Functional Characterization of Two Human Olfactory Receptors Expressed in the Baculovirus Sf9 Insect Cell System

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.

An Olfactory Receptor Pseudogene whose Function emerged in Humans

“…Experimentally, heterologous expression of a FLAG (DYKDDDDK polypeptide sequence) tagged receptor in insect cells confirmed an extracellular N-terminus 29 . This leads us to conclude that hOR17-210 is missing both the first and second TMs when compared to other mammalian ORs.…”

“…This genomic pseudogene sequence was identified earlier as possessing a two-nucleotide frame shift 28 . A cDNA clone of this frame-shifted sequence was subsequently shown to successfully initiate a G-protein mediated signal transduction cascade in the presence of a mixture of odorants (especially ketone compounds 29 ) commonly perceived by humans.…”

“…We also carried out computational docking studies using the preferred of the two models with selected odor ligands that are known to experimentally excite hOR17-210. 29 Figure 1a shows the results of a comparative sequence analysis between the cDNA sequence functionally studied and the pseudogene (OR1E3P) identified from the genome in the HORDE database 31 . The former is listed in GENBANK (http://www.ncbi.nih.gov/) under Accession Number AAC99555, the latter, Accession Number U53583.…”

An Olfactory Receptor Pseudogene whose Function emerged in Humans

“…Interestingly, the region of TM2 correctly predicted as a helical TM region for rat I7 is not predicted as a TM in hOR17-210 despite the apparent sequence similarities. Experiments showed that this region was indeed extracellular and not a TM 29 .…”

“…The sequence in entry AAC99555, when directly translated yields the functional OR. The bolded region in Figure 1c shows the cDNA cloned protein sequence that was used in our analysis and in the experimental functional studies 29 .…”