It is a basic characteristic of cells that they adapt when persistently stimulated. Thus, the extent to which second messengers and other intracellular signals are generated during activation of cell surface receptors does not remain constant, but with time tends to decline. In recent years, I have focussed on the adaptive responses that cells employ during activation of receptors (for example, muscarinic cholinergic receptors) that stimulate inositol 1,4,5-trisphosphate (InsP3) formation. In 1991, I discovered that persistent muscarinic stimulation of SH-SY5Y human neuroblastoma cells reduced the cellular complement of receptors for InsP3 and that this suppressed the primary intracellular function of InsP3 (Ca2+ mobilization from endoplasmic reticular stores). Subsequently, I showed that this down-regulation involved an acceleration of type l InsP3 receptor degradation. My long term goals are to define the mechanism and specificity of InsP3 receptor downregulation, to fully characterize its effects on cell function and to identify physiological, pathological or clinical situations in which it is significant. With regard to this application, my specific aims are as follows. Firstly, I will define the mechanism of type I InsP3 receptor down-regulation. Employing a specific antibody to quantitate receptors, I will use protease inhibitors to identify the activity responsible for the degradation, organelle perturbants to define the intracellular site of down-regulation, and transfected cells expressing mutated InsP3 receptors to define the regions of the receptor that are subject to proteolytic cleavage. Finally, to define the selectivity of the proteolytic pathway, I will establish whether other endoplasmic reticulum proteins are also down-regulated. Secondly, I will define the subtype specificity of InsP3 receptor down- regulation. To this end, I will raise antisera specific to type II and III receptors and to variants of the type I receptor and will examine which subtypes are subject to down-regulation. In parallel, I will use these antisera to determine the relative abundance of the different subtypes and will also examine explants of rat brain to assess the extent to which down-regulation occurs in systems more representative of the in vivo situation than cell lines. Thirdly, I will define the consequences of InsP3 receptor down-regulation on cell function. To measure this precisely, I will inhibit InsP3 receptor expression with antisense nucleic acids and will monitor effects on Ca2+ mobilization in intact cells. In parallel with these studies, I will use antisense nucleic acids to define the intracellular roles of type II and III receptors. The effects of inhibition of receptor expression, together with knowledge of the functions and relative abundance of the different Insp3 receptor subtypes, will reveal how cell function is altered by the InsP3 receptor down- regulation that results from persistent cell surface receptor activation. In summary, these studies will provide a comprehensive picture of the mechanism, specificity and significance of InsP3 receptor down-regulation.
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