The objectives of this proposal are to investigate molecular and functional properties of synaptic junctions (SJs) and alterations in these properties during synapse formation. Functional interactions will be examined between membrane surface glycoproteins and underlying components of the postsynaptic density (PSD). We plan to elucidate functional interactions between postsynaptic transmitter receptors and calmodulin-dependent protein kinase II (designated 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 and is assembled at developing synapses. We will determine 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 mPSDp and postsynaptic membrane proteins of hippocampal pyramidal neurons in culture. Neurons will be grown under conditions that favor (high density) or greatly limit (low density) the formation of SJs. Antibodies will be used as specific probes to study the molecular composition and functional properties of mature and developing synapses. Monoclonal antibodies have been obtained against antigens that are; (a) neuron-specific, (b) highly enriched in SJs, (c) cell-surface, and (d) developmentally regulated, i.e. appear during synapse formation. We will obtain a cDNA probe(s) for the mPSDp/CaM-kinase II. This will be used to; (a) identify mPSDp-specific mRNAs and measure their levels during brain development, and (b) obtain primary sequence information for mPSDp and determine its relationship to the 60 kDa subunit of CaM-kinase II. These studies will provide important information on the regulation of gene transcription and mRNA processing for this important protein kinase. The ultimate significance of these studies lies in their potential to provide explanations about; (1) molecular and cellular mechanisms that underlie synapse formation and the establishment of synaptic functions, (2) molecules that distinguish synapses from the remaining neuronal surface, and (3) relationships among molecular, functional and genetic properties of synaptic proteins in the CNS.
| Scholz, W K; Baitinger, C; Schulman, H et al. (1988) Developmental changes in Ca2+/calmodulin-dependent protein kinase II in cultures of hippocampal pyramidal neurons and astrocytes. J Neurosci 8:1039-51 |
