Team:TU-Delft/part1

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Introduction

Olfactory receptors

Animals sense their chemical environment through olfactory receptors (ORs). The olfactory receptors are a large group of proteins belonging to a subfamily of G protein-coupled receptors (GPCRs) that bind odorant ligands. If the receptor is activated by a ligand, the confirmation of the receptor is changed and there is an interaction with the α-subunit of the G-protein, Golf. This causes dissociation of the α-subunit from the Gβγ dimer, which causes signal propagation. Each OR recognizes multiple odorants and most odorants are recognized by several ORs [1]. Because of the sensitivity and selectivity of the of the olfactory system it can be for example of value in detection of environmental toxins [2], pharmaceutical screening and for diagnostics.

Yeast G protein-coupled receptors

In this project we choose to work with the budding yeast Saccharomyces cerevisiae as a host organism because it utilizes already a GPCR pathway. Furthermore S. cerevisiae has been successfully used for functional expression of GPCR’s [3,4], a lot of genomic tools are available, and it has a fully characterized genome. In S. cerevisiae two GPCR cascades have been identified: a glucose sensing pathway and a mating pathway [5]. There are two sexes of yeast cells, MATa and MATα. Whenever pheromones (small peptides) of the opposite sex are bound to the specific G-protein coupled receptors (Ste2 p or Ste3p), the MAP kinase cascade is turned on, leading to induction of mating genes such as FUS1 and growth arrest mediated by the FAR1 promoter. This mating response can be seen in the form of a morphological change, called shmoo formation. In figure an overview of the pheromone and glucose signaling pathways in S. cerevisiae is shown.

INTRODUCTION OF A NEW OLFACTORY RECEPTOR

Previously it was found that that the yeast pheromone signaling pathway can be coupled to a mammalian olfactory receptor. Minic et al. succeeded in functional express the rat 17 OR and its trafficking to the plasma membrane in S. cerevisiae. Between the three GPCRs that are known in S. cerevisiae, Ste2, Ste3 and Gpr1, the sequence similarity is limited. Except for pheromone receptors in Schizosaccharomyces pombe and Kluyveromyces lactis, Ste2 and Ste3 are largely unrelated in sequence to other GPCRs. [5]. Nevertheless, the yeast pheromone receptors can be functionally replaced by several mammalian GPCRs so that the pheromone pathway can be activated by the corresponding ligands [4].

Chimeric design

A major hindrance for functional expression of ORs has been that the receptors did not localize in the membrane or that the downstream coupling of the receptor to the Gα did not work properly. It has been shown that the rat olfactory receptor 17 (R17) that responds to octanal can be functionally expressed in many different cell types, including S. cerevisiae. [6]. Earlier research investigated on the question whether the RI7 sequence can be used to functionally express other ORs. Sequence analysis of ORs have shown that the N termini of the receptor are involved in plasma membrane localization, whereas the C termini generally define the specificity for G protein interaction [7]. Based on this observations Radhika et al. functionally expressed a chimeric OR with the N terminus and the C terminus of the RI7 sequence. A schematic picture is shown in figure.