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J. Biol. Chem., Vol. 281, Issue 8, 5050-5057, February 24, 2006

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Amino Acid Changes in Drosophila PS2PS Integrins That Affect Ligand Affinity^*

Thomas A. Bunch¹, Teresa L. Helsten, Timmy L. Kendall, Nikhil Shirahatti^¶, Daruka Mahadevan^¶, Sanford J. Shattil, and Danny L. Brower^||

From the Departments of Molecular and Cellular Biology, ^||Biochemistry, and ^¶Medicine, Arizona Cancer Center, Tucson, Arizona 85724 and the Department of Medicine, University of California San Diego, La Jolla, California 92093

We developed a ligand-mimetic antibody Fab fragment specific for Drosophila PS2PS 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 v3 ligand-mimetic antibody WOW-1. The specificity of PS2PS 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 PS metal ion-dependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the PS2 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 PS2 GFFNR GFANA mutation. A mutation in the PS I domain (PS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by PS2PS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin subunit plexin-semaphorin-integrin (PSI) and stalk domains have been shown to have activating properties. We found that complete deletion of the PS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because PS-PSI 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.

Received for publication, August 4, 2005 , and in revised form, December 21, 2005.

^* This work was supported by National Institutes of Health Grants R01GM42474 (to D. L. B.) and R01HL56595 (to S. J. S.) and American Cancer Society Grant 5P30CA023074 (to D. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data.

¹ To whom correspondence should be addressed: Dept. of Molecular and Cellular Biology, Arizona Cancer Center, Rm. 0977, 1515 N. Campbell Ave., Tucson, AZ 85724. Tel.: 520-621-3869; Fax: 520-626-3764; E-mail: tbunch{at}u.arizona.edu.

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