title: siRNA screen identifies the phosphatase acting on the G protein-coupled thyrotropin-releasing hormone receptor.
abstract: G protein-coupled receptors (GPCRs) are an ubiquitously expressed class of transmembrane proteins involved in the signal transduction of neurotransmitters, hormones and various other ligands. Their signaling output is desensitized by mechanisms involving phosphorylation, internalization, and dissociation from G proteins and resensitized by mechanisms involving dephosphorylation, but details about the phosphatases responsible are generally lacking. We describe here the use of an siRNA-based library to knock down expression of specific phosphatase subunits to identify protein phosphatase 1-α (PP1α) as important for the thyrotropin-releasing hormone (TRH) receptor. Inhibition of PP1α synthesis and overexpression of dominant negative PP1α preserved receptor phosphorylation under conditions favoring dephosphorylation, whereas overexpression of PP1α accelerated dephosphorylation. Knockdown of all three PP1 catalytic subunits inhibited TRH receptor phosphorylation much more powerfully than knockdown of PP1α alone, suggesting that different PP1 isoforms function redundantly. Knockdown of a structural subunit of PP2A, a second potential hit in the library screen, was ineffective. Calyculin A, a potent inhibitor of PP1 family phosphatases, strongly inhibited dephosphorylation of transfected TRH receptors and endogenous receptors in pituitary cells, but fostriecin, which is selective for PP2A family phosphatases, did not. We conclude that the PP1 class of phosphatases is essential for TRH receptor dephosphorylation.
authors.affiliation: Department of Science and Mathematics, National Technical Institute for the Deaf, Rochester Institute of Technology, Rochester, NY 14623, USA.
authors.forename: Austin U
authors.initials: AU
authors.lastname: Gehret
authors.forename: Patricia M
authors.initials: PM
authors.lastname: Hinkle
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D018025:Receptors, Thyrotropin-Releasing Hormone; D010744:Phosphoric Monoester Hydrolases; D019204:GTP-Binding Proteins