We conducted a genetic screen for mutations in myospheroid, the gene encoding the Drosophila betaPS integrin subunit, and identified point mutants in all of the structural domains of the protein. Surprisingly, we find that mutations in very strongly conserved residues will often allow sufficient integrin function to support the development of adult animals, including mutations in the ADMIDAS site and in a cytoplasmic NPXY motif. Many mutations in the I-like domain reduce integrin expression specifically when betaPS is combined with activating alphaPS2 cytoplasmic mutations, indicating that integrins in the extended conformation are unstable relative to the inactive, bent heterodimers. Interestingly, the screen has identified alleles that show gain-of-function characteristics in cell culture, but have negative effects on animal development or viability. This is illustrated by the allele mys(b58); available structural models suggest that the molecular lesion of mys(b58), V409>D, should promote the "open" conformation of the beta subunit I-like domain. This expectation is supported by the finding that alphaPS2betaPS (V409>D) promotes adhesion and spreading of S2 cells more effectively than does wild-type alphaPS2betaPS, even when betaPS is paired with alphaPS2 containing activating cytoplasmic mutations. Finally, comparisons with the sequence of human beta8 suggest that evolution has targeted the "mys(b58)" residue as a means of affecting integrin activity.

We have analyzed a set of new and existing strong mutations in the myospheroid gene, which encodes the betaPS integrin subunit of Drosophila. In addition to missense and other null mutations, three mutants behave as antimorphic alleles, indicative of dominant negative properties. Unlike null alleles, the three antimorphic mutants are synthetically lethal in double heterozygotes with an inflated (alphaPS2) null allele, and they fail to complement very weak, otherwise viable alleles of myospheroid. Two of the antimorphs result from identical splice site lesions, which create a frameshift in the C-terminal half of the cytoplasmic domain of betaPS. The third antimorphic mutation is caused by a stop codon just before the cytoplasmic splice site. These mutant betaPS proteins can support cell spreading in culture, especially under conditions that appear to promote integrin activation. Analyses of developing animals indicate that the dominant negative properties are not a result of inefficient surface expression, or simple competition between functional and nonfunctional proteins. These data indicate that mutations disrupting the C-terminal cytoplasmic domain of integrin beta subunits can have dominant negative effects in situ, at normal levels of expression, and that this property does not necessarily depend on a specific new protein sequence or structure. The results are discussed with respect to similar vertebrate beta subunit cytoplasmic mutations.

Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talin's role in regulating Drosophila ␣PS2␤PS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting ␣PS2␤PS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of ␣PS2␤PS with those of human ␣IIb␤3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of ␣PS2␤PS with those of ␣IIb␤3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type ␣IIb␤3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talin's integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila ␣PS2␤PS affinity because of structural features inherent in the ␣PS2␤PS extracellular and/or transmembrane domains. INTRODUCTIONIntegrin adhesion receptors couple the extracellular matrix with the actin cytoskeleton, allowing transmission of both mechanical force and biochemical signals across the plasma membrane (Hynes, 2002). The cytoskeletal protein talin, a product of the talin 1 (TLN1) gene, provides a key physical linkage between integrin ␤ cytoplasmic domains and F-actin (Critchley, 2004;Wiesner et al., 2005). Talin's N-terminal head region contains a FERM domain with a PTB subdomain capable of interacting with several proteins, including integrin ␤ cytoplasmic domains (Wegener et al., 2007). Talin's C-terminal rod domain contains binding sites for several cytoskeletal proteins, including actin (Calderwood, 2004;Critchley, 2004). Loss of talin in worms, flies, and mice leads to lethal phenotypes that closely resemble those caused by loss or mutation of integrins, indicating that talin's function overlaps that of integrins in the developing organism (Monkley et al., 2000;Brown et al., 2002;Brower, 2003;Cram et al., 2003;Wiesner et al., 2005). In fact, talin is essential for the normal development of organisms ranging in complexity from the multicellular slime mold Dictyostelium discoideum to vertebrates (Nuckolls et al., 1992;Albiges-Rizo et al., 1995;Bolton et al., 1997;Priddle et al., 1998;Tsujioka et al., 1999;Monkley et al., 2000;Cram et al., 2003). In vertebrates, it is also clear that talin plays a key role in regulating the affinity of integrins for adhesive ligands (Calderwood et al., 1999(Calderwood et al., , 2002Tadokoro et al., 2003;Ginsberg et al., 2005;Wegen...

We examined over 50 mutations in the Drosophila ␤PS integrin subunit that alter integrin function in situ for their ability to bind a soluble monovalent ligand, TWOW-1. Surprisingly, very few of the mutations, which were selected for conditional lethality in the fly, reduce the ligand binding ability of the integrin. The most prevalent class of mutations activates the integrin heterodimer. These findings emphasize the importance of integrin affinity regulation and point out how molecular interactions throughout the integrin molecule are important in keeping the integrin in a low affinity state. Mutations strongly support the controversial deadbolt hypothesis, where the CD loop in the ␤ tail domain acts to restrain the I domain in the inactive, bent conformation. Site-directed mutations in the cytoplasmic domains of ␤PS and ␣PS2C reveal different effects on ligand binding from those observed for ␣IIb␤3 integrins and identify for the first time a cytoplasmic cysteine residue, conserved in three human integrins, as being important in affinity regulation. In the fly, we find that genetic interactions of the ␤PS mutations with reduction in talin function are consistent with the integrin affinity differences measured in cells. Additionally, these genetic interactions report on increased and decreased integrin functions that do not result in affinity changes in the PS2C integrin measured in cultured cells.Integrin cell surface receptors are important for morphogenetic events in normal development and are also critical for adult and many disease-related processes as well (1). The integrin ␣␤ heterodimer is a dynamic receptor, whose activity is regulated by interactions with extracellular, intracellular, and other transmembrane proteins. This regulation can change the affinity of individual integrin molecules or alter the clustering of integrins such that their avidity is altered. Experiments utilizing conformation-specific antibodies, high resolution x-ray, NMR, small angle neutron scattering, molecular dynamics, steered molecular dynamics, and electron microscopy are elucidating how the ␣ and ␤ subunits are organized and change in response to activating conditions and ligand binding. One goal of the current work on integrin biology is to understand how the structural information gained on isolated integrins relates to integrin function in a cellular context. Therefore, models for integrin activation are being tested by site-directed mutagenesis (reviewed in Refs. 2-4).We have used a complementary approach of first identifying amino acids in the Drosophila integrin ␤PS subunit that alter its function in the whole organism and then examining the effects of these mutations at the cellular and molecular level. Previous work demonstrated that myospheroid (mys, encoding ␤PS integrin) hypomorphic mutations displayed stronger phenotypes at higher temperatures (5). Therefore, we screened for mutations in mys that produce viable flies at low temperatures but lethality at high temperatures. Because null alleles of mys are complet...

Decapentaplegic (Dpp) is a Drosophila member of the Transforming Growth Factor-beta (TGF-beta)/Bone Morphogenetic Protein (BMP) superfamily of growth factors. Dpp serves as a classical morphogen, where concentration gradients of this secreted factor control patterning over many cell dimensions. Regulating the level of Dpp signaling is therefore critical to its function during development. One type of molecule proposed to modulate growth factor signaling at the cell surface are integral membrane proteoglycans. We show here that division abnormally delayed (dally), a Drosophila member of the glypican family of integral membrane proteoglycans is required for normal Dpp signaling during development, affecting cellular responses to this morphogen. Ectopic expression of dally+ can alter the patterning activity of Dpp, suggesting a role for dally+ in modulating Dpp signaling strength. These findings support a role for members of the glypican family in controlling TGF-beta/BMP activity in vivo by affecting signaling at the cell surface.