Functional characterization of two human olfactory receptors expressed in the baculovirus Sf9 insect cell system. Matarazzo, V., Clot-Faybesse, O., Marcet, B., Guiraudie-Capraz, G., Atanasova, B., Devauchelle, G., Cerutti, M., Etiévant, P., & Ronin, C. Chemical Senses, 30(3):195--207, March, 2005. doi abstract bibtex Olfactory receptors (ORs) are the largest member of the G-protein-coupled receptors which mediate early olfactory perception in discriminating among thousands of odorant molecules. Assigning odorous ligands to ORs is a prerequisite to gaining an understanding of the mechanisms of odorant recognition. The functional expression of ORs represents a critical step in addressing this issue. Due to limitations in heterologous expression, very few mammal ORs have been characterized, and so far only one is from human origin. Consequently, OR function still remains poorly understood, especially in humans, whose genome encodes a restricted chemosensory repertoire compared with most mammal species. In this study, we have designed cassette baculovirus vectors to coexpress human OR 17-209 or OR 17-210 with either G(alpha olf) or G(alpha16) proteins in Sf9 cells. Each OR was found to be expressed at the cell surface and colocalized with both G(alpha) proteins. Using Ca2+ imaging, we showed that OR 17-209 and OR 17-210 proteins are activated by esters and ketones respectively. Odorant-induced calcium response was increased when ORs were coexpressed with G(alpha16) protein, whereas coexpression with G(alpha olf) abolished calcium signaling. This strategy has been found to overcome most of the limitations encountered when expressing an OR protein and has permitted odorant screening of functional ORs. Our approach could thus be of interest for further expression and ligand assignment of other orphan receptor proteins.
@article{ matarazzo_functional_2005,
title = {Functional characterization of two human olfactory receptors expressed in the baculovirus Sf9 insect cell system},
volume = {30},
issn = {0379-864X},
doi = {10.1093/chemse/bji015},
abstract = {Olfactory receptors ({ORs}) are the largest member of the G-protein-coupled receptors which mediate early olfactory perception in discriminating among thousands of odorant molecules. Assigning odorous ligands to {ORs} is a prerequisite to gaining an understanding of the mechanisms of odorant recognition. The functional expression of {ORs} represents a critical step in addressing this issue. Due to limitations in heterologous expression, very few mammal {ORs} have been characterized, and so far only one is from human origin. Consequently, {OR} function still remains poorly understood, especially in humans, whose genome encodes a restricted chemosensory repertoire compared with most mammal species. In this study, we have designed cassette baculovirus vectors to coexpress human {OR} 17-209 or {OR} 17-210 with either G(alpha olf) or G(alpha16) proteins in Sf9 cells. Each {OR} was found to be expressed at the cell surface and colocalized with both G(alpha) proteins. Using Ca2+ imaging, we showed that {OR} 17-209 and {OR} 17-210 proteins are activated by esters and ketones respectively. Odorant-induced calcium response was increased when {ORs} were coexpressed with G(alpha16) protein, whereas coexpression with G(alpha olf) abolished calcium signaling. This strategy has been found to overcome most of the limitations encountered when expressing an {OR} protein and has permitted odorant screening of functional {ORs}. Our approach could thus be of interest for further expression and ligand assignment of other orphan receptor proteins.},
language = {eng},
number = {3},
journal = {Chemical Senses},
author = {Matarazzo, Valéry and Clot-Faybesse, Olivier and Marcet, Brice and Guiraudie-Capraz, Gaëlle and Atanasova, Boriana and Devauchelle, Gérard and Cerutti, Martine and Etiévant, Patrick and Ronin, Catherine},
month = {March},
year = {2005},
pmid = {15741602},
keywords = {Animals, Baculoviridae, Calcium, Cell Line, {GTP}-Binding Protein alpha Subunits, Genomics, Humans, Insects, Microscopy, Confocal, Odors, Olfactory Receptor Neurons, Receptors, G-Protein-Coupled},
pages = {195--207}
}
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Consequently, OR function still remains poorly understood, especially in humans, whose genome encodes a restricted chemosensory repertoire compared with most mammal species. In this study, we have designed cassette baculovirus vectors to coexpress human OR 17-209 or OR 17-210 with either G(alpha olf) or G(alpha16) proteins in Sf9 cells. Each OR was found to be expressed at the cell surface and colocalized with both G(alpha) proteins. Using Ca2+ imaging, we showed that OR 17-209 and OR 17-210 proteins are activated by esters and ketones respectively. Odorant-induced calcium response was increased when ORs were coexpressed with G(alpha16) protein, whereas coexpression with G(alpha olf) abolished calcium signaling. This strategy has been found to overcome most of the limitations encountered when expressing an OR protein and has permitted odorant screening of functional ORs. 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Assigning odorous ligands to {ORs} is a prerequisite to gaining an understanding of the mechanisms of odorant recognition. The functional expression of {ORs} represents a critical step in addressing this issue. Due to limitations in heterologous expression, very few mammal {ORs} have been characterized, and so far only one is from human origin. Consequently, {OR} function still remains poorly understood, especially in humans, whose genome encodes a restricted chemosensory repertoire compared with most mammal species. In this study, we have designed cassette baculovirus vectors to coexpress human {OR} 17-209 or {OR} 17-210 with either G(alpha olf) or G(alpha16) proteins in Sf9 cells. Each {OR} was found to be expressed at the cell surface and colocalized with both G(alpha) proteins. Using Ca2+ imaging, we showed that {OR} 17-209 and {OR} 17-210 proteins are activated by esters and ketones respectively. Odorant-induced calcium response was increased when {ORs} were coexpressed with G(alpha16) protein, whereas coexpression with G(alpha olf) abolished calcium signaling. This strategy has been found to overcome most of the limitations encountered when expressing an {OR} protein and has permitted odorant screening of functional {ORs}. 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