The integrin family of adhesion receptors are essential for development and homeostasis in multicellular animals. Their adhesive roles are exceedingly diverse: from providing simple, stable adhesion between tissues to mediating dynamic cycles of attachment and release between a migrating cell and its substrate (Bokel and Brown, 2002
). This diversity in function is thought to be achieved, at least in part, by the expression of different, specialized integrins in different tissues (Hynes, 2002
). Identifying the structural elements that are responsible for the different functional properties amongst the integrin family is therefore an important goal.
Integrins are divalent-cation dependent, transmembrane heterodimers that mediate adhesion by binding directly to ligands in the extracellular matrix (ECM) and simultaneously linking to the cytoskeleton through adaptor molecules (Hynes, 2002
). In recent years, much advancement has been made towards the understanding of the structural basis of integrin activation and function. The determination of the 3D structure of αVβ3 integrin confirmed predictions: the αβ heterodimer is assembled into a globular, ovoid head that rests on two legs (Xiong et al., 2001
). Ligand-bound crystals of αVβ3 and αllbβ3 identified residues in both subunits that directly contact the ligand (Xiong et al., 2002
; Xiao et al., 2004
). These findings plus extensive mutational analysis and function-blocking antibody studies have led to a detailed map of sites important for integrin function (Humphries et al., 2003
). Despite these advances, many questions remain about integrin function within the intact organism. For example, a single integrin heterodimer can bind multiple extracellular ligands, so it is important to understand which receptor-ligand interactions are relevant in a particular tissue at a particular time. Similarly, each tissue expresses a unique pool of cytoplasmic factors, the combination of which may affect integrin activity. For these reasons it is difficult to predict how a mutation characterized in cell culture will affect integrin function in the intact animal.
Our interest is to understand how an integrin heterodimer achieves its numerous functions in different tissues during morphogenesis. In Drosophila
, the αPS2βPS integrin (also referred to as PS2 integrin) is required for a number of developmental processes including muscle attachment, midgut morphogenesis and adhesion between wing epithelia in the adult. The αPS2 subunit is most similar to mammalian α5, αV, and αIIb, and forms a heterodimer with the βPS subunit, the orthologue of β1 (Bokel and Brown, 2002
). Like its mammalian homologues, αPS2βPS binds Arg-Gly-Asp (RGD) peptides and RGD-containing extracellular ligands when expressed in cell culture (e.g. Graner et al., 1998
In a previous study we performed a number of genetic screens for mutations in the gene encoding the αPS2 subunit (called the inflated (if)
gene) (Bloor and Brown, 1998
). Out of the 35 mutant alleles examined, only 6 affected just a subset of αPS2βPS-dependent functions, and these fell into three phenotypic classes based on developmental phenotype and genetic behavior (). Three alleles (if17, if21
, and if35
) behaved as typical genetic hypomorphs: they displayed the full range of inflated
null embryonic phenotypes but each less severely. The ifSEF
allele specifically affected the muscles, while ifC2B
particularly affected midgut morphogenesis (Bloor and Brown, 1998
). Intriguingly, some inflated
mutations were able to complement each other genetically. Such interallelic complementation can occur if two mutations in the same gene affect two different subfunctions; an individual that is transheterozygous for the two alleles appears wild type because it retains normal activity for each subfunction. Transheterozygous flies carrying the ifSEF
allele in combination with either ifC2B
are fully viable, suggesting that these mutations affect distinct subfunctions.
Partial loss of function inflated alleles
In the current study, we utilized these unusual inflated mutants to explore how αPS2 structure relates to its function during different developmental events. A mutant in a residue predicted to directly contact the extracellular ligand led to the identification of a function for integrins in recruiting ECM proteins to muscle attachment sites (MAS). Other alleles led to the discovery that the amount of intracellular integrin-associated proteins, such as talin, that are recruited to integrin adhesive contacts is not simply regulated by the amount of integrin at the adhesive site.