BIAcore Analysis to Test Phosphopeptide-SH2 Domain Interactions

  • Frédéric Vély
  • Alain Trautmann
  • Eric Vivier
Part of the Methods in Molecular Biology book series (MIMB, volume 121)

Abstract

The elucidation of intracytoplasmic signaling pathways is a ctriical step in the precise understanding of cell biology. In this regard, the identification of intracytoplasmic domains, which are specialized in the transmission of biological messages, has been an enormous breakthrough (1). Among these transducing domains, the src homology 2 (SH2) domains are specialized in the interaction with the phosphorylated forms of tyrosine residues (2). SH2 domains are classically 100 amino acids long and the three-dimensional structure of several SH2-containing molecules has been resolved. As it is precisely controlled by a subtle balance between protein tyrosine kinases (which phosphorylate) and protein tyrosine phosphatases (which dephosphorylate), the phosphorylation on tyrosine residues is a highly versatile way of dictating the interaction between a given protein and an SH2-containing partner (3). The inhibitory receptors (IRs) for major histocompatibility complex (MHC) class Ia and Ib molecules expressed on NK cells, such as the killer cell inhibitory receptors (KIRs) and the CD94-NKG2A heterodimers, respectively, mediate their inhibitory function via intracytoplasmic motifs (IVLS)xYxx(LV), the immunoreceptor tyrosine-based inhibitory motifs (ITIMs) (34, 6). Upon engagement of these IRs, the tyrosine residue present in ITIMs is phosphorylated, and is therefore capable of interacting with two distinct SH2-containing proteins: the protein tyrosine phosphatases SHP-1 and SHP-2 . In this chapter, we provide protocols (1) to investigate the direct binding of recombinant soluble SH2 domains to phosphorylated peptides using surface plasmon reso-nance (SPR) technology, as well as (2) to measure the affinity constant that characterizes this interaction (7, 8, 9, 10, 11, 12, 13).

Keywords

Surfactant HPLC EDTA Tyrosine Aldehyde 

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Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • Frédéric Vély
    • 1
  • Alain Trautmann
    • 1
  • Eric Vivier
    • 2
  1. 1.Groupe Hospitalier Pitié-SalpétriéreLaboratoire d′Immunologie CellulaireParisFrance
  2. 2.Institut Universitaire de FranceMarseille-LuminyFrance

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