THE 270-KDA SPLICE VARIANT OF ERYTHROCYTE BETA-SPECTRIN (BETA-I-SIGMA-2) SEGREGATES IN-VIVO AND IN-VITRO TO SPECIFIC DOMAINS OF CEREBELLAR NEURONS

Spectrin isoforms arise from four distinct genes, three of which gener ate multiple alternative transcripts. With no biochemical restrictions on the assembly of alphabeta heterodimers, more than 25 distinct hete rodimeric spectrin species may exist. Whether (and why) this subtle bu t substantial diversity is realized in any single cell is unknown. To address this question, sequence-specific antibodies to alternatively s pliced regions of alpha- and beta-spectrin have been prepared. Reporte d here is the localization in rat cerebellar neurons at light and elec tron microscopic levels of an antibody against a unique sequence (beta ISIGMA2-A = PGQHKDGQKSTGDERPT) from the 270 kDa transcript of the red cell beta-spectrin gene (spectrin betaISIGMA2). In this version, the 3 ' sequence of erythroid beta-spectrin (betaISIGMA1) is replaced with a n alternative sequence that shares substantial homology with the 3' se quence of non-erythroid beta-spectrin (betaIISIGMA1). The antibody to betaISIGMA2-A stains a single protein band at 270 kDa, determined by w estern blotting, in both rat cerebellum and in cultured cerebellar gra nule cells, and does not react with betaIISIGMA1 spectrin (beta-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, cyto plasmic organelles and the postsynaptic densities of granular cells st ain strongly. Astrocytes are also stained. In all cells, plasma membra ne staining is confined to postsynaptic densities (PSD). The betaISIGM A2 isoform co-immunoprecipitates with non-erythroid alpha-spectrin (al phaIISIGMA), even though the distribution of alphaIISIGMA* within neu rons only partially overlaps that of betaISIGMA2. No hybrid betaISIGMA 2 and betaIISIGMA1 (beta-fodrin) spectrin complexes appear to exist. S pectrin betaISIGMA2 is also polarized in cultured rat cerebellar granu le cells, where it is abundant in cell bodies but not neurites. The ov erall distribution of betaISIGMA2 is as a subset of the distribution o f spectrins 240/235E previously detected with a generally reactive ery throcyte alphabeta-spectrin antibody. These findings establish the hig hly precise segregation of a beta-spectrin isoform to distinct cytopla smic and membrane surface domains, indicate that it is complexed (part ially) with non-erythroid alpha-spectrin, and demonstrate that cytoske letal targeting mechanisms are preserved in cultured granular cells. T he extreme concentration of betaISIGMA2 spectrin at the PSD and in sel ected cytoplasmic compartments suggests that unique isoforms of spectr in may play a pivotal role in organizing topographically defined clust ers of receptors or cytoplasmic protein complexes.