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

Timmy Kendall,Leona Mukai,Alison L Jannuzi,Thomas A Bunch

doi:10.1074/jbc.m111.254797

Abstract

We examined over 50 mutations in the Drosophila βPS 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 β tail domain acts to restrain the I domain in the inactive, bent conformation. Site-directed mutations in the cytoplasmic domains of βPS and αPS2C reveal different effects on ligand binding from those observed for αIIbβ3 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 βPS 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.

Highlights

Mutations in the MIDAS and SyMBS Domains Impair Ligand Binding—In the ␤ integrin subunit, two domains, MIDAS and SyMBS (or ligand-associated metal binding site (LIMBS)), have been identified as contributing to ligand binding
Supplemental figures are provided for each set of mutants that focus on Summary of mys mutant TWOW-1 binding, expression, and genetic interactions with rhea. This is a summary of the detailed data that can be found in supplemental Tables S1–S3 and S5
For mysG1, de1T825 refers to the carboxyl-terminal 21 amino acids deleted and replaced by 25 essentially random amino acids. mysb24 is a mutation of the single amino acid between I-EGF2 and I-EGF3. (For convenience, it has been placed in the I-EGF2 group)

Summary

Introduction

Mutations in the MIDAS and SyMBS Domains Impair Ligand Binding—In the ␤ integrin subunit, two domains, MIDAS and SyMBS (or ligand-associated metal binding site (LIMBS)), have been identified as contributing to ligand binding. Mutations that increase ligand binding are distributed in all domains, illustrating the importance of all domains in restraining the integrin in the low affinity (bent, head closed) conformation (Fig. 3C). Activation of the integrin by mutation of the long range disulfide bridge linking the PSI domain to the junction of the Hybrid and I-EGF (supplemental Fig. S2) suggests that this is the case [27].

Results

Conclusion