title: Peptide microarrays to probe for competition for binding sites in a protein interaction network.
abstract: Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins will greatly overestimate connectivity within the network. Here, we addressed the impact of intracellular complexity on signalling networks using microarrays that carried a collection of peptides binding to the GRB2 SH2 and SH3 domains. Binding patterns and affinities for the recombinant adaptor protein GRB2 were compared with the ones for the protein in cell lysates. Peptide microarrays were titrated with the histidine-tagged recombinant protein, cell lysates or mixtures of both. Indeed, for recombinant GRB2, binding was detected for more peptides than for GRB2 in cell lysates. Moreover, binding was also observed for poor binders. It was impossible to define affinity thresholds for the binding of the recombinant protein to enable a discrimination of physiologically relevant interactions. Titrations of recombinant protein with lysate confirmed competition as the basis for fewer interactions. Importantly, the methods presented here enable the description of physiologically relevant binding patterns for proteins of interest and the identification of those peptide motifs, which are most strongly affected by competition.
  
  
  CONCLUSIONS
  The biological significance of protein-protein interactions can only be addressed in a physiologically meaningful way in the presence of the endogenous proteome which may contain proteins that compete for binding sites. Using peptide microarrays, we here demonstrate for the adaptor protein GRB2 that this competition strongly reduces the number of interactions with other signalling proteins.
authors.affiliation: Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Sinzinger
authors.forename: Ivo R
authors.initials: IR
authors.lastname: Ruttekolk
authors.forename: Jolein
authors.initials: J
authors.lastname: Gloerich
authors.forename: Hans
authors.initials: H
authors.lastname: Wessels
authors.forename: Yi-Da
authors.initials: YD
authors.lastname: Chung
authors.forename: Merel J W
authors.initials: MJ
authors.lastname: Adjobo-Hermans
authors.forename: Roland
authors.initials: R
authors.lastname: Brock
mesh_terms: D020816:Amino Acid Motifs; D051380:GRB2 Adaptor Protein / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D040081:Protein Array Analysis / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D018909:src Homology Domains
chemical_list: D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D011994:Recombinant Proteins
keywords: Dissociation constant; Fluorescence correlation spectroscopy; GRB2; NanoLC–MS/MS; Peptide microarrays; SH2/SH3 domains