Primary Structure of the Brain c -Spectrin

We have determined the nucleotide sequence coding for the chicken brain a-spectrin. It is derived both from the cDNA and genomic sequences, comprises the entire coding frame, 5' and 3' untranslated sequences, and terminates in the poly(A)-tail. The deduced amino acid sequence was used to map the domain structure of the protein. The a-chain of brain spectrin contains 22 segments of which 20 correspond to the repeat of the human erythrocyte spectrin (Speicher, D. W., and V. T. Marchesi. 1984. Nature (Lond.). 311:177-180.), typically made of 106 residues. These homologous segments probably account for the flexible, rod-like structure of spectrin. Secondary structure prediction suggests predominantly a-helical structure for the entire chain. Parts of the primary structure are excluded from the repetitive pattern and they reside in the middle part of the sequence and in its COOH terminus. Search for homology in other proteins showed the presence of the following distinct structures in these nonrepetitive regions: (a) the COOH-terminal part of the molecule that shows homology with a-actinin, (b) two typical EF-hand (i.e., Ca:*-binding) structures in this region, (c) a sequence close to the EF-hand that fulfills the criteria for a calmodulin-binding site, and (d) a domain in the middle of the sequence that is homologous to a NH2-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. These regions are good candidates to carry some established as well as some yet unestablished functions of spectrin. Comparative analysis showed that a-spectrin is well conserved across the species boundaries from Xenopus to man, and that the human erythrocyte a-spectrin is divergent from the other spectrins. S PECTRIN is the major constituent of the cytoskeletal network underlying the plasma membrane (for a review see Marchesi, 1985). It was considered to be specific for red blood cells until spectrin-like proteins were detected immunologically in many types of cells (Goodman et al., 1981; Levine and Willard, 1981; Bennett et al., 1982; Burridge et al., 1982; Glenney et al., 1982a,b; Repasky et al., 1982; Kakiuchi et al., 1982; Lehto and Virtanen, 1983). At present we know several proteins related to the red blood cell spectrin. Their kinship has been primarily investigated by peptide mapping and immunological techniques (Repasky et al., 1982; Glenney et al., 1983; Glenney and Glenney, 1984a, b; Harris et al., 1985). These studies have shown that spectrins in different tissues occur as heterodimers and possess a common (c~, Mr from 230 to 260 kD) and a variant (/3 or % Mr from 220 to 260 kD) subunit (Lazarides and Nelson, 1985). The latter show a high degree of variation while the common subunits are much alike in different types of cells (Glenney et al., 1982b). The mammalian erythroid a-chain is, however, a deviant member of the family and diverges from the others by its immunological and structural properties (Glenney and Glenney, 1984a; Harris et al., 1985). The first spectrin-like molecule to be detected outside the realm of the red blood cells was found in brain (Levine and Willard, 1981; Goodman et al., 1981) and is also called fodrin (Levine and Willard, 1981) or calspectin (Kakiuchi et al., 1982). Recently it has been shown that mammalian brain contains two isoforms of spectrin (Lazarides and Nelson, 1983; Lazarides et al., 1984; Riederer et al., 1986; Virtanen et al., 1986), one located primarily in the axons and the other in the cell bodies and dendrites. Another spectrin-like protein that is more thoroughly characterized, was detected in the avian intestinal tissue (Glenney et al., 1982b). This terminal web (TW) 260/240-protein differs from the others by its location in the terminal web of the enterocytes, distant from the plasma membrane. First data on the primary structure of spectrin were presented by Speicher et al. (1983). They sequenced some tryptic peptides of the human erythrocyte spectrin o~and/3chains. The sequences revealed repetitive structure where each unit typically consists of 106 amino acids. However, the compiled fragmentary data cover only •48 % of the ~and 18 % of the/3-chain (Speicher and Marches i, 1984). The first cDNA sequence of a nonerythroid spectrin was determined by us for a clone isolated from a chicken gizzard expression library (Wasenius et al., 1985). This 1.5-kb sequence showed that the basic architecture of the erythroid spectrin (i.e., the multiply repeated 106-residue motif) is also found in the nonerythroid spectrins. This finding was confirmed by Birkenmeier et al. (1985). Later, McMahon et al. (1987), Leto et al. (1988), and Giebelhaus et al. (1987) have provided © The Rockefeller University Press, 0021-9525/89/01/79/15 $2.00 The Journal of Cell Biology, Volume 108, January 1989 79-93 79 on M ay 8, 2017 D ow nladed fom Published January 1, 1989