A protein skeleton lies beneath the plasma membrane of both erythroid and non-erythroid cells, including striated muscle. Little is known about how this skeleton assembles, and even less is known about its functions in nonerythroid cells. The principal aim of the proposed research is to answer the question of how cells regulate the assembly of spectrin, which is an abundant membrane skeleton protein composed of one alpha and one beta subunit. The approach will be to construct full-length cDNAs for each subunit, and to express functional spectrin mRNAs in vitro by transcription from SP6 vectors. To manipulate the process of spectrin assembly it would be useful to express spectrin from the SP6-generated mRNAs in a cell not actively synthesizing endogenous spectrin. Therefore, mRNA for the two subunits will be microinjected into Xenopus oocytes in the presence and absence of putative spectrin-binding sites, and in varying ratios of mRNA for each subunit. By following newly synthesized spectrin subunits by immunoprecipitation with monospecific antisera, we shall determine 1) whether spectrin which is synthesized in excess of the availability of its binding sites is preferentially degraded, as previously hypothesized (Moon and Lazarides, J,. Cell Biol. 98, 1984) and 2) whether assembly of the spectrin subunits in a 1:1 stoichiometry is independent of the ratio of synthesis of the subunits (op. cit.). Induceable constructs for each spectrin subunit will then be cotransfected into murine cells cultured in vitro, initially to further test the hypothesis that over-expression of spectrin will, in the absence of available binding sites, lead to degradation of excess subunits. Second, analysis of transfected cells will determine whether the levels of unassembled spectrin subunits feedback to regulate their synthesis, as proposed by several groups for regulation of tubulin synthesis. Finally, experiments will be initiated with these full-length cDNA constructs to determine the role of nonerythroid spectrin in the formation of specialized membrane-cytoskeletal domains in myogenic cells. The initial approach will be to use anti-sense RNA to induce phenotypic mutations in spectrin in myotubes. Collectively, these studies will determine some of the mechanisms by which cells regulate the synthesis of these membrane skeleton components, the mechanisms by which these polypeptides assemble with a specific stoichiometry, and after assembly has occurred, some of the functions of spectrin during myogenesis. Following the grant period mutagenesis of the full- length constructs will provide a powerful approach for identifying the functional domains of spectrin; defects in which may be causally linked to human hereditary hemolytic anemias (e.g., Lux, Semin. Hematol. 16, 1979).
