The genetic design of signaling cascades to record receptor activation

We have developed an experimental strategy to monitor protein interactions in a cell with a high degree of selectivity and sensitivity. A transcription factor is tethered to a membrane-bound receptor with a linker that contains a cleavage site for a specific protease. Activation of the receptor recruits a signaling protein fused to the protease that then cleaves and releases the transcription factor to activate reporter genes in the nucleus. This strategy converts a transient interaction into a stable and amplifiable reporter gene signal to record the activation of a receptor without interference from endogenous signaling pathways. We have developed this assay for three classes of receptors: G protein-coupled receptors, receptor tyrosine kinases, and steroid hormone receptors. Finally, we use the assay to identify a ligand for the orphan receptor GPR1, suggesting a role for this receptor in the regulation of inflammation.

[1]  S. A. Johnston,et al.  Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. , 1994, Analytical biochemistry.

[2]  R. Nicholas,et al.  HEK293 human embryonic kidney cells endogenously express the P2Y1 and P2Y2 receptors , 1997, Neuropharmacology.

[3]  J. Schlessinger,et al.  Cell Signaling by Receptor Tyrosine Kinases , 2000, Cell.

[4]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. Gustafsson,et al.  Differential response of estrogen receptor alpha and estrogen receptor beta to partial estrogen agonists/antagonists. , 1998, Molecular pharmacology.

[6]  W. Forssmann,et al.  Characterization of human circulating TIG2 as a ligand for the orphan receptor ChemR23 , 2003, FEBS letters.

[7]  T. Pawson,et al.  Signal transduction and growth control in normal and cancer cells. , 1994, Current opinion in genetics & development.

[8]  R. Lefkowitz,et al.  The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. , 2002, Journal of cell science.

[9]  G. Struhl,et al.  Nuclear Access and Action of Notch In Vivo , 1998, Cell.

[10]  M. Caron,et al.  Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. , 2000, The Journal of biological chemistry.

[11]  Darrell R. Abernethy,et al.  International Union of Pharmacology: Approaches to the Nomenclature of Voltage-Gated Ion Channels , 2003, Pharmacological Reviews.

[12]  Joseph Schlessinger,et al.  SH2 and PTB Domains in Tyrosine Kinase Signaling , 2003, Science's STKE.

[13]  M. Parmentier,et al.  The C-terminal Nonapeptide of Mature Chemerin Activates the Chemerin Receptor with Low Nanomolar Potency* , 2004, Journal of Biological Chemistry.

[14]  R. Axel,et al.  The isolation and sequence of the gene encoding T8: A molecule defining functional classes of T lymphocytes , 1985, Cell.

[15]  T. Pawson,et al.  Editorial overview: Signal transduction and growth control in normal and cancer cells , 1994 .

[16]  H. Heng,et al.  Cloning of human genes encoding novel G protein-coupled receptors. , 1994, Genomics.

[17]  J. Schlessinger.  Cell Signaling by Receptor Tyrosine Kinases , 2000, Cell.

[18]  P. Watson,et al.  Molecular diversity in the adenylylcyclase family. Evidence for eight forms of the enzyme and cloning of type VI. , 1992, The Journal of biological chemistry.

[19]  Marc Parmentier,et al.  Specific Recruitment of Antigen-presenting Cells by Chemerin, a Novel Processed Ligand from Human Inflammatory Fluids , 2003, The Journal of experimental medicine.

[20]  Raphael Kopan,et al.  Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain , 1998, Nature.

[21]  J. Olefsky,et al.  Evidence for a functional role of Shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor. , 1994, The Journal of biological chemistry.

[22]  V. Craig Jordan,et al.  International Union of Pharmacology. LXIV. Estrogen Receptors , 2006, Pharmacological Reviews.

[23]  Katarzyna Kalita,et al.  Estrogen receptor β , 2002 .

[24]  Tobias M. Fischer,et al.  Monitoring regulated protein-protein interactions using split TEV , 2006, Nature Methods.

[25]  T. Copeland,et al.  The P1' specificity of tobacco etch virus protease. , 2002, Biochemical and biophysical research communications.

[26]  T. O’Neill,et al.  Non-SH2 Domains within Insulin Receptor Substrate-1 and SHC Mediate Their Phosphotyrosine-dependent Interaction with the NPEY Motif of the Insulin-like Growth Factor I Receptor (*) , 1995, The Journal of Biological Chemistry.

[27]  M. Caron,et al.  A β-Arrestin/Green Fluorescent Protein Biosensor for Detecting G Protein-coupled Receptor Activation* , 1997, The Journal of Biological Chemistry.

[28]  M. Caron,et al.  Association of β-Arrestin with G Protein-coupled Receptors during Clathrin-mediated Endocytosis Dictates the Profile of Receptor Resensitization* , 1999, The Journal of Biological Chemistry.

[29]  Christine C. Hudson,et al.  The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors. , 2002, Assay and drug development technologies.

[30]  Xavier Deupi,et al.  Probing the β2 Adrenoceptor Binding Site with Catechol Reveals Differences in Binding and Activation by Agonists and Partial Agonists* , 2005, Journal of Biological Chemistry.