Interactions between dystrophin and the sarcolemma membrane.

Dystrophin serves as a link between the subsarcolemmal cytoskeleton and the extracellular matrix. The NH2 terminus attaches to the cytoskeleton, while the COOH terminus attaches to the dystrophin associated protein (DAP) complex, which can be separated into the dystroglycan, sarcoglycan, and syntrophin subcomplexes. While the function of each DAP is not known, the dystroglycan complex binds laminin in the extracellular matrix, and binds the dystrophin COOH terminus in vitro. The syntrophins also bind the dystrophin COOH terminus in vitro, but no evidence has been reported for an interaction between dystrophin and the sarcoglycans. Human mutations have been found in dystrophin, the sarcoglycans and laminin, all of which lead to various types of muscular dystrophy. We have been studying the dystrophin domains necessary for formation of a functional complex by generating transgenic mdx (dystrophin minus) mice expressing internally truncated dystrophins. These mice provide in vivo models to study the localization of truncated dystrophin isoforms, the association of the truncated proteins with the DAP complex, and the functional capacity of the assembled DAP complexes. Expression of a dystrophin deleted for most of the NH2-terminal domain in mdx mice leads to only a mild dystrophy, indicating that dystrophin can attach to the cytoskeleton by multiple mechanisms. Truncation of the central rod domain leads to normal DAP complex formation and almost fully prevents development of dystrophy. Deletion analysis of the COOH-terminal regions indicates that a broad cysteine-rich domain is indispensable for dystrophin function. This region coincides with the in vitro identified beta-dystroglycan binding domain. Mice lacking this latter domain express very low levels of the sarcoglycans, indicating that the sarcoglycan complex binds dystrophin via dystroglycan. All deletion constructs tested lead to normal expression of the syntrophins, indicating that syntrophin associates with the DAP complex via multiple binding partners.

Transgenic mdx mice expressing dystrophin with a deletion in the actin-binding domain display a "mild Becker" phenotype.

The functional significance of the actin-binding domain of dystrophin, the protein lacking in patients with Duchenne muscular dystrophy, has remained elusive. Patients with deletions of this domain (domain I) typically express low levels of the truncated protein. Whether the moderate to severe phenotypes associated with such deletions result from loss of an essential function, or from reduced levels of a functional protein, is unclear. To address this question, we have generated transgenic mice that express wild-type levels of a dystrophin deleted for the majority of the actin-binding domain. The transgene derived protein lacks amino acids 45-273, removing 2 of 3 in vitro identified actin interacting sites and part of hinge 1. Examination of the effect of this deletion in mice lacking wild-type dystrophin (mdx) suggests that a functional domain I is not essential for prevention of a dystrophic phenotype. However, in contrast to deletions in the central rod domain and to full-length dystrophin, both of which are functional at only 20% of wild-type levels, proteins with a deletion in domain I must be expressed at high levels to prevent a severe dystrophy. These results are also in contrast to the severe dystrophy resulting from truncation of the COOH-terminal domain that links dystrophin to the extracellular matrix. The mild phenotype observed in mice with domain I-deletions indicates that an intact actin-binding domain is not essential, although it does contribute to an important function of dystrophin. These studies also suggest the link between dystrophin and the subsarcolemmal cytoskeleton involves more than a simple attachment of domain I to actin filaments.

Forced expression of dystrophin deletion constructs reveals structure-function correlations.

Dystrophin plays an important role in skeletal muscle by linking the cytoskeleton and the extracellular matrix. The amino terminus of dystrophin binds to actin and possibly other components of the subsarcolemmal cytoskeleton, while the carboxy terminus associates with a group of integral and peripheral membrane proteins and glycoproteins that are collectively known as the dystrophin-associated protein (DAP) complex. We have generated transgenic/mdx mice expressing "full-length" dystrophin constructs, but with consecutive deletions within the COOH-terminal domains. These mice have enabled analysis of the interaction between dystrophin and members of the DAP complex and the effects that perturbing these associations have on the dystrophic process. Deletions within the cysteine-rich region disrupt the interaction between dystrophin and the DAP complex, leading to a severe dystrophic pathology. These deletions remove the beta-dystroglycan-binding site, which leads to a parallel loss of both beta-dystroglycan and the sarcoglycan complex from the sarcolemma. In contrast, deletion of the alternatively spliced domain and the extreme COOH terminus has no apparent effect on the function of dystrophin when expressed at normal levels. The proteins resulting from these latter two deletions supported formation of a completely normal DAP complex, and their expression was associated with normal muscle morphology in mdx mice. These data indicate that the cysteine-rich domain is critical for functional activity, presumably by mediating a direct interaction with beta-dystroglycan. However, the remainder of the COOH terminus is not required for assembly of the DAP complex.

Deletion analysis of the dystrophin-actin binding domain.

Three sequence motifs at the N-terminus of dystrophin have previously been proposed to be important for binding to actin. By analyzing a series of purified bacterial fusion proteins deleted for each of these sites we have demonstrated that none of the three are critical for dystrophin-actin interactions. Instead, our data suggest that sequences in the N-terminal 90 amino acids of dystrophin, excluding a conserved KTFT motif, contain the major site for interaction with actin.

A yeast gene (BEM1) necessary for cell polarization whose product contains two SH3 domains.

Cell polarization requires that a cellular axis or cell-surface site be chosen and that the cytoskeleton be organized with respect to it. Details of the link between the cytoskeleton and the chosen axis or site are not clear. Cells of the yeast Saccharomyces cerevisiae exhibit cell polarization in two phases of their life cycle, during vegetative growth and during mating, which reflects responses to intracellular and extracellular signals, respectively. Here we describe the isolation of two mutants defective specifically in cell polarization in response to peptide mating pheromones. The mutants carry special alleles (denoted bem1-s) of the BEM1 gene required for cell polarization during vegetative growth. Unlike other bem1 mutants, the bem1-s mutants are normal for vegetative growth. Complete deletion of BEM1 leads to the defect in polarization of vegetative cells seen in bem1 mutants. The predicted sequence of the BEM1 protein (Bem1p) reveals two copies of a domain (denoted SH3) that is found in many proteins associated with the cortical cytoskeleton and which may mediate binding to actin or some other component of the cell cortex. The sequence of Bem1p and the properties of mutants defective in this protein indicate that it may link the cytoskeleton to morphogenetic determinants on the cell surface.

Yeast BUD5, encoding a putative GDP-GTP exchange factor, is necessary for bud site selection and interacts with bud formation gene BEM1.

Cells of the yeast S. cerevisiae choose bud sites in an axial or bipolar spatial pattern depending on their cell type. We have identified a gene, BUD5, that resembles BUD1 and BUD2 in being required for both patterns; bud5- mutants also exhibit random budding in all cell types. The BUD5 nucleotide sequence predicts a protein of 538 amino acids that has similarity to the S. cerevisiae CDC25 product, an activator of RAS proteins that catalyzes GDP-GTP exchange. Two potential targets of BUD5 are known: BUD1 (RSR1) and CDC42, proteins involved in bud site selection and bud formation, respectively, that have extensive similarity to RAS. We also show that BUD5 interacts functionally with a gene, BEM1, that is required for bud formation. This interaction provides further support for the view that products involved in bud site selection guide the positioning of a complex necessary for bud formation.