ABSTRACT
Mammalian odorant receptors (ORs) are crucial for detecting a broad spectrum of odorants, yet their functional expression poses a significant challenge, often requiring Receptor-transporting proteins (RTPs). This study examines mouse Olfr733 and Olfr732, which, despite high homology, show different functional expression profiles in heterologous cell systems. Our research aimed to identify key amino acids impacting Olfr733's functional expression. We discovered that G112FBW3.40 and L148PBW4.49 (Ballesteros-Weinstein numbering in superscript) substitutions in Olfr732 markedly enhance its RTP-independent expression and ligand responsiveness, mirroring Olfr733. These substitutions, particularly Phe112 and Leu148, are crucial for aldehyde recognition and membrane localization in Olfr733, respectively. While Olfr732-type ORs are conserved across species, Olfr733-types, unique to specific rodents, appear to have evolved from Olfr732, with Pro148 enhancing membrane expression and aldehyde sensitivity. Mouse ORs with ProBW4.49 tend to exhibit improved membrane expression compared to their paralogs, especially when co-expressed with RTP1S. This study concludes that the Pro residue in the fourth transmembrane domain significantly contributes to the structural stability of certain olfactory receptors, highlighting the intricate molecular mechanisms underlying OR functionality and evolution.
Subject(s)
Aldehydes , Receptors, Odorant , Receptors, Odorant/metabolism , Receptors, Odorant/genetics , Receptors, Odorant/chemistry , Animals , Mice , Aldehydes/metabolism , Humans , Amino Acids/metabolism , HEK293 Cells , Amino Acid SequenceABSTRACT
Vertebrate animals detect odors through olfactory receptors (ORs), members of the G protein-coupled receptor (GPCR) family. Due to the difficulty in the heterologous expression of ORs, studies of their odor molecule recognition mechanisms have progressed poorly. Functional expression of most ORs in heterologous cells requires the co-expression of their chaperone proteins, receptor transporting proteins (RTPs). Yet, some ORs were found to be functionally expressed without the support of RTP (RTP-independent ORs). In this study, we investigated whether amino acid residues highly conserved among RTP-independent ORs improve the functional expression of ORs in heterologous cells. We found that a single amino acid substitution at one of two sites (NBW3.39 and 3.43) in their conserved residues (E and L, respectively) significantly improved the functional expression of ORs in heterologous cells. E3.39 and L3.43 also enhanced the membrane expression of RTP-dependent ORs in the absence of RTP. These changes did not alter the odorant responsiveness of the tested ORs. Our results showed that specific sites within transmembrane domains regulate the membrane expression of some ORs.