Date Published: December 27, 2012
Publisher: Public Library of Science
Author(s): Svenja Beckmann, Thomas Quack, Colette Dissous, Katia Cailliau, Gabriele Lang, Christoph G. Grevelding, Geoffrey N. Gobert. http://doi.org/10.1371/journal.pone.0052519
In all metazoa, the response of cells to molecular stimuli from their environment represents a fundamental principle of regulatory processes controlling cell growth and differentiation. Among the membrane-linked receptors mediating extracellular communication processes are integrin receptors. Besides managing adhesion to the extracellular matrix or to other cells, they arrange information flow into the cells by activating intracellular signaling pathways often acting synergistically through cooperation with growth factor receptors. Although a wealth of information exists on integrins in different model organisms, there is a big gap of knowledge for platyhelminths. Here we report on the in silico detection and reconstruction of α and β integrins from free-living and parasitic platyhelminths, which according to structural and phylogenetic analyses form specific clades separate from each other and from further metazoan integrins. As representative orthologs of parasitic platyhelminths we have cloned one beta-integrin (Smβ-Int1) and four alpha-integrins (Smα-Int1 – Smα-Int4) from Schistosoma mansoni; they were characterized by molecular and biochemical analyses. Evidence is provided that Smβ-Int1 interacts and co-localizes in the reproductive organs with known schistosome cellular tyrosine kinases (CTKs), of which the Syk kinase SmTK4 appeared to be the strongest interaction partner as shown by yeast two-hybrid analyses and coimmunoprecipitation experiments. By a novel RNAi approach with adult schistosomes in vitro we demonstrate for the first time multinucleated oocytes in treated females, indicating a decisive role Smβ-Int1 during oogenesis as phenotypically analyzed by confocal laser scanning microscopy (CLSM). Our findings provide a first comprehensive overview about platyhelminth integrins, of which the parasite group exhibits unique features allowing a clear distinction from the free-living groups. Furthermore, we shed first lights on the functions of integrins in a trematode model parasite, revealing the complexity of molecular processes involved in its reproductive biology, which may be representative for other platyhelminths.
Integrins are non-covalently linked heterodimeric transmembrane receptors, present on most eukaryotic cells. They integrate signals provided by the extracellular matrix with intracellular signaling pathways acting in both directions (inside-out signaling and outside-in) across the plasma membrane . Inside-out signaling is induced by conformational changes mediated by the cytoplasmic and transmembrane domains of integrins –. This subsequently influences ligand-binding affinities of the extracellular domains which, among others, regulates adhesion, cytoskeletal reorganization, cell proliferation, differentiation, or apoptosis as outside-in signaling processes –. Within metazoans integrins exhibit group-specific evolutionary differences in structure and subunit composition. Primitive bilateria have two integrin αβ-heterodimers, in Caenorhabditis elegans formed by one β- and two α-subunits . Vertebrates have an expanded set of integrins, e.g. mammals have 8 β- and 18 α-subunits, known to assemble 24 distinct integrins , . The extracellular domains of integrins recognize short peptide motifs as ligands, whose binding is specified by both subunits of a given αβ-heterodimer. The intracellular domains of integrins are small and comprise no intrinsic catalytic activity. Thus signals are transmitted through direct or indirect associations of the intracellular domains with other signaling molecules , , . Among these are cellular protein tyrosine kinases (CTKs) such as Src and Syk kinases, which have already been shown to play significant roles during outside-in signaling processes . While the primary function of integrins was previously assumed to be limited to adhesion processes, new evidence suggest integrins to synergistically cooperate with other molecules such as receptor tyrosine kinases (RTKs) in complex signaling pathways regulating growth and differentiation processes , , .
Using the S. mansoni genome data set  we identified sequences coding for four potential α-integrins as well as for one β-integrin, which were cloned and sequenced. In the genome data sets of the schistosome species S. haematobium and S. japonicum (Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium, 2009), the closely related trematode C. sinensis, the cestode E. multilocularis, and the free-living planarian S. mediterranea four α-integrin subunits (in S. mediterranea only three) and one β-integrin subunit could be also identified, which are similar in length and domain structures to the S. mansoni receptors. Smα-Int2 is the longest α-integrin and has, as Smα-Int1, a C-terminal transmembrane domain. As typical for α-integrins, the intracellular domains of Smα-Int1 and Smα-Int2 are short and lack conserved domains. Smα-Int3 is the smallest one containing also a transmembrane domain as Smα-Int4, which has only one α-integrin domain. Thus it may represent an archetype of this receptor class.