This renewal is a continuation of studies on the structure and function of the substance P (SP) receptor. The long term objective of this proposal is to understand, at the molecular level, the basis of agonist and antagonist specificity, receptor activation and downstream signaling events. Our approach will be to use photoreactive SP analogs containing p-benzoylphenylalanine substituted at different positions in the eleven amino acid sequence of SP. The site(s) of covalent attachment of these photoligands on the SP receptor will be determined by MALDI-mass spectrometric analysis of isolated photolabeled receptor fragments, which are generated by enzymatic and/or chemical cleavage of the receptor. These studies will aid in defining the SP binding pocket and orient the peptide within that pocket. We will develop benzophenone-containing derivatives of non-peptide SP antagonists as photoprobes of the antagonist binding domain(s). An understanding of the relationship between the binding sites for peptide agonist and the non-peptide antagonists forms the foundation for understanding underlying mechanisms of antagonism, which is essential for drug development. The availability of photoreactive peptide agonists, together with chemical crosslinking and immunodetection, provides us with tools to identify specific G proteins and other regulatory proteins which interact with the SP receptor. This approach will provide important information about the signal transduction machinery associated with the SP receptor. As it has been estimated that up to 60 percent of all medicines used today exert their effects through G protein signaling pathways, these studies may provide new and interesting information of basic clinical relevance. The peptide substance P has attracted considerable attention because of its multiple biological activities: as a neurotransmitter in the central, sensory and autonomic nervous systems; as an agent that causes contraction of smooth muscle in the gastrointestinal tract and as a mediator of inflammation and immune responses. SP has been implicated in a number of sensory and neurogenerative disorders. The information provided by our continuing effort should form the basis for understanding and development of novel SP receptor agonists and antagonists.
