cAMP and IP3 signaling pathways in HEK293 cells transfected with canine olfactory receptor genes.

Olfactory receptors (ORs) expressed at the cell surface of olfactory sensory neurons lining the olfactory epithelium are the first actors of events leading to odor perception and recognition. As for other mammalian ORs, few dog OR have been deorphanized, mainly because of the absence of good methodology and the difficulties encountered to express ORs at the cell surface. Within this work, our aim was 1) to deorphanize a large subset of dog OR and 2) to compare the implication of the 2 main pathways, namely the cAMP and inositol 1,4,5-triphosphate (IP3) pathways, in the transduction of the olfactory message. For this, we used 2 independent tests to assess the importance of each of these 2 pathways and analyzed the responses of 47 canine family 6 ORs to a number of aliphatic compounds. We found these ORs globally capable of inducing intracellular calcium elevation through the IP3 pathway as confirmed by the use of specific inhibitors and/or a cAMP increase in response to aldehyde exposure. We showed that the implication of the cAMP or/and IP3 pathway was dependent upon the ligand-receptor combination rather than on one or the other partner. Finally, by exposing OR-expressing cells to the 21 possible pairs of C6-C12 aliphatic aldehydes, we confirmed that some odorant pairs may have an inhibitory or additive effect. Altogether, these results reinforce the notion that odorant receptor subfamilies may constitute functional units and call for a more systematic use of 2 complementary tests interrogating the cAMP and IP3 pathways when deorphanizing ORs.

IL-7 induces tyrosine phosphorylation of clathrin heavy chain.

IL-7 induction of protein tyrosine phosphorylation was examined in an IL-7-dependent thymocyte cell line, D1, which was generated from a p53-/- mouse. Anti-phosphotyrosine antibody was used both to immunoprecipitate and Western blot, and showed that IL-7 induced tyrosine phosphorylation of a protein with a molecular weight of approximately 200 kDa. The P200 band was purified by reversed-phase high-performance liquid chromatography. Amino acid sequencing by mass spectrometry revealed three peptides identical to rat clathrin heavy chain (CHC) 1 (192 kDa), and this was confirmed by blotting with an anti-clathrin antibody. Stimulation of normal pro-T cells by IL-7 showed an increased tyrosine phosphorylation of clathrin heavy chain. Tyrosine phosphorylation of clathrin heavy chain was strongly induced by IL-7 and to a lesser extent by IL-4, while no effect could be observed with the cytokines IL-2, IL-9 and IL-15, whose receptors share the gammac chain. Phosphorylation of clathrin heavy chain was found to be sensitive to Jak3 inhibitors but not to Src inhibitors. Clathrin is involved in internalization of many receptors, and its phosphorylation by IL-7 stimulation may affect the internalization of the IL-7 receptor.

IL-7 withdrawal induces a stress pathway activating p38 and Jun N-terminal kinases.

IL-7 delivers survival signals to cells at an early stage in lymphoid development. In the absence of IL-7, pro-T cells undergo programmed cell death, which has previously been associated with a decline in Bcl-2 and translocation of Bax from cytosol to mitochondria. A new, earlier feature of IL-7 withdrawal was identified using an IL-7-dependent thymocyte line. We observed that withdrawal of IL-7 induced increased expression of jun and fos family member genes including c-jun, junB, junD, c-fos and fra2. This transient response peaked 3-4 h after IL-7 was withdrawn and resulted in increased DNA-binding activity of AP-1 and in a change in the composition of the Jun/Fos family dimers shown by electrophoretic mobility shift and supershift assays. Induction of jun and fos genes and the increased DNA-binding activity of AP-1 were attributable to the phosphorylation-induced activation of the stress kinases p38 and JNK and were blocked by the chemical kinase inhibitors SB203580 and SB202190. The stress response contributed to cell death following IL-7 withdrawal as shown by blocking the activity of the stress (MAP) kinases or by blocking the production of c-Jun and c-Fos using antisense oligonucleotides.

Activation of SRE and AP1 by olfactory receptors via the MAPK and Rho dependent pathways.

Whereas the activation of MAPKs (mitogen activated kinases) and Rho dependant pathways by GPCR (G protein coupled receptors) has been the subject of many studies, its implication in the signalling of olfactory receptors, which constitute the largest GPCR family, has been far less analysed. Using an in vitro heterologous system, we showed that odorant activated ORs activate SRE containing promoters via the ERK pathway. We also demonstrated that RhoA and Rock kinases but not Rac were involved in ORs-induced SRE/SRF activation and that AP1 was activated, via JNK and p38 MAPKinase. Using real time PCR we found that mOR23, RnI7 and CfOR12A07 induced elevated levels of transcription factors ELK-4, srf, c-fos and c-jun mRNAs whereas mOREG induced an elevated transcription levels of c-fos and c-jun mRNA only. We showed also that odorant activated ORs stimulate the downstream MAPKs and Rho pathways in primary cultures of rat olfactory sensory neurons (OSNs). Similar results were also obtained with OE (olfactory epithelium) extracts prepared from rats exposed to odorants in vivo. Finally, we showed the important role of the AKT and MAPK signalling pathways in OSNs survival. Taken together, these data provide direct evidence that the binding of odorants onto their ORs activates the MAPK and Rho signalling pathways that are involved in OSNs survival events. This suggests that these pathways could be implicated in the regulation of OSNs homeostasis.

Functional analysis of a subset of canine olfactory receptor genes.

In this paper, we explored the level complexity of the combinatorial olfactory code that allows mammals with a repertoire of about thousand putatively active olfactory receptors encoded in their genomes to recognize and identify a much larger repertoire of odorant molecules. To that end, we cloned 38 canine OR genes belonging to the same OR gene family and transiently expressed them in a subclone of embryonic human kidney cells (HEK293) permanently expressing the G(olf) subunit. Using a Ca(2+) imaging approach, we established for example that as many as 26 out of the 38 cloned OR elicited a Ca(2+) response when exposed to octanal, whereas 10 responded to nonanal, other aldehydes providing intermediate responses. Altogether, these results demonstrated that the combinatorial code is quite complex in support to the highly developed sense of olfaction demonstrated by dogs.