Identification of Integrin β Subunit Mutations That Alter Affinity for Extracellular Matrix Ligand

Abstract

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...

Zasp regulates integrin activation

“…Recently several point mutations in the extracellular domain of bPS integrin were identified that increase the affinity of aPS2bPS integrin binding to the extracellular matrix and cause lethality owing to this increase in affinity (Kendall et al, 2011). Lethality associated with some of these mutations can be suppressed by removing one copy of talin, confirming the role of talin as an integrin activator (Kendall et al, 2011).…”

“…We therefore tested the genetic interaction of two of these mutants (b44, I375F in the ADMIDAS and b30, I298F in b-I domain) with Zasp. Removing one copy of Zasp increases viability of b44 from 33% viable mutant males to 54%, n51445 [21% to 59% for rhea 2 (Drosophila talin) (Kendall et al, 2011)] and it increases viability of b30 from 41% to 57%, n51516 [3% to 44% for rhea 2 (Kendall et al, 2011)]. This suggests that both talin and Zasp are involved in modulating integrin affinity in Drosophila.…”

“…Lethality associated with some of these mutations can be suppressed by removing one copy of talin, confirming the role of talin as an integrin activator (Kendall et al, 2011). We therefore tested the genetic interaction of two of these mutants (b44, I375F in the ADMIDAS and b30, I298F in b-I domain) with Zasp.…”

“…Bunch, Arizona Cancer Center, Tucson, AZ, USA) (Kendall et al, 2011) and Dmef2-Gal4 from the Bloomington Drosophila Stock Center. For Fig.…”

“…Integrins are the major family of ECM adhesion receptors, and regulation of integrin affinity for ligand (integrin activation) is a key control point in cell adhesion, migration and assembly of the ECM (Calderwood, 2004). Consistent with its central role in these fundamental processes, perturbation of integrin activation in model organisms impairs viability and disrupts development, tissue formation, morphogenesis, cell trafficking and mechanosensing (Czuchra et al, 2006;Kendall et al, 2011;Martin-Bermudo et al, 1998;Millon-Frémillon et al, 2008;Pines et al, 2011;.…”