RTP1S And RTP2 Play Divergent Roles In The Functional Expression Of Odorant Receptors

Odorant receptors(ORs)detect and discriminate odorants at the ciliary surface of olfactory sensory neurons(OSNs),and the activation of the downstream signaling pathway is considered as the first step of olfactory perception in mammals.The functional expression of ORs involves correct protein folding in the endoplasmic reticulum(ER),transportation through the Golgi apparatus to the cell-surface membrane and finally the functional response to cognate ligands.Currently,in vitro deorphanization of odorant receptors are often carried out in heterologous cell lines of non-olfactory origins;however,in these cells,OR proteins are retained in the ER and unable to be trafficked to the plasma membrane,resulting in OR degradation and loss of function.Receptor transporting protein(RTP)family members,RTP1 S 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 RTP1 S that are important for different stages of OR trafficking,odorant-mediated responses,and interaction with ORs.The exact roles of RTP2 and the significance of the requirement of the seemingly redundant coexpression of the two RTP proteins in vivo have received less attention in the past.Here we attempted to dissect the functional differences between RTP1 S and RTP2 using a HEK293 T cell-based OR heterologous expression system.We first compared the response of 51 known receptor-ligand responses including a set of 24 ORs tested against 28 cognate ligands with the different types of RTP proteins coexpressed.Unlike RTP1 S,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 RTP1 S for a subset of the tested ORs in 34 response pairs.Moreover,RTP2 reduced the number of responsive ligands for the same subset of ORs.We also isolated another set of 17 receptor-ligand pairs where both of RTP1 S and RTP2 could significantly promote their response levels;in addition,coupling with RTP1 S or RTP2 showed no significant difference in ligand selectivity for these ORs.Coexistence of the two RTPs always elicited hypo-additive effects for all of the screened receptor-ligand pairs,pointing to a possible competitive interaction between the two RTPs.A protein-protein interaction analysis also supported positive interactions among OR,RTP1 S,and RTP2.Further cell-surface and permeabilized immunocytochemical studies revealed that OR and the coexpressed RTP1 S proteins were retained in the Golgi when co-transfected with RTP2,indicating that RTP1 S and RTP2 could play different roles in the OR trafficking process.Finally,we characterized the function of a longer form of RTP2—RTP2 isoform b,with an extra 20 amino acids in the N-terminal compared to the original RTP2.When RTP2 isoform b was co-expressed with RTP1 S,there was a much more significant efficacy in promoting the functional expression of ORs than RTP2.RTP1 S and RTP2 isoform b showed synergistic effect in enhancing the odorantmediated response levels of certain ORs.As a follow-up study to the structure-function analysis of RTP1 S,this study further characterizes the functional differences and linkages between RTP1 S and RTP2 in the functional expression of ORs.In general,the role of RTP1 S is dominant in promoting the odorant-mediated response and the cell-surface expression of ORs.RTP2 diminishes the functional interaction between ORs and RTP1 S by competitively binding these two molecules,resulting in limited response levels and decreased number of ligands of ORs.RTP2 shows inhibitory effect on the function of RTP1 S in transporting ORs to the plasma membrane,possibly due to the fact that RTP1 S and RTP2 play distinct roles in ORs trafficking process,and RTP2 may be more potent in transporting ORs from the ER to the Golgi apparatus than transporting from the Golgi to the cell membrane.The investigation of the functional differentiation between RTP1 S and RTP2 showing divergent mechanisms underlying the two chaperone molecules would aid in the understanding of the requirement of the coexpression of the two molecules in vivo and could shed light on the mechanism of ORs.Furthermore,we suggest that the co-expression of RTP1 S and RTP2 isoform b may help to optimize the current ORs heterologous expression system for OR deorphanization.