**1. Introduction**

Olfaction is an essential sensation for animals to continue carrying out their daily life activities while experiencing changes in the surrounding environment. Mammalian olfactory receptors (ORs) are expressed on the cell surface of olfactory sensory neurons (OSNs) to detect and discriminate the vast number of odors in the environment [1]. ORs form the largest and most diverse family of G protein-coupled receptors (GPCRs) [2]. There are more than 400 and 1200 functional ORs in the olfactory sensory neurons of humans and mice, respectively [3–5]. It is thought that this diverse odor recognition of ORs has made it possible for olfaction to discriminate odors, which are a mixture of various volatile molecules, with high sensitivity [6,7]. A major difficulty in characterizing the function of these mammalian ORs is that most ORs are not exported to the plasma membrane and are instead retained in the endoplasmic reticulum (ER) in nonolfactory cells, making in vitro characterization extremely challenging [2]. Consequently, functional analysis of ORs is far behind that of other canonical Class A GPCRs. Therefore, the flexible and selective odor molecule recognition mechanism of ORs is almost unknown. To fill this gap in knowledge,

**Citation:** Fukutani, Y.; Nakamura, Y.; Muto, N.; Miyanaga, S.; Kanemaki, R.; Ikegami, K.; Noguchi, K.; Ohsawa, I.; Matsunami, H.; Yohda, M. Hot Spot Mutagenesis Improves the Functional Expression of Unique Mammalian Odorant Receptors. *Int. J. Mol. Sci.* **2022**, *23*, 277. https:// doi.org/10.3390/ijms23010277

Academic Editors: Masoud Jelokhani-Niaraki and Edgar Soria-Gomez

Received: 19 November 2021 Accepted: 24 December 2021 Published: 28 December 2021

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it is necessary to develop a method for improving OR expression without changing the function of ORs. The use of a Rho tag or IL6 tag attached to the N-terminus of ORs has been suggested as a method due to the technology's ability to promote the enhancement of surface expression of ORs [8–10]. Additionally, co-expression of chaperone proteins, Hsp70t or Ric-8B, is effective for the functional expression of some ORs [11,12]. However, both modifications only enhance the expression of a limited number of ORs.

Heterologous expression of many ORs is achieved with the assistance of receptor transporting protein (RTP) 1S and RTP2 [13–17]. These are single transmembrane proteins, a class of chaperones, that mediate the transport of ORs into the plasma membrane. However, the detailed mechanism of the membrane trafficking of ORs by RTPs is unknown. Interestingly, a small subset of ORs can localize to the cell membrane without RTP1 and RTP2 in heterologous cells and function even in double knockout mice lacking RTP1S and RTP2 [18]. We previously demonstrated the importance of a specific amino acid residue within transmembrane domain 4 (G4.53) for the functional expression of OR in heterologous cells. Poor cell surface expression seems to correlate with structural instability [19].

This study investigated whether mammalian OR expression could be improved by replacing an amino acid with one that is highly conserved in a well-expressed OR (RTPindependent OR). We also tried to identify important amino acid residues that contribute to the functional expression of mammalian ORs.
