BRAIN ALPHA-ERYTHROID SPECTRIN - IDENTIFICATION, COMPARTMENTALIZATION, AND BETA-SPECTRIN ASSOCIATIONS

Using isoform and subunit specific antibodies we have determined the p resence, localization, and beta spectrin associations of alpha erythro id spectrin, alpha SpI Sigma, as well as alpha non-erythroid spectrin , alpha SpII Sigma 1, in mouse brain. Peptide specific antibodies agai nst unique sequences within the beta SpII Sigma 1, non-erythroid beta spectrin isoform, and within beta SpI Sigma 1, erythrocyte beta spectr in isoform were used to compare the immunolocalization of beta spectri n subunit isoforms with that of alpha spectrin subunit isoforms and to immunoprecipitate spectrin tetramers in order to identify the subunit components by immunoblot analysis. The specificity and sensitivity of antibodies for isoform specific alpha and beta subunits was determine d by immunodot and immunoblot methods. Immunohistochemical analyses in dicated that beta SpI Sigma 2 is located in neuronal somata and dendri tes in mouse cerebellum. beta SpII Sigma 1 is located in the medullary layer, chiefly composed of axonal tracts. Parallel immunohistochemica l analysis with antibodies for the alpha and beta spectrin isoforms re vealed that antibodies specific for the alpha subunit of erythrocyte s pectrin (alpha SpI Sigma 1) localized antigen to the somata and dendri tes of cerebellar granule cell neurons, a pattern similar to that for the localization of the erythroid beta subunit (beta SpI Sigma 2). In contrast antibodies specific for the non-erythroid alpha subunit (alph a SpII Sigma 1) localized antigen to axons in the cerebellum correspon ding to the pattern for the non-erythroid beta subunit (beta SpII Sigm a 1). The distinct localization of antigens by antisera which recogniz e either the alpha subunit of red blood cell spectrin or the alpha sub unit of non-erythroid brain spectrin, together with the correspondence of their localization with appropriate beta subunits, clearly indicat e that brain contains at least two species of spectrin each with disti nct alpha and beta subunits. Immunoprecipitation experiments of cerebe llar extracts using beta spectrin peptide specific antibodies followed by immunoblotting analysis confirmed the association of an erythroid alpha subunit isoform with a beta erythroid subunit isoform, as well a s the association of non-erythroid alpha and beta subunits. In additio n the immunoblot analysis of the immunoprecipitated material suggested there are minor populations of various hybrid tetramers in brain cons isting of mixed erythroid and non-erythroid subunits. In summary these data collectively demonstrate that in mouse brain there are at least two alpha spectrin subunits, one erythroid alpha SpI Sigma and one no n-erythroid alpha SpII Sigma 1; these associate with an erythroid beta SpI Sigma 1, and a non-erythroid beta SpII Sigma 1 in the cerebellum of mouse. Although mixed tetramers occur, spectrin tetramers occur pre dominantly as (alpha SpI Sigma/beta SpI Sigma 1)(2), a purely erythro id tetramer, and as (alpha SpII Sigma 1/beta SpI Sigma 1)(2), a purely non-erythroid tetramer.