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Originally published In Press as doi:10.1074/jbc.M603782200 on July 26, 2006

J. Biol. Chem., Vol. 281, Issue 39, 28993-29001, September 29, 2006
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Argos Mutants Define an Affinity Threshold for Spitz Inhibition in Vivo*

Diego Alvarado, Damon Runyon Fellow supported by Damon Runyon Cancer Research Fellowship DRG-1884-05{ddagger}1, Timothy A. Evans§2, Raghav Sharma§3, Mark A. Lemmon{ddagger}, and Joseph B. Duffy§4

From the {ddagger}Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6059 and the §Department of Biology, Indiana University, Bloomington, Indiana 47405

Argos, a secreted antagonist of Drosophila epidermal growth factor receptor (dEGFR) signaling, acts by sequestering the activating ligand Spitz. To understand how different domains in Argos contribute to efficient Spitz sequestration, we performed a genetic screen aimed at uncovering modifiers of an Argos misexpression phenotype in the developing eye. We identified a series of suppressors mapping to the Argos transgene that affect its activity in multiple developmental contexts. These point mutations map to both the N- and C-terminal cysteine-rich regions, implicating both domains in Argos function. We show by surface plasmon resonance that these Argos mutants are deficient in their ability to bind Spitz in vitro. Our data indicate that a mere ~2-fold decrease in KD is sufficient to compromise Argos activity in vivo. This effect could be recapitulated in a cell-based assay, where a higher molar concentration of mutant Argos was needed to inhibit Spitz-dependent dEGFR phosphorylation. In contrast, a ~37-fold decrease in the binding constant nearly abolishes Argos activity in vivo and in cellular assays. In agreement with previously reported computational studies, our results define an affinity threshold for optimal Argos inhibition of dEGFR signaling during development.


Received for publication, April 20, 2006 , and in revised form, July 24, 2006.

* This work was supported by National Science Foundation Research Grant NSF IBN-0131707 (to J. B. D.), National Institutes of Health Grant RO1-CA079992 (to M. A. L.), and Department of Defense Breast Cancer Research Program Grant W81XWH-05-1-0289 (to M. A. L.). 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.

2 Supported by National Institutes of Health Predoctoral Training Grant GM-007757.

3 Recipient of an Howard Hughes Medical Institute Capstone Award.

1 To whom correspondence may be addressed. Tel.: 215-898-3411; E-mail: dalvarad{at}mail.med.upenn.edu. 4To whom correspondence may be addressed. Present address: Dept. of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA 01609. Tel.: 508-831-5579; E-mail: jduffy{at}wpi.edu.


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