We recently identified a new integrin co-activator, Z-band Alternatively Spliced PDZ containing protein (Zasp), a member of the PDZ and LIM containing protein family. Zasp is a member of the Z-line-associated proteins known for its role in the assembly of muscle contractile machinery, through binding of its N-terminal PDZ domain to α-actinin while its C-terminal LIM domains interact with Protein kinase C (PKC). Zasp depletion is neonatal lethal and mutations in the Zasp gene are linked to cardiac and skeletal muscle myopathies known as “Zaspopathies”. Most of these mutations target the internal domain of Zasp and do not affect Zasp co-localization with α-actinin, nor do they affect protein stability. More recently, Zasp was implicated in the assembly of integrin adhesion sites and was shown to co-localize with βPS integrin in myotendinous junctions in Drosophila. Initial observations in Zasp knockout flies show an embryonic muscle detachment phenotype. In these flies, the αPS2 integrin co-localizes with talin but still separates from the ECM ligand, tiggrin, indicating that Zasp regulates integrin adhesion. The partial rescue of this defect obtained through the expression of the talin FERM domain is indicative of a Zasp-dependent integrin activation defect. We confirmed this hypothesis through our established flow cytometry-based activation assay by measuring activation of α5β1 integrin, which is most similar to αPS2/βPS in Drosophila. Here, we demonstrated that Zasp co-activates α5β1 integrin in the presence of the talin FERM domain. Our investigation points to an involvement of the Zasp internal domain in integrin activation, and we suggest Zasp-mediated integrin activation to be β1 integrin specific due to the fact that Zasp fails to co-activate αIIbβ3 integrin under the same conditions in our assay. The combination of structural biology, biochemistry and cell biology techniques previously used for kindlin characterization will also be applied to understand the detailed molecular requirements for Zasp-mediated integrin activation. The corroboration of the genetic interaction of Zasp and integrins in Drosophila by our in vitro data implies a conserved molecular mechanism through which Zasp regulates β1 integrin activation. Furthermore, members of this protein family co-localize with β1 integrins and promote cell attachment and migration in breast malignant cells. The investigation of the β1 integrin-specific regulation by Zasp will be addressed using chimeric integrins, assessing integrin activation in different cell types and testing various splicing-generated forms of these proteins. The molecular mechanism through which PDZ-LIM containing proteins may activate β1 integrins is thought to be different than kindlin. In fact, our preliminary data show that Zasp binds to β1 integrin tails and this interaction is LIM domains-mediated; however, the Zasp co-operative effect with the talin FERM domain does not require LIM domains.
