BIOLOGICAL OVERVIEW

Integrins play a crucial role in cell motility, cell proliferation and cell survival. The evolutionarily conserved LIM protein PINCH is postulated to act as part of an integrin-dependent signaling complex. The molecular architecture of PINCH (Particularly Interesting New Cysteine-Histidine rich protein), which consists exclusively of multiple LIM domains (Hobert, 1999) suggests that it may function as a platform for the docking and/or productive juxtaposition of proteins involved in integrin signaling. In order to evaluate the role of PINCH in integrin-mediated cellular events, function of PINCH in Drosophila melanogaster was directly tested in vivo. The steamer duck (stck) alleles, that were first identified in a screen for potential integrin effectors (Prout, 1997), represent mutations in Drosophila pinch. stck mutants die during embryogenesis, revealing a key role for PINCH in development. Muscle cells within embryos that have compromised PINCH function display disturbed actin organization and cell-substratum adhesion. Mutation of stck also causes failure of integrin-dependent epithelial cell adhesion in the wing. Consistent with the idea that PINCH could contribute to integrin function, PINCH protein colocalizes with ßPS integrin at sites of actin filament anchorage in both muscle and wing epithelial cells. Furthermore, it is shown that integrins are required for proper localization of PINCH at the myotendinous junction. Integrin-linked kinase (Ilk), is also essential for integrin function. Drosophila PINCH and Ilk are complexed in vivo and are coincident at the integrin-rich muscle-attachment sites in embryonic muscle. Interestingly, Ilk localizes appropriately in stck mutant embryos, therefore the phenotypes exhibited by the stck mutants are not attributable to mislocalization of Ilk. These results provide direct genetic evidence that PINCH is essential for Drosophila development and is required for integrin-dependent cell adhesion (Clark, 2003).

Cell adhesion to the extracellular matrix (ECM) is required for tissue architecture and can have dramatic effects on cell behavior. Integrins are transmembrane, heterodimeric receptors that comprise the primary recognition sites for binding to ECM. alpha- and ß-integrin subunits possess large extracellular domains that form a binding interface for specific ECM components. The cytoplasmic domains of integrins tether actin filaments, and recruit a wide array of proteins involved in signal transduction. Proteins that associate either directly or indirectly with integrin cytoplasmic tails may also contribute to activation of the ligand binding capacity of the integrins, thus modulating integrin adhesive function by an 'inside-out' signaling mechanism (Clark, 2003).

One cytoplasmic protein that has been postulated to play a role in integrin function is PINCH, a protein comprising five tandemly arrayed LIM domains (Rearden, 1994). LIM domains are double zinc-finger structures that serve as protein-binding interfaces; therefore, PINCH probably functions as a molecular scaffold that supports the assembly of a multi-protein complex at sites of integrin enrichment. In agreement with this notion, biochemical studies of human PINCH have identified Integrin-Linked Kinase (Ilk) as a binding partner for the first LIM domain of PINCH (Tu, 1999), and the SH2-SH3 adaptor protein NCK2 as a partner for the fourth LIM domain (Tu, 1998). Although the complete binding partner repertoire of PINCH remains to be elucidated, the colocalization of PINCH with integrins and its capacity to bind Ilk and NCK2 provided the first hints that PINCH might play a role in recruitment of regulatory factors to integrin-rich sites (Wu, 1999; Wu, 2001) and may thus contribute to integrin function (Clark, 2003).

Further support for the view that PINCH is essential for integrin function came from studies in which PINCH expression in C. elegans was compromised by RNA interference. Developing embryos that are deficient in PINCH display a paralyzed-at-twofold (PAT) phenotype, similar to that observed in integrin mutants (Hobert, 1999). In spite of the comparable developmental arrest when either integrin or PINCH function is compromised in the worm, this phenotypic description did not provide mechanistic insight into the relationship between PINCH and integrins. Recently, however, it was demonstrated that expression of a dominant-negative form of PINCH in tissue culture cells results in compromised cell adhesion (Zhang, 2002c). These findings are consistent with the view that PINCH is required for integrin-dependent cell adhesion. However, because the LIM domain is a conserved structural feature found in many modular proteins, it is essential that conclusions from studies using dominant-negative tools be confirmed using a loss-of-function strategy where specificity is insured (Clark, 2003).

Analysis of the cellular and developmental consequences of mutations in Drosophila pinch illustrates that PINCH is essential for integrin-dependent cell adhesion events in embryos and adults and reveals that PINCH is required to stabilize membrane-cytoskeletal linkages at sites of cell-substratum anchorage (Clark, 2003).

The genetic analysis of PINCH function has led to four main conclusions: (1) Drosophila PINCH is encoded by the stck locus and is essential for embryonic development and maintenance of tissue architecture; (2) PINCH is necessary for stable actin-membrane anchorage in muscle and contributes to integrin-dependent adhesion in muscle cells and epithelial cells; (3) integrins are required for the stable association of PINCH with muscle-attachment sites; and (4) the lethal stck mutant phenotype cannot be attributed to mislocalization of the PINCH-binding partner, Ilk, whose recruitment to muscle-attachment sites appears normal in stck mutant embryos (Clark, 2003).

Genetic analyses of the roles of integrins in Drosophila have clearly highlighted the importance of integrins for adhesion and signaling in vivo. Drosophila PINCH is colocalized with integrins in both muscle and epithelial cells. Integrins retain the capacity to accumulate at muscle-attachment sites in stck mutants, illustrating that PINCH does not have an obligatory role in the proper processing and membrane targeting of integrins in vivo. The integrin staining in stck mutants does lack the high degree of order and lateral registration observed in wild-type embryos. In the Drosophila system, it is difficult to distinguish whether this modest disorganization simply reflects the underlying disturbance of the musculature or if it is revealing some contribution of PINCH to maintenance of spatially restricted integrin localization. In C. elegans embryos in which PINCH function is compromised by unc-97 mutation (Hobert, 1999), both integrin and vinculin spread laterally beyond their normal zones of accumulation in dense plaques, suggesting a role for PINCH in clustering of adhesive junction components in this system (Clark, 2003).

Interestingly, PINCH depends on the presence of integrins for its stable accumulation at muscle-attachment sites. Several other proteins, including Talin, Ilk, Myosin II and Short stop colocalize with ßPS integrin at Drosophila muscle-attachment sites. These proteins display variable levels of dependence on integrins for their localization. Like Talin, a well-established integrin effector, PINCH depends on the presence of integrins for its concentration at muscle-attachment sites. The reliance of PINCH and Talin on integrins for their spatially restricted accumulation in muscle emphasizes their connection to the integrin receptors (Clark, 2003).

Integrins must establish links to both extracellular determinants and to intracellular cytoskeletal elements in order to support strong adhesion. Examination of the cellular defects in stck mutant muscle suggests that PINCH contributes to the stabilization of actin-membrane linkages at integrin-rich adhesion sites. In a stck mutant muscle cell, the actin filaments lose their linear organization and eventually accumulate in clumps at one end of the cell. These defects are interpreted to mean that a primary consequence of disturbed PINCH function is a destabilization of the linkage between the actin cytoskeleton and the muscle membrane; it appears that the actin-membrane attachments in stck mutants lack the mechanical strength to remain intact during cyclic muscle contraction. Because integrin functionality relies on the ability of the receptors to establish a transmembrane link between the cytoskeletal elements and the extracellular matrix, reduced substratum attachment strength and/or stability might also be expected to occur if membrane cytoskeletal linkages were compromised. Consistent with this prediction, loss of adhesion is evident in the stck^17-/- wing cell clones and, to some extent, in muscles of stck mutant embryos (Clark, 2003).

The molecular architecture of PINCH suggests that it may function as a platform for the docking and/or productive juxtaposition of protein partners. Ilk, a binding partner of PINCH, is thus a candidate to collaborate with PINCH in the stabilization of integrin-cytoskeletal linkages. Consistent with the view that PINCH and Ilk cooperate to promote stable actin anchorage at sites of integrin-mediated adhesion, the phenotypes that result from compromised function of either protein in Drosophila are very similar (Zervas, 2001; Clark, 2003). Moreover, PINCH and Ilk are colocalized in Drosophila embryos and are recovered in a protein complex isolated from embryos by immunoprecipitation. Drosophila PINCH also interacts directly with Ilk using two-hybrid methods (J. L. Kadrmas, S. M. Pronovost and M. C. Beckerle, unpublished, cited in Clark, 2003). These latter results are consistent with findings reported previously for vertebrate PINCH and Ilk (Li, 1999; Tu, 1999). PINCH and Ilk also colocalize at actin-membrane anchorage sites in C. elegans muscle, and elimination of either gene product was shown to produce a paralyzed at twofold stage (PAT) phenotype similar to that seen for ß-integrin mutants (Hobert, 1999; Mackinnon, 2002). Collectively, results in both invertebrate and vertebrate systems illustrate that the capacity to form a PINCH/Ilk complex has been conserved through evolution (Clark, 2003).

Given the fact that Ilk and PINCH colocalize, co-precipitate and have similar loss of function phenotypes, it is possible that disturbed PINCH function could adversely affect Ilk localization and that such mislocalization might account for the stck mutant phenotype. To explore this possibility the localization of Ilk was examined in stck mutant embryos; Ilk was found to be unperturbed in its ability to accumulate at muscle-attachment sites, even when a dramatic lethal phenotype is evident in stck mutant embryos. As noted above, ßPS integrin also accumulates at muscle-attachment sites in stck mutant embryos. These findings illustrate that the proper localization of integrin and Ilk is not sufficient to stabilize actin membrane linkages at sites of integrin-dependent adhesion, and define PINCH as a critical component of the molecular machinery necessary for the tethering of actin to the integrin-rich membranes (Clark, 2003).

The demonstration that single ilk and stck mutants both display deficiencies in integrin-dependent processes illustrates that neither PINCH nor Ilk is sufficient on its own to support full integrin function. It is possible that PINCH acts as a positive regulator of Ilk function, either by modulating Ilk function by direct binding or by recruitment of an Ilk-modifying factor. Alternatively, Ilk may activate some PINCH function that is crucial for stabilization of actin-membrane linkages. Finally, a PINCH-Ilk protein complex may be a key component of the platform necessary for the recruitment of other proteins required to achieve stable actin-membrane associations. In this regard, it is of interest that PINCH and Ilk can be recovered in a complex with the Ilk-binding partner, CH-IlkBP, a calponin domain-containing protein related to Affixin and Actopaxin that could provide the link to actin filaments (Tu, 2001; Yamaji, 2001; Nikolopoulos, 2002). Because the localization of Drosophila PINCH is dependent on integrins, the establishment of PINCH-Ilk complexes at muscle-attachment sites is not be supported in the absence of integrin function. This dependence of PINCH localization on integrins could provide a means to couple integrin adhesive function to its role in cytoskeletal anchorage (Clark, 2003).

In vertebrate cells, PINCH and Ilk appear to be mutually dependent on each other for their localization to integrin-rich focal adhesions (Zhang, 2002b). However, as noted above, despite their ability to interact with each other, PINCH and Ilk show distinct requirements for their recruitment to specific subcellular domains in Drosophila. In particular, it is shown that PINCH requires functional integrins for its localization to muscle-attachment sites, whereas it has previously been demonstrated that Drosophila Ilk fails to bind integrins directly and localizes normally in an integrin mutant (Zervas, 2001). Rather than employing an association with integrins, Ilk may rely on a protein such as Paxillin for its targeting to integrin-rich sites (Nikolopoulos, 2001). Although Drosophila PINCH requires integrins for its stable accumulation at muscle-attachment sites, there is no evidence that PINCH can associate directly with integrin cytoplasmic domains, therefore additional proteins probably act as a bridge (Clark, 2003).

GENE STRUCTURE

cDNA clone length - 1399

Bases in 5' UTR - 145

Exons - 6

Bases in 3' UTR - 246

PROTEIN STRUCTURE

Amino Acids -

Structural Domains

The C. elegans PINCH homolog UNC-97, encoded by the open reading frame (GenBank/EMBL/DDBJ accession number AF035583), consists entirely of 5 LIM domains. Database searches revealed the presence of highly related proteins from other species, including the human PINCH protein, another predicted C. elegans protein, termed pin-2 (for PINCH-related gene 2), and several ESTs from mice, humans, and Drosophila. A Drosophila EST contains the entire open reading frame (GenBank/EMBL/ DDBJ accession number AF078907). Like the other family members, Drosophila PINCH is a LIM-only protein comprised of five LIM domains and contains a significant sequence identity (on the order of 60%) to Unc-97 in each of its LIM modules (Hobert, 1999).

This new family of LIM proteins is named the PINCH family after its founding member, PINCH (Rearden, 1994). The PINCH family can be clearly distinguished from other multiple LIM domain-containing subfamilies and is defined by the following features: (a) all sequenced members consist exclusively of five LIM domains; (b) the spacing between the Zn-coordinating amino acids, a variable parameter in different LIM domain proteins, is with two exceptions invariant in the PINCH family; and (c) the Zn-coordinating amino acids in each LIM domain are with two exceptions invariant. Most notably, the first Zn finger of the last LIM domain of all PINCH family members is characterized by the highly unusual replacement of a His in the Cys-Cys-His-Cys LIM domain consensus motif by a Cys residue (Hobert, 1999).

date revised: 20 June 2003

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