Receptor-transporting protein (RTP) family members play divergent roles in the functional expression of odorant receptors

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.

Fig 1. RTP1S and RTP2 differ in their abilities to promote the functional activation of ORs.

(A-B) normalized luciferase activities of concentration gradients of 28 odorants from 0 μM to 30, 300 or 320 μM tested against 24 ORs with different combinations of RTPs co-transfected, including RTP1S (red), RTP2 (green), and a combination of the two (purple) in HEK293T cells. An “OR only” negative control is co-transfected with the empty pCI vector (blue). The x-axis represents molar odorant concentrations on a logarithmic scale. The y-axis represents normalized luciferase activity shown as mean ± S.E.M. (N = 3). The scatter diagrams (inset) depict the functional effect induced by the combination of RTP1S and RTP2 compared to RTP1S or RTP2 alone (Materials and methods and S1 Fig). The x-axis represents the value of the parameter τ_RTP1S, which represents the ratio of the OR response level when co-transfected with RTP1S to the algebraic sum of individual OR response levels when co-transfected with single RTPs. The y-axis represents the value of the parameter σ, with σ = 1 dividing the diagram into hyper-addition and hypo-addition sections. The responses at lower concentrations that did not elicit OR activation were not plotted.

Fig 2. RTP1S and RTP2 show no significant difference in OR ligand selectivity.

Normalized luciferase activities of 4 representative ORs from Category 1 (MOR180-1 and MOR203-1) and Category 2 (MOR23-1 and MOR256-17), transfected alone (blue) or co-transfected with different combinations of RTPs, including RTP1S (red), RTP2 (green), and a combination of the two (purple), and tested against various ligands at a fixed concentration of 100 μM. The x-axis represents normalized luciferase activity shown as mean ± S.E.M. (N = 6).

Fig 3. OR, RTP1S, and RTP2 interact with each other in HEK293T cells.

Left, interactions among MOR258-5 (Category 1), RTP1S and RTP2. Right, interactions among MOR23-1 (Category 2), RTP1S and RTP2. First and second panels, protein lysates of HEK293T cells transfected with Flag-tagged OR and/or HA-tagged RTP1S and Flag-tagged/HA-tagged RTP2 and blotted with anti-Flag or anti-HA antibody. Third panels, co-immunoprecipitation of Flag-tagged proteins with anti-HA antibody. Fourth panels, co-immunoprecipitation of HA-tagged proteins with anti-Flag antibody. Refer to S4 Fig for the original blots. IB, immunoblot; IP, immunoprecipitation. The asterisks indicate non-specific bands.

Fig 4. RTP1S and RTP2 play divergent roles in OR trafficking.

(A-B) permeabilized staining were performed to examine the subcellular localizations of MOR180-1 (Category 1) and MOR23-1 (Category 2) when they were transfected alone or cotransfected with different RTPs or a combination of the two RTPs. Left panels, red signals represent the localization of OR proteins. Middle panel, green signals represent staining of organelles; first row, staining of ER with calnexin; second row, staining of Golgi with wheat germ agglutinin. Right panels, yellow signals represent OR proteins merged with the corresponding organelle. Blue signals in the second row are DAPI nuclear staining. Scale bar, 100 μm. (C-D) quantification of the cell-surface expression and subcellular localizations of the 4 tested ORs and RTPs. Columns represent the cells of each counting session where the OR or RTP proteins were localized in the ER, the Golgi, or at the cell surface. The y-axis represents the cell numbers of each counting session where the OR or RTP proteins were localized in ER, Golgi, or at the cell surface, shown as mean ± S.E.M. (N = 3). Paired two-tailed t test was used to compare the localizations in certain organelle or at the cell surface when different combinations of OR and RTP were cotransfected. * P < 0.05, ** P < 0.01.