenna and maxillary palpus. These results are consistent with a role for AgOr7 in both olfaction and gustation in An. gambiae and raise the possibility that AgOr7 orthologs may also be of general importance to both modalities of chemosensation in other insects.

Transmembrane organization of the Bacillus subtilis chemoreceptor McpB deduced by cysteine disulfide crosslinking.

The Bacillus subtilis chemoreceptor McpB is a dimer of identical subunits containing two transmembrane (TM) segments (TM1, residues 17-34: TM2, residues 280-302) in each monomer with a 2-fold axis of symmetry. To study the organization of the TM domains, the wild-type receptor was mutated systematically at the membrane bilayer/extracytoplasmic interface with 15 single cysteine (Cys) substitutions in each of the two TM domains. Each single Cys substitution was capable of complementing a null allele in vivo, suggesting that no significant perturbation of the native tertiary or quaternary structure of the chemoreceptor was introduced by the mutations. On the basis of patterns of disulfide crosslinking between subunits of the dimeric receptor, an alpha-helical interface was identified between TM1 and TM1' (containing residues 32, 36, 39, and 43) and between TM2 and TM2' (containing residues 276, 277, 280, 283 and 286). Pairs of cysteine substitutions (positions 34/280 and 38/273) in TM1 and TM2 were used to further elucidate specific contacts within a monomer subunit, enabling a model to be constructed defining the organization of the TM domain. Crosslinking of residues that were 150-180 degrees removed from position 32 (positions 37, 41, and 44) suggested that the receptors may be organized as an array of trimers of dimers in vivo. All crosslinking was unaffected by deletion of cheB and cheR (loss of receptor demethylation/methylation enzymes) or by deletion of cheW and cheV (loss of proteins that couple receptors with the autophosphorylating kinase). These findings indicate that the organization of the transmembrane region and the stability of the quaternary complex of receptors are independent of covalent modifications of the cytoplasmic domain and conformations in the cytoplasmic domain induced by the coupling proteins.

Neuroepithelial cells and associated innervation of the zebrafish gill: a confocal immunofluorescence study.

Peripheral chemoreceptors responsive to hypoxia have been well characterized in air-breathing vertebrates, but poorly in water-breathers. The present study examined the distribution of five populations of neuroepithelial cells (NECs), putative O(2) chemoreceptors, and innervation patterns in the zebrafish gill using whole-mounts and confocal immunofluorescence. Nerve bundles and fibers of the gill were labeled with zn-12 (a zebrafish-specific neuronal marker) and SV2 antisera and NECs were characterized by serotonin (5-HT) immunoreactivity (IR), SV2-IR and the purinoceptor P2X(3)-IR. A zn-12-IR nerve bundle extended the length of the gill filament and gave rise to a nerve plexus surrounding the efferent filament artery (eFA) and a rich network of fibers that innervated both serotonergic and nonserotonergic NECs of the filament and lamellar epithelium. Three populations of serotonergic, SV2-IR neurons intrinsic to the gill filaments are described, one of which provided innervation to NECs of the filament epithelium. Degeneration of nerve fibers in gill arches maintained in explant culture for 2 days revealed the extrinsic origin of nerve fibers of the plexus and lamellae and the innervation of filament NECs by both intrinsic and extrinsic fibers. Intrinsic innervation surrounding the eFA survived in explant cultures, suggesting a mechanism of local vascular control within the gill. In addition, NECs survived in explants after degeneration of extrinsic nerve fibers. Thus, NECs of the zebrafish gill are organized in a manner reminiscent of O(2) chemoreceptors of mammalian vertebrates, suggesting a role in respiratory regulation.

Localization of gonadotropin-releasing hormone in the central nervous system and a peripheral chemosensory organ of Aplysia californica.

Gonadotropin-releasing hormone (GnRH) is a neurohormone crucial for the regulation of reproductive and neural functions in vertebrates. Recent discoveries of GnRH immunoreactivity (IR) in a number of invertebrates raised the possibility that GnRH may be an ancient molecule that had arisen before the emergence of Phylum Chordata. We previously demonstrated the presence of a GnRH IR similar to the mammalian (m) and tunicate I (tI) forms of GnRH in the hemolymph and ovotestis of an opisthobranch mollusk, Aplysia californica; however, the presence of GnRH in the central nervous system (CNS) of A. californica could not be detected with the available antisera against various forms of chordate GnRH. In the present study, we performed immunohistochemistry (IHC) to localize the presence of GnRH in the CNS and a peripheral chemosensory organ, the osphradium, of A. californica. A newly generated antiserum against tI-GnRH revealed the strong expression of GnRH IR in neurons of all CNS ganglia. A notable asymmetry in immunostaining was detected in the left and right abdominal hemiganglia. The CNS is rich in tI-GnRH immunoreactive neurons but lacks mGnRH IR, whereas the osphradium contains abundant mGnRH immunoreactive neurons but lacks tI-GnRH IR. The extract of CNS failed to stimulate the release of LH from mouse pituitary, demonstrating that the A. californica GnRH IR is structurally different from what is required to bind and activate mammalian GnRH receptor. Together, these results indicate the presence of at least two distinct GnRH systems in A. californica. The presence of GnRH in the osphradium is consistent with the long-standing anatomical relationship between GnRH and the chemosensory system observed in vertebrates.

Bioinformatics-based identification of chemosensory proteins in African Malaria Mosquito, Anopheles gambiae.

Chemosensory proteins (CSPs) are identifiable by four spatially conserved Cysteine residues in their primary structure or by two disulfide bridges in their tertiary structure according to the previously identified olfactory specific-D related proteins. A genomics- and bioinformatics-based approach is taken in the present study to identify the putative CSPs in the malaria-carrying mosquito, Anopheles gambiae. The results show that five out of the nine annotated candidates are the most possible Anopheles CSPs of A. gambiae. This study lays the foundation for further functional identification of Anopheles CSPs, though all of these candidates need additional experimental verification.

Modeling the transmembrane domain of bacterial chemoreceptors.

Bacterial chemoreceptors signal across the membrane by conformational changes that traverse a four-helix transmembrane domain. High-resolution structures are available for the chemoreceptor periplasmic domain and part of the cytoplasmic domain but not for the transmembrane domain. Thus, we constructed molecular models of the transmembrane domains of chemoreceptors Trg and Tar, using coordinates of an unrelated four-helix coiled coil as a template and the X-ray structure of a chemoreceptor periplasmic domain to establish register and positioning. We tested the models using the extensive data for cross-linking propensities between cysteines introduced into adjacent transmembrane helices, and we found that many aspects of the models corresponded with experimental observations. The one striking disparity, the register of transmembrane helix 2 (TM2) relative to its partner transmembrane helix 1, could be corrected by sliding TM2 along its long axis toward the periplasm. The correction implied that axial sliding of TM2, the signaling movement indicated by a large body of data, was of greater magnitude than previously thought. The refined models were used to assess effects of inter-helical disulfides on the two ligand-induced conformational changes observed in alternative crystal structures of periplasmic domains: axial sliding within a subunit and subunit rotation. Analyses using a measure of disulfide potential energy provided strong support for the helical sliding model of transmembrane signaling but indicated that subunit rotation could be involved in other ligand-induced effects. Those analyses plus modeled distances between diagnostic cysteine pairs indicated a magnitude for TM2 sliding in transmembrane signaling of several angstroms.

A putative pheromone receptor gene is expressed in two distinct olfactory organs in goats.

Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.