Application): Sigma receptors are membrane proteins that have attracted much interest because of their high affinity for several different drug classes. They are also potentially involved in the modulation of neurotransmitter action, endocrine and immune function. Multiple types of signal receptors have been defined, based primarily on their pharmacological characteristics. Sigma type 1 and type 2 are the most well defined subtypes. Sigma type 1 has recently been cloned and purified, and its functional role in the cell is currently under investigation by several laboratories. Sigma type 2 or other suggested receptor subtypes are proposed to exist based only on their pharmacological differentiation from sigma type 1 and from other known receptor families. This is because other receptor subtypes still remain to be cloned or purified. In view of the possible involvement of sigma receptors in the nervous, endocrine, and immune systems, a projected long-term goal for the structural identification of receptor subtypes will aid in better definition the functional role of sigma receptors. The focus of this present proposal is to more clearly characterize, localize, and differentiate other sigma receptor subtypes, specifically sigma 2, from sigma 1. The current proposal is based on previous studies in the PI's laboratory which demonstrate additional biochemical evidence of this subtype. The working hypothesis is that sigma 2 is a distinctly different receptor from sigma 1. This difference is based on the solubility, localization, pharmacological distinction, and molecular weight of sigma 2. In order to test the hypothesis, the following series of experiments will be undertaken: (1) examine several peripheral tissues for sigma 2 activity; (2) perform subcellular fractionation studies on several peripheral tissues in an effort to localize sigma 2 activity; (3) solubilize appropriate subcellular fractions with most abundant sigma 2 activity; and (4) examine ligands that may be used in the specific identification of the sigma 2 or other receptor subtypes. Established techniques for pharmacological distinction between sigma receptors will be used to determine and compare the existence and density of sigma 1 and 2 in specific aims 1, 2, and 3.
Specific aim 4 will include modification of some current sigma specific ligands for fluorescence or photoaffinity labeling of the sigma 2 receptor. The localization and differential solubilization of sigma 2 from sigma 1 would provide a means to later investigate the role sigma 2 receptors play in the cell membrane.
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