Characterization of Guanylate Kinase-Associated Protein, a Postsynaptic Density Protein at Excitatory Synapses That Interacts Directly with Postsynaptic Density-95/Synapse-Associated Protein 90

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Abstract
The structure of central synapses is poorly understood at the molecular level. A recent advance came with the identification of the postsynaptic density-95 (PSD-95)/synapse-associated protein 90 family of proteins as important mediators of the synaptic clustering of certain classes of ion channels. By yeast two-hybrid screening, a novel protein termed guanylate kinase-associated protein (GKAP) has been isolated that binds to the GK-like domain of PSD-95 (Kim et al., 1997). Here we present a detailed characterization of GKAP expression in the rat brain and report the cloning of a novel GKAP splice variant. By Northern blot, GKAP mRNAs (4, 6.5, and 8 kB) are expressed predominantly in the rat brain. Byin situhybridization, GKAP is expressed widely in neurons of cortex and hippocampus and in the Purkinje and granule cells of the cerebellum. On brain immunoblots, two prominent bands of 95 and 130 kDa are detected that correspond to products of short and long N-terminal splice variants of GKAP. Two independent GKAP antibodies label somatodendritic puncta in neocortical and hippocampal neurons in a pattern consistent with synaptic elements. Immunogold electron microscopy reveals GKAP to be predominantly postsynaptic and present at asymmetric synapses and in dendritic spines. The distribution of GKAP immunogold particles is uniform in the lateral plane of the PSD but peaks in the perpendicular axis ∼20 nm from the postsynaptic membrane. In cultured hippocampal neurons GKAP immunoreactive puncta colocalize with the AMPA receptor subunit Glu receptor 1 but not with the GABAAreceptor subunits β2 and β3. Thus GKAP is a widely expressed neuronal protein localized specifically in the PSD of glutamatergic synapses, consistent with its direct interaction with PSD-95 family proteins.