Studies will investigate molecular, cellular and functional properties of synaptic junctions (SJs) and alterations in these properties during synapse formation. Functional interactions between substrate phosphoproteins and underlying components of the postsynaptic density (PSD) will be examined. Experiments will examine functional interactions between transmitter receptors and calmodulin-dependent protein kinase II (CaM-kinase II), both of which are concentrated in SJs. Studies will focus on SJ-associated CaM-kinase II (of which the major PSD protein (mPSDp) is the major subunit) and its endogenous substrate proteins; how this dynamic system functions, how CaM-kinase II is regulated through autophosphorylaton, and how constituent molecules are assembled at developing synapses. We will examine the in situ state of phosphorylation of SJ proteins and determine if the phosphorylation of these proteins or CaM-kinase II is altered by membrane depolarization or neurotransmitters. Highly specific antibodies will be used to examine the expression and localization of CaM-kinase II and substrate phosphoproteins in hippocampal pyramidal neurons is culture. Differentiating neurons are grown under conditions that favor (high density) or greatly limit (low density) synapse formation. Antibodies will be used as specific probes to study molecular and cellular properties of mature and developing synapses in isolated pyramidal neurons. A cDNA probe(s) for the mPSDp/CaM-kinase II will be obtained. cDNAs will be used to; (a) identify mPSDp-specific mRNA and measure its levels during brain development, (b) obtain primary sequence information about functional domains of the mPSDp and determine its relationship to the 60 kDa subunit of CaM-kinase II, and (c) determine the number of genes that code for CaM-kinase II. These studies will provide important information about the regulation of gene transcription and mRNA processing for this protein kinase. The ultimate significance of these studies lies in their potential to provide explanations about; (A) molecular and cellular mechanisms that underlie synapse formation and the establishment of synaptic functions, (b) the role(s) of calmodulin- dependent protein phosphorylation in regulating synaptic properties, and (C) relationships among molecular, functional and genetic properties of CaM-kinase II in mature and developing brain.
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