title: Amino acid changes in Drosophila alphaPS2betaPS integrins that affect ligand affinity.
abstract: We developed a ligand-mimetic antibody Fab fragment specific for Drosophila alphaPS2betaPS integrins to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the alphavbeta3 ligand-mimetic antibody WOW-1. The specificity of alphaPS2betaPS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the betaPS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the alphaPS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn2+. Although Mn2+ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn2+ could be increased further by the alphaPS2 GFFNR --> GFANA mutation. A mutation in the betaPS I domain (betaPS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by alphaPS2betaPS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin beta subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. We found that complete deletion of the betaPS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because betaPS-DeltaPSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system.
authors.affiliation: Department of Molecular and Cellular Biology, Arizona Cancer Center, Tucson, Arizona 85724, USA. tbunch@u.arizona.edu
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Bunch
authors.forename: Teresa L
authors.initials: TL
authors.lastname: Helsten
authors.forename: Timmy L
authors.initials: TL
authors.lastname: Kendall
authors.forename: Nikhil
authors.initials: N
authors.lastname: Shirahatti
authors.forename: Daruka
authors.initials: D
authors.lastname: Mahadevan
authors.forename: Sanford J
authors.initials: SJ
authors.lastname: Shattil
authors.forename: Danny L
authors.initials: DL
authors.lastname: Brower
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics*; D004492:Edetic Acid / Q000737:chemistry; D039001:Integrin alpha Chains / Q000737:chemistry* / Q000235:genetics*; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D016023:Integrins / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008345:Manganese / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D029721:Drosophila Proteins; D039001:Integrin alpha Chains; D039302:Integrin alphaVbeta3; D016023:Integrins; D008024:Ligands; D009842:Oligopeptides; C082188:if protein, Drosophila; D008345:Manganese; C047981:arginyl-glycyl-aspartic acid; D004492:Edetic Acid