The 270 kDa splice variant of erythrocyte - spectrin ( I 2 ) segregates in vivo and in vitro to specific domains of cerebellar neurons

Spectrin isoforms arise from four distinct genes, three of which generate multiple alternative transcripts. With no biochemical restrictions on the assembly of heterodimers, more than 25 distinct heterodimeric spectrin species may exist. Whether (and why) this subtle but substantial diversity is realized in any single cell is unknown. To address this question, sequence-specific antibodies to alternatively spliced regions of and spectrin have been prepared. Reported here is the localization in rat cerebellar neurons at light and electron microscopic levels of an antibody against a unique sequence ( I 2-A = PGQHKDGQKSTGDERPT) from the 270 kDa transcript of the red cell -spectrin gene (spectrin I 2). In this version, the 3 sequence of erythroid -spectrin ( I 1) is replaced with an alternative sequence that shares substantial homology with the 3 sequence of non-erythroid -spectrin ( II 1). The antibody to I 2-A stains a single protein band at 270 kDa, determined by western blotting, in both rat cerebellum and in cultured cerebellar granule cells, and does not react with II 1 spectrin ( -fodrin). This antibody stains the dendritic spines of Purkinje cells in the molecular layer, and is concentrated at postsynaptic densities (PSDs) adjacent to synapsin I (which is confined to the presynaptic membrane). The soma of Purkinje cells do not stain. In the granular layer, cytoplasmic organelles and the postsynaptic densities of granular cells stain strongly. Astrocytes are also stained. In all cells, plasma membrane staining is confined to postsynaptic densities (PSD). The I 2 isoform co-immunoprecipitates with non-erythroid -spectrin ( II ), even though the distribution of II within neurons only partially overlaps that of I 2 No hybrid I 2 and II 1 ( -fodrin) spectrin complexes appear to exist. Spectrin I 2 is also polarized in cultured rat cerebellar granule cells, where it is abundant in cell bodies but not neurites. The overall distribution of I 2 is as a subset of the distribution of spectrins 240/235E previously detected with a generally reactive erythrocyte spectrin antibody. These findings establish the highly precise segregation of a -spectrin isoform to distinct cytoplasmic and membrane surface domains, indicate that it is complexed (partially) with non-erythroid spectrin, and demonstrate that cytoskeletal targeting mechanisms are preserved in cultured granular cells. The extreme concentration of I 2 spectrin at the PSD and in selected cytoplasmic compartments suggests that unique isoforms of spectrin may play a pivotal role in organizing topographically defined clusters of receptors or cytoplasmic protein complexes.