Immunoprecipitation Assay to Study Enzyme Receptor Interactions in Transfected Cells
Transcription
Programmed death-1, PD-1, a cell surface receptor protein, contains two cytoplasmic tyrosine motifs — the inhibitory motif, ITIM, and the switch motif, ITSM. Ligand binding induces ITIM and ITSM phosphorylation and tyrosine phosphatase protein SHP2 recruitment to the cytoplasmic domain of PD-1, triggering a downstream signaling cascade.
To study SHP2-PD-1 interactions via a co-immunoprecipitation assay, obtain an adherent epithelial cell culture. These cells express green fluorescent protein, GFP-tagged wild-type PD-1 with phosphorylated ITIM and ITSM. Obtain epithelial cells expressing GFP-tagged mutant PD-1, wherein ITIM or ITSM mutations inhibit their phosphorylation.
Add lysis buffer containing detergent and sodium orthovanadate. The detergents lyse the cells, causing GFP-tagged PD-1 protein release. Sodium orthovanadate blocks released phosphatases, thus, preventing ITIM and ITSM dephosphorylation.
Centrifuge the lysate. Transfer the protein-containing supernatant into fresh tubes. Add anti-GFP antibody-coated agarose beads. The GFP-tagged PD-1 proteins bind to the anti-GFP antibodies.
Incubate with the SHP2 suspension. The bead-bound PD-1-GFP binds SHP2 via the phosphorylated ITIM and ITSM.
Add Laemmli buffer and boil so that the reducing agents and detergents in the buffer denature the PD-1-GFP-SHP2 protein complexes. Centrifuge and collect the supernatant containing the denatured protein complexes. Perform SDS-PAGE and western blot.
Wild-type and ITIM-mutated PD-1 interacts with SHP2. ITSM mutants fail to bind SHP2, indicating that the PD1-SHP2 interaction requires phosphorylated ITSM.
