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As the discovery of orexin illustrates, the identification of native ligands for orphan GPCRs or other receptors are seminal events in understanding biological pathways. A hypothesis that we would like to explore is that sleep homeostasis may involve certain orphan GPCRs and their ligands, but we have no idea which ones. We intend to attempt a combined genomics/proteomics approach to this problem. First, we will conduct DNA microarray experiments to determine which GPCRs are expressed in sections of mouse brains believed to be relevant to sleep homeostasis. We anticipate that this will constitute a group of 200-400 proteins. We will then construct a “GPCR chip” containing these proteins. Specifically, we will express most or all of these GPCRs fused covalently to their cognate Ga protein, then attach these proteins to a surface in an active form. Robust functional assays will then be developed to screen for agonists and reverse agonists of these immobilized GPCR-Ga fusion proteins. Next, we will construct a large collection of mammalian expression vectors that encode peptides or proteins known or predicted bioinformatically to be extracellular regulatory molecules (hormones, etc.). These putative regulatory molecules will be expressed in cells capable of carrying out the appropriate processing events and export. The extracellular fluid will be collected and applied to the GPCR chip to determine which, if any, GPCRs are activated by the ligand. We hope that this process will result in the characterization of ligands for many current orphan GPCRs. These findings and the chip itself will then help us in subsequent experiments designed to shed light on the molecular basis of sleep homeostasis, as is discussed in a later section.

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Last Update: Friday, August 8, 2003.