BETA-II-SPECTRIN (FODRIN) AND BETA-I-SIGMA-2-SPECTRIN (MUSCLE) CONTAIN NH2-TERMINAL AND COOH-TERMINAL MEMBRANE ASSOCIATION DOMAINS (MAD1 AND MAD2)

Central to spectrin's function is its association with the plasma memb rane. The linking proteins ankyrin and protein 4.1 partly mediate this association, and their interactions with spectrin are well understood . Both beta I (erythrocyte) and beta II (fodrin, beta(G)) spectrin als o associate with unknown protein receptors in crude membrane preparati ons by ankyrin and protein 4.1 independent mechanisms. As a first step to understanding this interaction, kinetic and equilibrium assays hav e been used to monitor which regions of beta I and beta II spectrin in hibit the binding of purified (125)-labeled bovine brain spectrin to d emyelinated and NaOH-stripped bovine brain membranes. A series of 19 r ecombinant proteins spanning the entire sequence of beta II spectrin, including an alternatively spliced NH2-terminal isoform (beta II Sigma 2 spectrin), were prepared as glutathione S-transferase fusion protei ns. Also prepared were peptides representing the alternatively spliced COOH-terminal domain found in beta I Sigma 2 spectrin (''muscle spect rin''). Two distinct sequence motifs inhibited the binding of native b rain spectrin. Membrane association domain 1 (MAD1) was repre sented i n all fusion peptides that included spectrin repeat 1. These peptides slowed the kinetics of brain spectrin binding and inhibited up to 46% of the maximal binding under the conditions of these assays (apparent K-i less than or equal to 0.2 mu m). Peptides representative of repeat s 2-17 of beta II spectrin were devoid of inhibitory activity. The sec ond membrane association domain (MAD2) was identified in penultimate C OOH-terminal sequences (domain III) of both beta II and beta I Sigma 2 spectrin. These sequences were absent in beta I Sigma 1 (erythrocyte) spectrin. MAD2 competitively inhibited over 80% of brain spectrin bin ding in these assays, with an apparent K-i, less than or equal to 0.1 mu M. Direct binding studies confirmed that both MAD1 and MAD2 peptide s associated with membranes with affinities comparable to their inhibi tion constants. Sequence comparisons suggest that MAD1 is created by t he insertion of two non-homologous sequence motifs into repeat 1, exte nding it from 106 to 122 amino acids. Similarly, MAD2 encompasses a pu tative site of beta gamma-heterotrimeric G-protein binding called the pleckstrin homology domain, and MAD2 may in fact be the pleckstrin hom ology domain although this has not been rigorously proven. Collectivel y these studies identify two novel functional motifs in spectrin that mediate ankyrin independent association with membranes. We hypothesize that these motifs and their still to be discovered ligands play a pri mary role in the nascent assembly and stabilization of an ordered and polarized spectrin skeleton.