ABSTRACT
Heterologous functional expression system for odorant receptors (ORs) is essential for investigating the structure-activity relationship (SAR) of various ligands. Different systems that coexpressed ORs with different G-protein alpha subunits (Galpha) demonstrated inconsistent effects on weak agonists and antagonists, but retained original relative sensitivities to potent agonists. In order to maintain the binding specificity of Galpha to ORs, we constructed a chimeric Galpha(15_olf), which contained the Galpha(15) sequence with the conserved C-terminal region of Galpha(olf). The Ca(2+) responses of the HEK293 cells that coexpressed OR-S6 with Galpha(15_olf) were more robust and reproducible compared to those of cells that coexpressed OR-S6 with Galpha(15). Furthermore, Galpha(15) sometimes induced unstable Ca(2+) responses that limited the accuracy of quantitative comparison of peak responses. Our results showed that a heterologous expression system that coexpressed ORs with Galpha(15_olf) and receptor transporting proteins was suitable for SAR analysis of various ligands.
Subject(s)
Gene Expression Regulation/physiology , Receptors, Odorant/genetics , Receptors, Odorant/metabolism , Amino Acid Sequence , Analysis of Variance , Calcium/metabolism , Calcium Signaling/genetics , Cell Line, Transformed , GTP-Binding Protein alpha Subunits/chemistry , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits/metabolism , Gene Expression Regulation/genetics , Humans , Protein Binding , Structure-Activity Relationship , Transfection/methodsABSTRACT
Human papillomaviruses infect epithelia but little is known about the nature of cell surface receptors interacting with the viral particles. It has been proposed that glycosaminoglycans and integrins may be involved in the attachment process. In the present study, the putative interactions of virus-like particles of human papillomavirus type 11 (HPV11), which present a tropism for nasopharyngeal epithelia, with olfactory and taste receptors expressed in the human lingual epithelium were studied. The L1 protein of HPV11 was produced in insect cells. The presence of L1 virus-like particles was analyzed by ELISA using monoclonal antibodies specific for full-size particles and by electron microscopy. Using immunofluorescence, it was observed that virus-like particles interacted with taste buds from murine tongue, with the tagged human olfactory receptor hJCG5 expressed in HEK-293 but not with the tagged taste receptor hT2R4. This therefore suggests that hJCG5 may be involved in the adsorption process of HPV11 to lingual epithelium serving as a so-called "adsorption-adhesive molecule."
Subject(s)
Epithelium/metabolism , Human papillomavirus 11/metabolism , Receptors, G-Protein-Coupled/metabolism , Tongue/metabolism , Virion/metabolism , Animals , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Line , Fluoroimmunoassay , Humans , Insecta , Mice , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Receptors, Odorant/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Taste Buds/metabolismABSTRACT
Olfactory-like receptors (OLRs) have been shown previously to be expressed in adult and foetal human tongue. This prompted us to verify the hypothesis if OLRs were involved in taste perception. In the present work, the mouse orthologs of OLR genes expressed in adult and/or foetal human tongue were identified. Analysis of their genomic localization and of their primary sequence features using bioinformatics did not reveal any shared remarkable characteristic. The expression of eight of these orthologs (S25/mJCG1, K42, mJCG2, mJCG3, P2/mJCG5, P3/mJCG6, mT09m/mJCG9, K21/mTPCR85 and mTPCR06) was studied in three types of mouse papillae as well as in the olfactory epithelium. It was found that all of them are expressed in olfactory epithelium and that only three of them (S25/mJCG1, K42/mJCG2 and mTPCR06) are expressed in papillae. However, despite many efforts it was impossible to detect without ambiguity the presence of OLR mRNAs in taste receptor cells nor in surrounding tissues by in-situ hybridization. Hence, the studied OLRs very likely play in taste papillae other roles than taste perception.
