Def-6, a guanine nucleotide exchange factor for Rac1, interacts with the skeletal muscle integrin chain alpha7A and influences myoblast differentiation.

Integrin alpha7beta1 is the major laminin binding integrin receptor of muscle cells. The alpha7 chain occurs in several splice isoforms, of which alpha7A and alpha7B differ in their intracellular domains only. The fact that the expression of alpha7A and alpha7B is tightly regulated during skeletal muscle development suggests different and distinct roles for both isoforms. However, so far, functional properties and interacting proteins were described for the alpha7B chain only. Using a yeast two-hybrid screen, we have found that Def-6, a guanine nucleotide exchange factor for Rac1, binds to the intracellular domain of the alpha7A subunit. The specificity of the Def-6-alpha7A interaction has been shown by direct yeast two-hybrid binding assays and coprecipitation experiments. This is the first description of an alpha7A-specific and -exclusive interaction, because Def-6 did not bind to any other tested integrin cytoplasmic domain. Interestingly, the binding of Def-6 to alpha7A was abolished, when cells were cotransfected with an Src-related kinase, which is known to phosphorylate Def-6 and stimulate its exchange activity. We found expression of Def-6 was not only restricted to T-lymphocytes as described thus far but in a more widespread manner, including different muscle tissues. In cells, Def-6 is seen in newly forming cell protrusions and focal adhesions, and its localization partially overlaps with the alpha7A integrin receptor. C2C12 myoblasts overexpressing Def-6 show a delay of Rac1 inactivation during myogenic differentiation and abnormal myotube formation. Thus, our data suggest a role for Def-6 in the fine regulation of Rac1 during myogenesis with the integrin alpha7A chain guiding this regulation in a spatio-temporal manner.

The binding of Mss4 to alpha-integrin subunits regulates matrix metalloproteinase activation and fibronectin remodeling.

In four independent yeast two-hybrid screens with the integrin alpha-subunits alpha3A, alpha6A, alpha7A, and alpha7B, we identified the Mss4 protein, a nucleotide exchange factor for exocytic Rab GTPases, as a novel integrin interacting protein. We have previously shown that it binds to the conserved KXGFFKR region of integrin alpha-subunits located directly beneath the cell membrane. Here we show that the binding site for integrins on Mss4 is overlapping with those for Rab GTPases. Functional analysis of the Mss4/integrin interaction revealed its importance for activation of matrix metalloproteinases (MMPs) and remodeling of secreted extracellular matrix (ECM) proteins. The exocytosis of all the proteins analyzed, however, was unaffected. Furthermore, our data suggest that Mss4 drives the coordinated action of the MT1-MMP/integrin protein complex, thus regulating the presence and activation of MT1-MMP at newly formed filopodia and lamellipodia. This in turn facilitates the conversion of pro-MMPs to MMPs, resulting in cleavage and remodeling of ECM proteins. C2C12 myoblasts with stably down-regulated Mss4 showed a disturbed fibronectin remodeling during differentiation, resulting in malfunctioned myotube formation.

AtSUC8 and AtSUC9 encode functional sucrose transporters, but the closely related AtSUC6 and AtSUC7 genes encode aberrant proteins in different Arabidopsis ecotypes.

Three members of the Arabidopsis sucrose transporter gene family, AtSUC6-AtSUC8 (At5g43610; At1g66570; At2g14670), share a high degree of sequence homology in their coding regions and even in their introns and in their 5'- and 3'-flanking regions. A fourth sucrose transporter gene, AtSUC9 (At5g06170), which is on the same branch of the AtSUC-phylogenetic tree, shows only slightly less sequence homology. Here we present data demonstrating that two genes from this subgroup, AtSUC6 and AtSUC7, encode aberrant proteins and seem to represent sucrose transporter pseudogenes, whereas AtSUC8 and AtSUC9 encode functional sucrose transporters. These results are based on analyses of splice patterns and polymorphic sites between these genes in different Arabidopsis ecotypes, as well as on functional analyses by cDNA expression in baker's yeast. For one of these genes, AtSUC7 (At1g66570), different, ecotype-specific splice patterns were observed in Wassilewskija (Ws), C24, Columbia wild type (Col-0) and Landsberg erecta (Ler). No incorrect splicing and no sequence polymorphism were detected in the cDNAs of AtSUC8 and AtSUC9, which encode functional sucrose transporters and are expressed in floral tissue. Finally, promoter-reporter gene plants and T-DNA insertion lines were analyzed for AtSUC8 and AtSUC9.