Radiolabel‐transfer cross‐linking demonstrates that protein 4.1 binds to the N‐terminal region of β spectrin and to actin in binary interactions

Abstract

Erythrocyte protein 4.1 plays a major role in stabilizing the spectrin-actin junction of the erythrocyte membrane skeleton. The particular sites on spectrin responsible for the binding of actin and protein 4.1 have not been specifically defined, although the general region of the 'tail' end, opposite the self-association site, has been deduced by electron microscopy. Using a photoactivatable, radiolabel-transfer cross-linker, l-[N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl]-4-(N-hydroxysuccinimidyl)-succinate, we have determined that the binding site for protein 4.1 on spectrin resides in the N-terminal region of j spectrin within a sequence homologous to the actinbinding region of c1 actinin. Moreover, this technique provided clear evidence for a direct binding interaction between actin filaments and protein 4.1 that was confirmed by rapid-sedimentation assays. In summary, use of radiolabel-transfer cross-linking has enabled assignment of the protein-4. I-binding site on erythrocyte spectrin and has identified a previously ill-defined binary interaction between protein 4.1 and F-actin.The erythrocyte membrane skeleton is composed primarily of spectrin, actin, proteins 4.1, 4.9 and adducin. The skeleton is attached to the lipid bilayer by spectrin's interaction with ankyrin (syndcin; protein 2.1) [l -41 which binds to the transmembrane protein 3 [5. 61. A second membrane attachment occurs via spcctrin's interaction with protein 4.1, which in turn binds to the transmembrane glycoproteins, glycophorin A [7], glycophorin C [S] and protein 3 [9]. Spectrin is a highly elongated heterodimer, composed of intertwined c1 and p chains (proteins 1 and 2, respectively), aligned in antiparallel fashion with respect to the amino-termini. Spectrin dimers self-associate head-to-head to form tetramers and higher oligomers [ 10 -121. Spectrin also binds to F-actin [ 13 -151 and this interaction is greatly enhanced by protein 4.1 [16][17][18][19]. Protein 4.9 [20] and adducin [21, 22) may serve as actinbundling proteins and the latter protein also enhances spectrin-actin binding.Spectrin structure was initially defined as a linear sequence of large peptides, called domains, separated by protease-sensitive regions [23, 241. Later studies determined that spectrin consisted of a series of homologous 106-amino-acid repeats arranged in triple ci helices [25]. When the cDNA for spectrin was isolated and sequenced, it became evident that spectrin is a member of a supergene family that includes c1 actinin and dystrophin [26 -321. The amino-terminal region of p spectrin and the carboxy-terminal region of ci spectrin contain nonrepetitive sequence with a high degree of similarity to the amino-and carboxy-terminal regions of the actin-binding protein, a actinin [33, 341. The specific sites on spectrin responsible for binding ankyrin or for self-association have been localized to particular proteolytic peptides by in vitro binding studies [35], and these sites are located near the 'head' corresponding to the Nterminal c1 chain/C-terminal p chain...

Spectrins and AnkyrinB Constitute a Specialized Paranodal Cytoskeleton

“…In agreement, a mutant of Caspr lacking its intracellular domain was not retained properly on the axolemma at the paranodal junction (Gollan et al, 2002). Caspr interacts with protein 4.1B (Gollan et al, 2002;Denisenko-Nehrbass et al, 2003), and 4.1 family members bind to the N terminus of ␤-spectrins (Becker et al, 1990(Becker et al, , 1993. ␤II spectrin and ␣II spectrin interact as antiparallel heterodimers, and these heterodimers associate further to form heterotetramers.…”

Beta spectrin kissimmee: a spectrin variant associated with autosomal dominant hereditary spherocytosis and defective binding to protein 4.1.

“…We had also previously shown that the binding site for protein 4.1 was located within the NH2-terminal region of (3 spectrin before the beginning of the spectrin repeat structure ( 12). We therefore decided to amplify this region of the :3 spectrin cDNA.…”

Defining of the Minimal Domain of Protein 4.1 Involved in Spectrin-Actin Binding

“…Binary interaction between spectrin and actin is characterized by a weak binding (K a ϳ 5 ϫ 10 ) (3,5). While the interaction between protein 4.1 and F-actin was initially found to be weak (5), more recent studies suggest a cooperative mechanism for this interaction (6). 1 In contrast, protein 4.1 binds strongly to spectrin (K a ranging from ϳ0.5 to 86 ϫ 10 6 M Ϫ1 ) (7)(8)(9)(10).…”