Heteromeric MAPPIT: a novel strategy to study modification-dependent protein-protein interactions in mammalian cells

Abstract

We recently reported a two-hybrid trap for detecting protein-protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait-prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the (beta)c chain and its modifying enzyme to the GM-CSFRalpha chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFbeta/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells.

Figure 1

Layout of the heteromeric MAPPIT method. A representation of the different components and mechanism of the cytokine receptor-based interaction trap is shown. (A) The homodimeric MAPPIT approach is presented (see also 13). (B) The heteromeric MAPPIT principle. The ligand-binding domain in (B) is from a heteromeric cytokine receptor (with subunits CR1 and CR2), which replaces the erythropoietin receptor (EpoR) from (A). The bait, ‘B’ is fused to the CR1 chain whereas its modifying enzyme, ‘E’ is fused to the CR2 chain. Ligand-induced activation of the receptor-associated JAK tyrosine kinase puts the receptor complex in a ‘stand-by’ mode without induction of detectable reporter activity. No STAT3 recruitment and activation can occur due to the Y1138F mutation in the cytosolic domain of the LR. Additional Y985/1077F mutations eliminate adapter and/or negative feedback mechanisms. Complementation is induced upon interaction between the modified bait (with a black dot representing the modification) and prey, ‘P’, which leads to recruitment of the C-terminal part of gp130 containing four functional STAT3-binding sites. Subsequent STAT3 phosphorylation and activation induces luciferase activity under control of the rPAP1 promoter. Functional STAT3 recruitment sites are presented as black boxes; positions of the Y→F mutations within the LR-F3 are shown as open boxes. Hinge regions, drawn as shaded boxes, preceding the prey, bait and modifying enzyme provide additional flexibility in the chimeric polypeptides. (C) Dose–response curves of heteromeric chimeric receptors. HEK293T cells were transiently transfected with different receptor chimeras: 1, EpoR-LR; 2, IL3Rα-LR + βc-LR; 3, IL5Rα-LR + βc-LR; 4, GMRα-LR + βc-LR; 5, IL3Rα-LR; 6, IL5Rα-LR; 7, GMRα-LR. Bars and error bars represent mean values of the luciferase measurements and SD, respectively. LR, cytoplasmic domain of the wild-type leptin receptor. The βc-LR chain alone was stimulated with all the different ligands at a concentration of 10 ng/ml and showed no response in all cases tested (data not shown).

Figure 2

Detection of phosphoserine-dependent Smad interactions using the heteromeric MAPPIT procedure. (A) HEK293T cells were transfected with plasmids encoding: 1, βc-LRF3+2L-CA-ALK6, GM-CSFRα-LRF3+ 2L-Smad5 and gp130-Smad4; 2, βc-LRF3+2L-CA-ALK6, GM-CSFRα-LRF3+2L-Smad5 S→A (a mutant construct wherein critical serines of Smad5 are replaced by alanines) and gp130-Smad4; 3, βc-LRF3+2L (no CA-ALK6), GM-CSFRα-LRF3+2L-Smad5 and gp130-Smad4; 4, βc-LRF3+ 2L-CA-ALK6, GM-CSFRα-LRF3+2L (no Smad5) and gp130-Smad4; 5, βc-LRF3+2L-CA-ALK6, GM-CSFRα-LRF3+2L-Smad5 and gp130-SVT (no Smad4); 6, βc-LRF3+2L-CA-ALK6, GM-CSFRα-LRF3+2L-Smad5 and Smad4 (no gp130). Average values for relative luciferase activities (x-fold increase, luciferase values obtained from stimulated cells with respect to values derived from untreated cells) are shown. (B) HEK293T cells were transfected with plasmids encoding: 1, GM-CSFRα-LRF3+1L-CA-ALK4, βc-LRF3+1L-Smad3 and gp130-Smad4; 2, GM-CSFRα-LRF3+1L-CA-ALK4, βc-LRF3+1L-Smad3 S→A and gp130-Smad4. (C) HEK293T cells were transfected with plasmids encoding: 1, GM-CSFRα-LRF3+1L-CA-ALK4, βc-LRF3+1L-Smad3, and gp130-Smad4; 2, GM-CSFRα-LRF3+1L-CA-ALK4 and βc-LRF3+1L-Smad3 S→A (critical serines of Smad3 mutated to alanines) and gp130-Smad4; 3, mock control with empty vector. Cells were either left untreated (–) or were stimulated with 10 ng/ml GM-CSF (+). α phospho-Smad3, serine phosphorylated Smad3; α leptin receptor, the chimeric chain detected using an anti-leptin receptor antibody; α phospho-JAK2, phosphorylated JAK2; α JAK2, total JAK2; α phospho-STAT3, phosphorylated STAT3; α STAT3, total STAT3.

Figure 3

Heteromeric MAPPIT using cells stably expressing the chimeric receptor chains. (A) An isogenic cell pool stably expressing the GM-CSFRα-LRF3+1L-CA-ALK4 and βc-LRF3+1L-Smad3 receptor chains was selected and transfected with plasmids encoding: 1, gp130-Smad4; 2, Smad4 (no gp130); 3, gp130-SVT (no Smad4); 4, gp130-Smad4 combined with βc-LRF3+1L-Smad3 S→A mutant; 5, gp130-Smad4 combined with GM-CSFRα-LRF3+1L (no ALK4); 6, gp130-Smad4 combined with Smad4 (no gp130 chain). Average values for relative luciferase activities (x-fold increase, stimulated versus untreated) are shown. (B and C) FACS analyses, with filled curves representing parental HEK293-16 cells and open curves representing the selected isogenic cell pool expressing both receptor chimeras. (B) and (C) show the expression of the βc-LRF3+1L-Smad3 and the GM-CSFRα-LRF3+1L-CA-ALK4 chains, respectively.