Although it has been known for some time that olfactory receptors (ORs) reside in spermatozoa, the function of these ORs is unknown. Here, we identified, cloned, and functionally expressed a previously undescribed human testicular OR, hOR17-4. With the use of ratiofluorometric imaging, Ca2+ signals were induced by a small subset of applied chemical stimuli, establishing the molecular receptive fields for the recombinantly expressed receptor in human embryonic kidney (HEK) 293 cells and the native receptor in human spermatozoa. Bourgeonal was a powerful agonist for both recombinant and native receptor types, as well as a strong chemoattractant in subsequent behavioral bioassays. In contrast, undecanal was a potent OR antagonist to bourgeonal and related compounds. Taken together, these results indicate that hOR17-4 functions in human sperm chemotaxis and may be a critical component of the fertilization process.

1 TRPM8 (CMR1) is a Ca 2 þ -permeable channel, which can be activated by low temperatures, menthol, eucalyptol and icilin. It belongs to the transient receptor potential (TRP) family, and therefore is related to vanilloid receptor type-1 (VR1, TRPV1). We tested whether substances which are structurally related to menthol, or which produce a cooling sensation, could activate TRPM8, and compared the responses of TRPM8 and VR1 to these ligands. 2 The effects of 70 odorants and menthol-related substances on recombinant mouse TRPM8 (mTRPM8), expressed in HEK293 cells, were examined using a FLIPR s assay. In all, 10 substances (linalool, geraniol, hydroxycitronellal, WS-3, WS-23, FrescolatMGA, FrescolatML, PMD38, CoolactP and Cooling Agent 10) were found to be agonists. 3 The EC 50 values of the agonists defined their relative potencies: icilin (0.270.1 mM)4FrescolatML (3.371.5 mM) 4 WS-3 (3.771.7 mM) (À)menthol (4.171.3 mM) frescolatMAG (4.871.1 mM) 4 cooling agent 10 (672.2 mM) (þ )menthol (14.471.3 mM) 4 PMD38 (3171.1 mM) 4 WS-23 (4477.3 mM) 4 Coolact P (66720 mM) 4 geraniol (5.971.6 mM) 4 linalool (6.772.0 mM) 4 eucalyptol (7.772.0 mM) 4 hydroxycitronellal (19.672.2 mM). 4 Known VR1 antagonists (BCTC, thio-BCTC and capsazepine) were also able to block the response of TRPM8 to menthol (IC 50 : 0.871.0, 3.571.1 and 1871.1 mM, respectively). 5 The Ca 2 þ response of hVR1-transfected HEK293 cells to the endogenous VR1 agonist Narachidonoyl-dopamine was potentiated by low pH. In contrast, menthol-and icilin-activated TRPM8 currents were suppressed by low pH. 6 In conclusion, in the present study, we identified 10 new agonists and three antagonists of TRPM8. We found that, in contrast to VR1, TRPM8 is inhibited rather than potentiated by protons.

Olfactory receptors (ORs) are expressed not only in the sensory neurons of the olfactory epithelium, where they detect volatile substances, but also in various other tissues where their potential functions are largely unknown. Here, we report the physiological characterization of human OR51E2, also named prostate-specific G-protein-coupled receptor (PSGR) due to its reported up-regulation in prostate cancer. We identified androstenone derivatives as ligands for the recombinant receptor. PSGR can also be activated with the odorant ␤-ionone. Activation of the endogenous receptor in prostate cancer cells by the identified ligands evoked an intracellular Ca 2؉ increase. Exposure to ␤-ionone resulted in the activation of members of the MAPK family and inhibition of cell proliferation. Our data give support to the hypothesis that because PSGR signaling could reduce growth of prostate cancer cells, specific receptor ligands might therefore be potential candidates for prostate cancer treatment.Excessive signaling by G-protein-coupled receptors (GPCRs) 3 such as endothelin A receptor (1), bradykinin 1 receptor (2), follicle-stimulating hormone receptor (3), and thrombin receptor (4, 5) is known to occur in prostate cancers due to strong overexpression of the respective receptors. Activation of some of these GPCRs results in androgen-independent androgen receptor activation, thus promoting the transition of prostate cancer cells from an androgen-dependent to an androgen-independent state (6, 7).The prostate-specific G-protein-coupled receptor (PSGR) is a class A GPCR that was initially identified as a prostate-specific tumor biomarker (8 -10). It is specifically expressed in prostate epithelial cells, and its expression increases significantly in human prostate intraepithelial neoplasia and prostate tumors, suggesting that PSGR may play an important role in early prostate cancer development and progression (9, 11). Although expression of the human PSGR was found to be prostate-specific (10, 12), mRNA can also be detected in the olfactory zone and the medulla oblongata of the human brain (12). Human PSGR shares 93% amino acid homology to the respective mouse and rat homologues, which are also expressed in the brain (12). Interestingly, PSGR has numerous sequence motifs in common with the large superfamily of olfactory receptors (ORs), which build the largest class of human GPCRs and allow the recognition of a wide range of structurally diverse molecules in the nasal epithelium (13-15). Recently, also the steroid hormones androstenone and androstadienone were identified as OR ligands (16). In addition to their role in the sensory neurons of the nose, ORs have been found in different tissues throughout the body (17,18). Their function(s) in these extranasal locations are questionable except for in a few cases where functional studies have been performed in spermatozoa (19,20) and in enterochromaffin cells of the gastrointestinal tract (21).Here, we report the identification of steroid ligands of heterologously expressed PSGR...

Olfactory receptors (ORs) provide the molecular basis for the detection of volatile odorant molecules by olfactory sensory neurons. The OR supergene family encodes G-protein coupled proteins that belong to the seven-transmembrane-domain receptor family. It was initially postulated that ORs are exclusively expressed in the olfactory epithelium. However, recent studies have demonstrated ectopic expression of some ORs in a variety of other tissues. In the present study, we conducted a comprehensive expression analysis of ORs using an extended panel of human tissues. This analysis made use of recent dramatic technical developments of the so-called Next Generation Sequencing (NGS) technique, which encouraged us to use open access data for the first comprehensive RNA-Seq expression analysis of ectopically expressed ORs in multiple human tissues. We analyzed mRNA-Seq data obtained by Illumina sequencing of 16 human tissues available from Illumina Body Map project 2.0 and from an additional study of OR expression in testis. At least some ORs were expressed in all the tissues analyzed. In several tissues, we could detect broadly expressed ORs such as OR2W3 and OR51E1. We also identified ORs that showed exclusive expression in one investigated tissue, such as OR4N4 in testis. For some ORs, the coding exon was found to be part of a transcript of upstream genes. In total, 111 of 400 OR genes were expressed with an FPKM (fragments per kilobase of exon per million fragments mapped) higher than 0.1 in at least one tissue. For several ORs, mRNA expression was verified by RT-PCR. Our results support the idea that ORs are broadly expressed in a variety of tissues and provide the basis for further functional studies.

Members of the superfamily of transient receptor potential (TRP) channels are proposed to play important roles in sensory physiology. As an excitatory ion channel TRPA1 is robustly activated by pungent irritants in mustard and garlic and is suggested to mediate the inflammatory actions of environmental irritants and proalgesic agents. Here, we demonstrate that, in addition to pungent natural compounds, Ca 2؉ directly gates heterologously expressed TRPA1 in whole-cell and excised-patch recordings with an apparent EC 50 of 905 nM. Pharmacological experiments and site-directed mutagenesis indicate that the N-terminal EFhand calcium-binding domain of the channel is involved in Ca 2؉ -dependent activation. Furthermore, we determine Ca 2؉ as prerequisite for icilin activity on TRPA1. The transient receptor potential channel A1 (TRPA1)2 is a member of the superfamily of TRP channels. In mammals, A1 forms its own subfamily and is distinguished from other TRP channels by the presence of ϳ14 ankyrin repeats in its N terminus (1). TRPA1 was initially described as a cold sensitive nonselective cation channel (2), but it also functions as a ligand-gated channel in heterologous expression systems and sensory neurons (3). The pungent ingredients in mustard (allyl isothiocyanate, AITC) and garlic (allicin) robustly activate TRPA1 currents (4 -6). In addition, TRPA1 appears to be regulated by phospholipase C (PLC)-coupled receptors, suggesting that channel opening can be mediated by second messengers (4,7,8).TRPA1 expression was first described in a subset of sensory neurons of dorsal root and trigeminal ganglia that contribute to nociception and co-express calcitonin gene related peptide, substance P and TRPV1 (2, 4). Recent studies on TRPA1 deficient mice support a role of the channel in inflammatory pain and sensation of noxious cold (8,9). A model suggests TRPA1 activation by bradykinin, a potent algogenic substance released due to tissue injury and inflammation, in two possible ways: through PLC-mediated increases in intracellular Ca 2ϩ or other metabolites (e.g. diacylglycerol) and via Ca 2ϩ influx through TRPV1 (9). Whether an increase in intracellular Ca 2ϩ is sufficient to activate TRPA1 is still debated (4, 7), but several findings indicate a role of Ca 2ϩ on TRPA1 function. It was shown that extracellular Ca 2ϩ enhances the current rate and magnitude of AITC-induced currents (4, 10). Furthermore, Ca 2ϩ is thought to be responsible for fast channel closure (10). In addition, single channel recordings of heterologously expressed TRPA1 revealed an AITC-induced conductance, which is reduced in the presence of Ca 2ϩ (10). Together these reports emphasize the importance of Ca 2ϩ for TRPA1 function. Very recently the existence of a putative EF-hand calciumbinding domain (EF-hand CBD) at the N terminus of TRPA1 was reported (11). The EF-hand CBD is the most common motif among Ca 2ϩ -binding sites of a large number of Ca 2ϩ -interacting proteins (12). The classical EF-hand is a helix-loophelix motif that coordinates the Ca 2ϩ...