title: Argos mutants define an affinity threshold for spitz inhibition in vivo.
abstract: 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 approximately 2-fold decrease in K(D) 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 approximately 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.
authors.affiliation: Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6059, USA. dalvarad@mail.med.upenn.edu
authors.forename: Diego
authors.initials: D
authors.lastname: Alvarado
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Evans
authors.forename: Raghav
authors.initials: R
authors.lastname: Sharma
authors.forename: Mark A
authors.initials: MA
authors.lastname: Lemmon
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Duffy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004330:Drosophila; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors* / Q000235:genetics*; D066246:ErbB Receptors / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism / Q000502:physiology*; D005786:Gene Expression Regulation; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000502:physiology*; D017956:Photoreceptor Cells, Invertebrate / Q000000981:diagnostic imaging / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014463:Ultrasonography
chemical_list: D029721:Drosophila Proteins; D005136:Eye Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C075638:aos protein, Drosophila; C076026:spi protein, Drosophila; D004815:Epidermal Growth Factor; D066246:ErbB Receptors