The sense of smell plays an important role in vertebrate behavior. In man it is crucial for the assessment of food quality, for the avoidance of harmful substances and for social interactions. The understanding of molecular components int he olfactory mechanism constitutes a major challenge for sensory research. previously, we have studied functional membrane proteins in olfactory cilia, that generate, amplify and modulate odorant signals. In the last three year period our laboratory has begun to explore the nature of olfactory receptors, the key membrane proteins that recognize odors and initiate the sensory amplification cascade. A major emphasis has been placed on a program aimed at elucidating the role of such proteins in human olfaction. The present proposal is based on two important developments in the pI's laboratory: 1) the discovery of an olfactory receptor gene cluster on human chromosome 17, believed to be typical of more than a dozen such clusters ina the human genome, coding for a repertoire of several hundred proteins; 2) the first successful purification and lipid reconstitution of an olfactory receptor protein.
The aim of the proposal is to utilize such results for an in-depth exploration of the human olfactory receptor gene repertoire. Dozens of such genes will be mapped, cloned and fully sequenced. Those found to be suitable, will be incorporated in the appropriate recombinant DNA vectors and abe expressed in mammalian cells. Overexpression of olfactory receptor genes ina the specialized baculovirus vector will allow the preparation of pure olfactory receptor proteins in large amounts. Such proteins will be further analysed for their ability to activate the olfactory transduction machinery. This will be done with the fidelity and accuracy attained only when receptors are studied in pure form, isolated from the interference caused by hundreds of other proteins present in the cell's membrane. A novel direction of research, making use of the availability of pure olfactory receptor protein, is the application of random peptide display library screens to discover """"""""odorant mimics"""""""". These could then be used to identify proper odorants for selected human olfactory receptors. A complementary approach to be implemented is the use of a high sensitivity luciferase reporter gene to monitor the slightest changes in the olfactory second messenger cascade, a method that in other systems has proved useful in studying membrane receptors coupled to th cyclic AMP pathway. The establishment of all the above technologies will then make it possible to screen for and identify the specific odorants recognized by some of the human olfactory receptors, and to analyze olfactory specificity by computer modelling. In combination with parallel research in the PI's laboratory, in which individual genetic differences in the human olfactory receptor repertoire are investigated, the present results may shed light ont he mechanisms underlying human olfactory sensory deficits.
