MAPPIT (Mammalian Protein–Protein Interaction Trap) Analysis of Early Steps in Toll-Like Receptor Signalling

Abstract Binding of GH to its receptor induces rapid phosphorylation of conserved tyrosine motifs that function as recruitment sites for downstream signaling molecules. Using mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, we mapped the binding sites in the GH receptor for signal transducer and activator of transcription 5 (STAT5) a and b and for the negative regulators of cytokine signaling cytokine-inducible Src-homology 2 (SH2)-containing protein (CIS) and suppressor of cytokine signaling 2 (SOCS2). Y534, Y566, and Y627 are the major recruitment sites for STAT5. A non-overlapping recruitment pattern is observed for SOCS2 and CIS with positions Y487 and Y595 as major binding sites, ruling out SOCS-mediated inhibition of STAT5 activation by competition for shared binding sites. More detailed analysis revealed that CIS binding to the Y595, but not to the Y487 motif, depends on both its SH2 domain and the C-terminal part of its SOCS box, with a critical role for the CIS Y253 residue. This functional divergence of the two CIS/SOCS2 recruitment sites is also observed upon substitution of the Y+1 residue by leucine, turning the Y487, but not the Y595 motif into a functional STAT5 recruitment site.

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MAPPIT: a versatile tool to study cytokine receptor signalling

Biochemical Society Transactions ◽

10.1042/bst0361448 ◽

2008 ◽

Vol 36 (6) ◽

pp. 1448-1451 ◽

Cited By ~ 4

Author(s):

Irma Lemmens ◽

Sam Lievens ◽

Jan Tavernier

Keyword(s):

Protein Interactions ◽

Mammalian Cells ◽

Cytokine Receptor ◽

Bait Protein ◽

Protein Protein Interaction ◽

Receptor Signalling ◽

Versatile Tool ◽

Two Hybrid ◽

Interaction Trap ◽

Mammalian Protein

MAPPIT (mammalian protein–protein interaction trap) is a cytokine receptor-based two-hybrid method that operates in intact mammalian cells. A bait is fused C-terminally to a STAT (signal transducer and activator of transcription) recruitment-deficient receptor, whereas the prey is linked to functional STAT-binding sites. When bait and prey interact a ligand-dependent complementation of the STAT recruitment deficiency occurs, leading to activation of a STAT-responsive reporter. MAPPIT is very well suited to study protein interactions involving activated cytokine receptors as the technique allows modification of the bait protein in a physiologically optimal environment.

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MAPPIT (MAmmalian Protein–Protein Interaction Trap) as a tool to study HIV reverse transcriptase dimerization in intact human cells

Journal of Virological Methods ◽

10.1016/j.jviromet.2008.06.021 ◽

2008 ◽

Vol 153 (1) ◽

pp. 7-15 ◽

Cited By ~ 14

Author(s):

Els Pattyn ◽

Delphine Lavens ◽

José Van der Heyden ◽

Annick Verhee ◽

Sam Lievens ◽

...

Keyword(s):

Reverse Transcriptase ◽

Protein Interaction ◽

Human Cells ◽

Hiv Reverse Transcriptase ◽

Protein Protein Interaction ◽

Interaction Trap ◽

Mammalian Protein

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Straightforward Protein-Protein Interaction Interface Mapping via Random Mutagenesis and Mammalian Protein Protein Interaction Trap (MAPPIT)

International Journal of Molecular Sciences ◽

10.3390/ijms20092058 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2058 ◽

Cited By ~ 3

Author(s):

Laurens Vyncke ◽

Delphine Masschaele ◽

Jan Tavernier ◽

Frank Peelman

Keyword(s):

Protein Interaction ◽

Protein Interactions ◽

Posttranslational Modifications ◽

Random Mutagenesis ◽

Interaction Mechanism ◽

Protein Protein Interaction ◽

Interaction Sites ◽

Multiple Partners ◽

Interaction Trap ◽

Mammalian Protein

The MAPPIT (mammalian protein protein interaction trap) method allows high-throughput detection of protein interactions by very simple co-transfection of three plasmids in HEK293T cells, followed by a luciferase readout. MAPPIT detects a large percentage of all protein interactions, including those requiring posttranslational modifications and endogenous or exogenous ligands. Here, we present a straightforward method that allows detailed mapping of interaction interfaces via MAPPIT. The method provides insight into the interaction mechanism and reveals how this is affected by disease-associated mutations. By combining error-prone polymerase chain reaction (PCR) for random mutagenesis, 96-well DNA prepping, Sanger sequencing, and MAPPIT via 384-well transfections, we test the effects of a large number of mutations of a selected protein on its protein interactions. The entire screen takes less than three months and interactions with multiple partners can be studied in parallel. The effect of mutations on the MAPPIT readout is mapped on the protein structure, allowing unbiased identification of all putative interaction sites. We have thus far analysed 6 proteins and mapped their interfaces for 16 different interaction partners. Our method is broadly applicable as the required tools are simple and widely available.

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