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

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.

[1]  A. Sorkin,et al.  Interaction of EGF receptor and Grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy , 2000, Current Biology.

[2]  J. Tavernier,et al.  Neutralizing monoclonal antibodies can potentiate IL‐5 signaling , 2001, European journal of immunology.

[3]  J. Tavernier,et al.  Identification of the Y985 and Y1077 motifs as SOCS3 recruitment sites in the murine leptin receptor , 2000, FEBS letters.

[4]  R. Heim,et al.  Using GFP in FRET-based applications. , 1999, Trends in cell biology.

[5]  A. Varshavsky,et al.  Split ubiquitin as a sensor of protein interactions in vivo. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[6]  I. Wilson,et al.  Erythropoietin receptor activation by a ligand-induced conformation change. , 1999, Science.

[7]  T. Hunter,et al.  The Protein Kinase Complement of the Human Genome , 2002, Science.

[8]  J. Darnell,et al.  The role of STATs in transcriptional control and their impact on cellular function , 2000, Oncogene.

[9]  M. Goumans,et al.  Expression of type I and type IB receptors for activin in midgestation mouse embryos suggests distinct functions in organogenesis , 1995, Mechanisms of Development.

[10]  H. Blau,et al.  Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Ian Walker,et al.  Structure of the Complete Extracellular Domain of the Common β Subunit of the Human GM-CSF, IL-3, and IL-5 Receptors Reveals a Novel Dimer Configuration , 2001, Cell.

[12]  M. Osborne,et al.  The Yeast Tribrid System—Genetic Detection of trans-phosphorylated ITAM-SH2-Interactions , 1995, Bio/Technology.

[13]  H. Lodish,et al.  Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[14]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[15]  S. Elledge,et al.  Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions , 1997, Molecular and cellular biology.

[16]  Y. Umezawa,et al.  Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing. , 2001, Analytical chemistry.

[17]  H. Mizuguchi,et al.  IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. , 2000, Molecular therapy : the journal of the American Society of Gene Therapy.

[18]  K. Isselbacher,et al.  A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: application to interaction screening. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[19]  P. Heinrich,et al.  Orientational Constraints of the gp130 Intracellular Juxtamembrane Domain for Signaling* , 2002, The Journal of Biological Chemistry.

[20]  A. Aronheim,et al.  The Ras recruitment system, a novel approach to the study of protein–protein interactions , 1998, Current Biology.

[21]  H. Lodish,et al.  The erythropoietin receptor cytosolic juxtamembrane domain contains an essential, precisely oriented, hydrophobic motif. , 2001, Molecular cell.

[22]  Jeffrey L. Wrana,et al.  Signal Transduction by the TGF-β Superfamily , 2002, Science.

[23]  J. Tavernier,et al.  Comparison of leptin- and interleukin-6-regulated expression of the rPAP gene family: evidence for differential co-regulatory signals. , 2002, European cytokine network.

[24]  I. Wilson,et al.  Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation. , 1999, Science.

[25]  L. Nelles,et al.  SIP1, a Novel Zinc Finger/Homeodomain Repressor, Interacts with Smad Proteins and Binds to 5′-CACCT Sequences in Candidate Target Genes* , 1999, The Journal of Biological Chemistry.

[26]  Jan Tavernier,et al.  Design and application of a cytokine-receptor-based interaction trap , 2001, Nature Cell Biology.

[27]  Ursula Klingmüller,et al.  Self assembly of the transmembrane domain promotes signal transduction through the erythropoietin receptor , 2001, Current Biology.

[28]  J. Vandekerckhove,et al.  A sensitive and versatile bioassay for ligands that signal through receptor clustering. , 2000, Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research.

[29]  J. Joung,et al.  A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions. , 2000, Proceedings of the National Academy of Sciences of the United States of America.