*Article* **Hot Spot Mutagenesis Improves the Functional Expression of Unique Mammalian Odorant Receptors**

**Yosuke Fukutani 1,\*, Yuko Nakamura 1, Nonoko Muto 1, Shunta Miyanaga 1, Reina Kanemaki 1, Kentaro Ikegami 1, Keiichi Noguchi 2, Ikuroh Ohsawa 3, Hiroaki Matsunami 4 and Masafumi Yohda 1**


**Abstract:** 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.

**Keywords:** odorant receptor; chemosensory; membrane traffic; heterologous expression
