Using isoform and subunit specific antibodies we have determined the presence, localization, and beta spectrin associations of alpha erythroid spectrin, alpha SpI sigma*, as well as alpha non-erythroid spectrin, alpha SpII sigma 1, in mouse brain. Peptide specific antibodies against unique sequences within the beta SpII sigma 1, non-erythroid beta spectrin isoform, and within beta SpI sigma 1, erythrocyte beta spectrin isoform were used to compare the immunolocalization of beta spectrin 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 determined by immunodot and immunoblot methods. Immunohistochemical analyses indicated that beta SpI sigma 2 is located in neuronal somata and dendrites in mouse cerebellum. beta SpII sigma 1 is located in the medullary layer, chiefly composed of axonal tracts. Parallel immunohistochemical analysis with antibodies for the alpha and beta spectrin isoforms revealed that antibodies specific for the alpha subunit of erythrocyte spectrin (alpha SpI sigma 1) localized antigen to the somata and dendrites 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 (alpha SpII sigma 1) localized antigen to axons in the cerebellum corresponding to the pattern for the non-erythroid beta subunit (beta SpII sigma 1). The distinct localization of antigens by antisera which recognize either the alpha subunit of red blood cell spectrin or the alpha subunit of non-erythroid brain spectrin, together with the correspondence of their localization with appropriate beta subunits, clearly indicate that brain contains at least two species of spectrin each with distinct alpha and beta subunits. Immunoprecipitation experiments of cerebellar 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 as the association of non-erythroid alpha and beta subunits. In addition the immunoblot analysis of the immunoprecipitated material suggested there are minor populations of various hybrid tetramers in brain consisting 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 non-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.(ABSTRACT TRUNCATED AT 400 WORDS)
