Protein Interactions

Primary Drosophila embryo cells from postgastrulation embryos differentiate when cultured on a substratum of Drosophila laminin or human vitronectin. Tiggrin, and a fusion protein of Tiggrin (residues 1,891-2,161) that contains the RGD binding site have been tested as substrata. Excellent differentiation of several different cell types occurs; e.g. abundant multinucleate myotubes and neurites form and both hemocytes and clusters of epidermal cells are present after 18 hours of culture at 22 degrees C. On control coverslips coated with bovine serum albumin very limited differentiation is observed (Fogerty, 1994).

The finding of an RGD cell attachment motif in Tiggrin, which conspicuously colocates with both alphaPS integrins at muscle apodemes and at Z-bands, emphasized the need to test Tiggrin as an integrin ligand. To do this, the ability of Tiggrin-coated substratum to mediate the spreading of Drosophila S2 cells that have been transformed with genes for alphaPS2 and betaPS integrin chains was assayed. A pair of alternative RNA splice variants codes for two forms of the alphaPS2 integrin chain that differ by 25 amino acids near the putative ligand binding site. Correspondingly, two transformed S2 cell lines were created: HSPS2(C), which expresses the alphaPS2 chain that contains the additional 25 amino acids, and HSPS2(m8), a line in which these same 25 amino acids (coded for by exon 8) are missing. Both cell lines also express the betaPS chain and make comparable amounts of integrins. Tiggrin serves as a substratum for alphaPS2betaPS integrin-mediated cell spreading. HSPS2(C) cells show no spreading when applied to tissue culture plastic that has been blocked with milk solids to prevent nonspecific interactions. When the plastic is first coated with Tiggrin, these cells show extensive cell spreading. Similar but less extensive spreading is seen for HSPS2(m8) cells. The untransformed S2 cells show no spreading on Tiggrin, demonstrating that the observed spreading is mediated by the integrins expressed from the transgenes. Spreading of both cell lines is abolished when a monoclonal antibody that blocks integrin-mediated aggregation of Drosophila cells is added to the spreading medium. In contrast, the control CF.6G11 antibody, which binds to betaPS integrin chains on cells but does not block their function, has no effect on cell spreading. Tiggrin that was eluted from an SDS-PAGE band also promotes cell adhesion and spreading, providing further evidence that the active cell adhesive component is Tiggrin and not some chromatographically unresolved contaminant. The fusion protein that contains 270 amino acids from the C-terminal region of Tiggrin (residues 1,891-2,161), including the RGD sequence, but that lacks both of the putative LRE cell attachment sequences and the central repeat domain, also promotes adhesion and spreading of >65% of HSPS2(m8) cells and >85% of HSPS2(C) cells. The amounts of intact purified Tiggrin or Tiggrin fusion protein required to promote spreading are similar. A control fusion protein, derived from the antisense strand of the cDNA that codes for the RGD-containing fusion protein, has no effect on cell attachment and spreading. Cell spreading was quantitated on surfaces coated with 1 µg/ml or 5 µg/ml tiggrin solutions. Tiggrin mediates the spreading of a high percentage of cells expressing either alphaPS2betaPS integrin. However, on plastic coated with 1 µg/ml Tiggrin the HSPS2(C) cells spread better than the HSPS2(m8) cells (Fogerty, 1994).

To test the functional significance of Tiggrin's RGD sequence in these cell spreading experiments, the effects of increasing concentrations of an added, soluble RGD peptide (GRGDSP) was tested. This peptide inhibits spreading of both HSPS2(C) and HSPS2(m8) cells on tiggrin. Inhibition of HSPS2(m8) cells is achieved with much lower levels of soluble peptide than is seen for the HSPS2(C) cells. This is probably due to the lower initial levels of spreading seen for the HSPS2(m8) cells in the absence of any peptide. The control RGE peptide (GRGESP) has relatively little effect on cell spreading (Fogerty, 1994).

Drosophila cells expressing PS2 integrins spread on Tiggrin purified from Drosophila cells or Tiggrin fusion proteins expressed in bacteria. This spreading is inhibitable by RGD peptides, which suggests that the RGD sequence in Tiggrin is critical for the integrin-tiggrin interaction (Fogerty, 1994). This hypothesis was further tested in cultured cells and the whole fly. Using in vitro mutagenesis the sequence encoding RGD in Tiggrin was changed to encode LGA. Fusion proteins containing the final 333 amino acids of Tiggrin (of a total of 2186 amino acids) were produced in bacteria. These proteins are identical with the exception of having RGD in one case and LGA in the other (TIG-RGD and TIG-LGA respectively). A standard cell spreading assay tested the ability of each fusion protein to promote PS2 integrin-mediated cell spreading. There are four potential forms of the PS2 integrin that are generated by alternative splicing of mRNA encoding the alphaPS2 and betaPS subunits All four forms were tested for their ability to interact with TIG-RGD and TIG-LGA. The TIG-RGD readily promotes cell spreading (Fogerty, 1994).

To test the importance of the RGD sequence in the whole organism, an RGD-to-LGA mutant tiggrin transgene (UASTig LGA ) was constructed and this was introduced into flies. This mutant transgene fails to rescue tiggrin mutant flies to the levels of the wild-type transgene. It shows little rescue of the muscle defects observed in dissected larvae, as muscles are often missing and gaps are observed between muscles 9 and 10. However, measurement of these gaps indicates that they are intermediate in distance between wild-type, or UASTig + rescued, larvae. UASTig LGA-rescued tig x /tig x larvae have muscle contraction waves that are twice as fast as tig x mutants; however, this is still much slower than wild-type or tig x flies rescued by UASTig +. Thus, Tiggrin lacking an RGD sequence shows partial activity in these two assays. Lethality can be rescued by the UASTig LGA up to a maximum of 30%. tig x /tig A1 and tig x /tig O2 mutants are also rescued much more efficiently by wild-type tiggrin trangenes as compared with tig LGA transgenes (Bunch, 1998).

In the Drosophila embryo, the correct association of muscle cells with their specific ectodermally derived tendon cells, also known as epidermal muscle attachment or EMA cells, isachieved through reciprocal interactions between these two distinct cell types. Vein, a neuregulin-like factor secreted by the approaching myotube, activates the EGF-receptor signaling pathway within the tendon cells to initiate tendon cell differentiation. kakapo is expressed in the tendons and is essential for muscle-dependent tendon cell differentiation. Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. The expression of delilah, stripe, and beta1 tubulin is induced in the epidermal attachment cells as a result of the EGF-receptor pathway activation by the neuregulin-like growth factor, Vein. Vein is secreted by mesodermal cells underlying the EMA cells. Vein protein localization is restricted to the muscle-tendon junctional site in wild-type embryos. However, in kak mutant embryos, Vein protein is not localized and appears rather diffuse. This altered pattern of Vein may explain the multiple number of cells expressing delilah and stripe: since Vein is not strictly localized at a given muscle-tendon junction site, it apparently weakly activates the EGF-receptor pathway in neighboring cells as well. It is presumed that the only cells that can respond to the ectopic Vein protein are the competent population of EMA cells, defined by the early expression of stripe. These cells express stripe during early developmental stages in a muscle-independent manner and normally lose their stripe expression by stage 16 of embryonic development. When these competent EMA cells receive the muscle-derived Vein signal, the expression of stripe and delilah is reactivated. It appears that only this population of cells is capable of responding to Vein, since the pattern of the ectopic Stripe- or Delilah-expressing cells in the kak mutant embryos resembles that of the early population of Stripe-expressing cells. The reduced levels of beta1 tubulin mRNA in the mutant tendon cells may also result from the abnormal pattern of Vein localization, since lower levels of Vein may not be sufficient to induce maximal beta1 tubulin expression. It therefore appears that the primary defect in kak mutant embryos stems from the lack of Vein accumulation at the muscle-tendon junctional site (Strumpf, 1998).

How could this intracellular protein affect the localization of Vein at the extracellular matrix surrounding the EMA cell? At least two possibilities, which are not mutually exclusive, are considered. The first is the association of Kak with the unique cytoskeletal network of the EMA cell, which is critical for the cell's polarized organization. Tendon cell polarity may be essential for maintaining the characteristic junctional complexes formed between the basal surfaces of the EMA cell and the muscle cells. The space between these junctional complexes contains many extracellular matrix proteins, some of which may possess a Vein binding function. Impaired tendon cell polarity may lead to the loss of the putative Vein-binding component(s). Alternatively, Kak may be associated with a transmembrane protein(s) responsible for Vein localization either by direct binding or by association with additional extracellular matrix components that may directly bind Vein. Immunoprecipitation experiments with anti-Kak antibody indicated that Kakapo forms protein complexes containing the extracellular protein Tiggrin. These results favor the latter possibility that Kak is directly associated with protein complexes that may be important for Vein binding. The reduced amount of electron-dense material observed at the muscle-tendon junction site in the kak mutant embryos described in Prokop, et al. (1998b) is in agreement with both mechanisms mentioned above (Strumpf, 1998).

Drosophila integrins have been expressed on the surfaces of cultured cells and tested for adhesion and spreading on various matrix molecules. PS1 integrin (Multiple edematious wings) is a laminin receptor, PS1 and PS2 (inflated) integrins promote cell spreading on two different Drosophila extracellular matrix molecules, laminin and Tiggrin: PS1 on laminin, and PS2 on Tiggrin. The differing ligand specificities of these two integrins, combined with data on the in vivo expression patterns of the integrins and their ligands, lead to a model for the structure of integrin-dependent attachments in the pupal wings and embryonic muscles of Drosophila. Specifically it is thought that PS2 integrins are restricted to cells of the presumptive ventral layer of the wing and PS1 is restricted to the presumptive dorsal cells. Clones of wing tissue mutant for the PS2 integrin result in blisters on the ventral, but not the dorsal wing surface. It is not known, however, how the opposing cell layers are linked extracellularly. It is proposed that both laminin and Tiggrin are involved in these linkages. Either Tiggrin and laminin bind to one another or bind via other, as yet unidentified ECM molecules (Gotwals, 1994).

Splice variants of the Drosophila PS2 integrins differentially interact with RGD-containing fragments of the extracellular proteins tiggrin, ten-m, and D-laminin 2

Two new potential ligands of the Drosophila PS2 integrins have been characterized by functional interaction in cell culture. These potential ligands are a new Drosophila laminin alpha2 chain encoded by the wing blister locus and Ten-m, an extracellular protein known to be involved in embryonic pattern formation. As with previously identified PS2 ligands, both contain RGD sequences, and RGD-containing fragments of these two proteins (DLAM-RGD and TENM-RGD) can support PS2 integrin-mediated cell spreading. In all cases, this spreading is inhibited specifically by short RGD-containing peptides. As previously found for the PS2 ligand tiggrin (and the tiggrin fragment TIG-RGD), TENM-RGD induces maximal spreading of cells expressing integrin containing the alphaPS2C splice variant. This is in contrast to DLAM-RGD, which is the first Drosophila polypeptide shown to interact preferentially with cells expressing the alphaPS2 m8 splice variant. The betaPS integrin subunit also varies in the presumed ligand binding region as a result of alternative splicing. For TIG-RGD and TENM-RGD, the beta splice variant has little effect, but for DLAM-RGD, maximal cell spreading is supported only by the betaPS4A form of the protein. Thus, the diversity in PS2 integrins due to splicing variations, in combination with diversity of matrix ligands, can greatly enhance the functional complexity of PS2-ligand interactions in the developing animal. The data also suggest that the splice variants may alter regions of the subunits that are directly involved in ligand interactions, and this is discussed with respect to models of integrin structure (Graner, 1998full text of article).

Tiggrin: Biological Overview | Developmental Biology | Effects of Mutation | References

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