Structural and Signaling Proteins at the Synapse: Dystroglycan & Insulin Receptor Tyrosine Kinase Substrate p58/53: a Dissertation

Abstract

The synapse is the primary locus of cell-cell communication in the nervous system. The elaboration of a functional synapse requires both a specialized structure and an efficient communication system. For my thesis work, I studied proteins implicated in each of these functions: the structural molecules dystroglycan and dystrophin, and the signaling elements Insulin Receptor Substrate p58/53 and insulin receptor. The α/β-dystroglycan complex, believed to be the heart of cellmatrix adhesion in muscle and other tissues, provides a link between dystrophin, a cytoskeletal protein at the base of the muscle cell's Dystrophin Associated Protein Complex, and the extracellular matrix. In addition, dystrophin is found at central synapses, tightly associated with the postsynaptic density. The absence of dystrophin and the secondary loss of its associated proteins causes the genetic disease Duchenne Muscular Dystrophy. DMD affects both muscle and brain, causing a severe muscular dystrophy and lower IQs than control groups. In the first portion of my thesis work, I sought to determine the role of dystroglycan, dystrophin's peripheral partner, at central synapses. I probed Northern blots of brain regions to delineate the distribution of brain β-dystroglycan mRNA and to uncover any β-dystroglycan-related transcripts in brain. Then, using subcellular brain fractions, and cultured hippocampal neurons, I determined that whereas α-dystroglycan is associated with central synapses, β-dystroglycan is not. This discovery is surprising, and differs from the finding that dystrophin and α- and β-dystroglycan colocalize at the presynaptic membrane of retinal photoreceptors. In the course of the above mentioned work, using the anti-β-dystroglycan antiserum Ab98, I discovered a pair of proteins that were tightly associated with the postsynaptic density. These polypeptides of 58 kDa and 53 kDa (p58/53) were highly enriched in postsynaptic density (PSD) fractions from rat cerebral cortex, hippocampus, and cerebellum. In pursuit of a potential synapse-specific dystroglycan relative, I purified p58 and p53 by a combination of hydrophobic interaction chromatography and two-dimensional gel electrophoresis. Mass spectroscopy and peptide microsequencing revealed that p58/53 is identical to the insulin receptor tyrosine kinase substrate p58/53 (IRSp53). Whereas IRSp58/53 has no significant homology to β-dystroglycan other than the one span of peptides that confers its antibody cross-reactivity, its localization to the PSD newly implicates insulin signaling at synapses. Analysis of IRSp58/53 mass profiles, peptides, and mRNA indicated that IRSp58 and IRSp53 are the product of the same coding sequence. Immunolocalization showed that IRSp58/53 is expressed in the synapserich molecular layer of the cerebellum. Immunostaining of cultured hippocampal neurons showed that both IRSp58/53 and insulin receptor are highly concentrated at synapses. Like IRSp58/53, insulin receptors are a component of the PSD fraction. Together, these data suggest that the synapse is a specialized site for insulin signaling in the brain.