Monolayers were washed twice with HEPES/HBSS and then blocked with HBSS containing 4% bovine serum albumin for 15 min at 37C. concentrations of TSH causes persistent phosphoinositide and cAMP signaling that is not dependent on internalization. To our knowledge, this is the first demonstration of persistent activation by any G protein-coupled receptor (GPCR) via the Gq pathway and of two G protein-mediated pathways by any GPCR. Introduction Although G protein-coupled receptors (GPCRs) are able to signal independently of G proteins, the major pathways for GPCR signaling involve coupling of the activated receptor to one or more G proteins (Woehler and Ponimaskin, 2009). The human thyrotropin [thyroid-stimulating hormone (TSH)] receptor (TSHR) has been shown to couple to several G proteins (Laugwitz et al., 1996), including the SAPKK3 stimulatory G protein (Gs), which activates adenylyl cyclase to produce cAMP (cAMP pathway), and Gq/11, which activates phospholipase C to produce inositol-1,4,5-trisphosphate (I-1,4,5-P3) (phosphoinositide pathway). The Gs-mediated stimulation of cAMP formation has been regarded as the Tiplaxtinin (PAI-039) principal intracellular signaling mechanism mediating the action of TSH. However, recently Kero et al. (2007) demonstrated that the Gq/G11-mediated signaling pathway is required for TSH-induced thyroid hormone synthesis and release in the adult and that the lack of Gq/G11 leads to hypothyroidism. Additional support for an essential physiological role of Gq/G11 Tiplaxtinin (PAI-039) proteins in mediating the regulation of thyroid function was demonstrated by a TSHR germline mutation that preferentially affected the phosphoinositide pathway (Grasberger et al., 2007). Until recently, it was believed that GPCRs with dissociable agonists signal transiently and that the signaling pathway was rapidly desensitized by several mechanisms including receptor internalization (Hausdorff et al., 1990). However, over the last 2 years, it was shown that three GPCRsTSHR (Calebiro et al., 2009; Neumann et al., 2010a); the parathyroid hormone receptor (Ferrandon et al., 2009), which also couples to Gs; and the sphingosine-1-phosphate receptor (Mullershausen et al., 2009), which couples to Gi to decrease cAMP productionexhibit persistent signaling even after the agonist has been removed. With these receptors, persistent signaling has been found to last for more than several hours. In this study, we sought to determine whether the TSHR signaled persistently via the phosphoinositide pathway. It was shown that higher concentrations of TSH are required to stimulate I-1,4,5-P3 production than cAMP production Tiplaxtinin (PAI-039) (Van Sande et al., 1990). By using high concentrations of TSH, we were able to stimulate cAMP and phosphoinositide signaling simultaneously. We found that TSHR exhibits persistent activation of the phosphoinositide pathway as it does the cAMP pathway and that persistent phosphoinositide signaling occurs independently of internalization. We also show that persistent signaling can be caused by a small-molecule agonist of TSHR and by a thyroid-stimulating antibody. Materials and Methods Cell Culture and Transfection. The Tiplaxtinin (PAI-039) generation Tiplaxtinin (PAI-039) of a HEK-EM293 cell lines stably expressing human TSHR (HEK-TSHR cells) or human TRH receptor were described previously (Engel et al., 2006; Neumann et al., 2009). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 10 g/ml streptomycin and 250 g/ml hygromycin B (both from Invitrogen, Carlsbad, CA) at 37C in a humidified 5% CO2 incubator. Cells were transiently transfected with -arrestin-2 (Arr2) or K44A (both kindly provided by Dr. Marc Caron, Duke University Medical Center, Durham, NC), Gq-YFP chimera (Hughes et al., 2001) (kindly provided by Dr. Catherine Berlot, Weis Center for Research, Geisinger Clinic, Danville, PA) in 24-well plates (7.5 104 cells/well) with 0.2 g of DNA/well or on poly(d-lysine)-coated coverglass.