Abstract
Receptor transporting protein (RTP) family members, RTP1S and RTP2, are accessory proteins to mammalian odorant receptors (ORs). They are expressed in the olfactory sensory neurons and facilitate OR trafficking to the cell-surface membrane and ligand-induced responses in heterologous cells. We previously identified different domains in RTP1S that are important for different stages of OR trafficking, odorant-mediated responses, and interaction with ORs. However, the exact roles of RTP2 and the significance of the requirement of the seemingly redundant co-expression of the two RTP proteins in vivo have received less attention in the past. Here we attempted to dissect the functional differences between RTP1S and RTP2 using a HEK293T cell-based OR heterologous expression system. When a set of 24 ORs were tested against 28 cognate ligands, unlike RTP1S, which always showed a robust ability to support odorant-mediated responses, RTP2 had little or no effect on OR responses and exhibited a suppressive effect over that of RTP1S for a subset of the ORs tested. RTP1S and RTP2 showed no significant difference in OR ligand selectivity and co-transfection with RTP2 increased the detection threshold for some ORs. A protein-protein interaction analysis showed positive interactions among OR, RTP1S, and RTP2, corroborating the functional linkages among the three molecules. Finally, further cell-surface and permeabilized immunocytochemical studies revealed that OR and the co-expressed RTP1S proteins were retained in the Golgi when co-transfected with RTP2, indicating that RTP1S and RTP2 could play different roles in the OR trafficking process. By examining the functional differentiations between the two RTP family members, we provided a molecular level explanation to the suppressive effect exerted by RTP2, shedding light on the divergent mechanisms underlying the RTP proteins in regulating the functional expression of ORs.
Highlights
Detecting and discriminating a large number of volatile chemicals is one of the essential survival skills for animals in nature
In these pairs, co-transfection with a combination of RTP1S and RTP2 showed a lower level of response compared to RTP1S alone, indicating a suppressive effect of RTP2 when co-expressed with RTP1S (Fig 1A)
We thoroughly investigated the functional differentiation between the two Receptor transporting protein (RTP) family members, RTP1S and RTP2, which are expressed in olfactory sensory neurons (OSN) and facilitate odorant receptors (ORs) function
Summary
Detecting and discriminating a large number of volatile chemicals is one of the essential survival skills for animals in nature. This ability is determined by the odorant receptors (OR) distributed at the ciliary surface of olfactory sensory neurons (OSN). Similar to some of the other GPCRs, precise trafficking of the OR proteins, involving the synthesis from the endoplasmic reticulum (ER) and the transport to the cell-surface membrane, is critical for OR function [6]. Cultured cell lines of non-olfactory origins differ from native OSNs to the extent that OR proteins are retained in the ER and unable to be trafficked to the plasma membrane, resulting in OR degradation and loss of function [8, 9]
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