Persistent signaling by thyrotropin-releasing hormone receptors correlates with G-protein and receptor levels
ABSTRACT G-protein-coupled receptors with dissociable agonists for thyrotropin, parathyroid hormone, and sphingosine-1-phosphate were found to signal persistently hours after agonist withdrawal. Here we show that mouse thyrotropin-releasing hormone (TRH) receptors, subtypes 2 and 1(TRH-R2 and TRH-R1), can signal persistently in HEK-EM293 cells under appropriate conditions, but TRH-R2 exhibits higher persistent signaling activity. Both receptors couple primarily to Gα(q/11). To gain insight into the mechanism of persistent signaling, we compared proximal steps of inositolmonophosphate (IP1) signaling by TRH-Rs. Persistent signaling was not caused by slower dissociation of TRH from TRH-R2 (t(1/2)=77 ± 8.1 min) compared with TRH-R1 (t(1/2)=82 ± 12 min) and was independent of internalization, as inhibition of internalization did not affect persistent signaling (115% of control), but required continuously activated receptors, as an inverse agonist decreased persistent signaling by 60%. Gα(q/11) knockdown decreased persistent signaling by TRH-R2 by 82%, and overexpression of Gα(q/11) induced persistent signaling in cells expressing TRH-R1. Lastly, persistent signaling was induced in cells expressing high levels of TRH-R1. We suggest that persistent signaling by TRHRs is exhibited when sufficient levels of agonist/receptor/G-protein complexes are established and maintained and that TRH-R2 forms and maintains these complexes more efficiently than TRH-R1.
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ABSTRACT: The pituitary receptor for thyrotropin-releasing hormone (TRH) is a calcium-mobilizing G protein-coupled receptor (GPCR) that signals through Gq/11, elevating calcium, and activating protein kinase C. TRH receptor signaling is quickly desensitized as a consequence of receptor phosphorylation, arrestin binding, and internalization. Following activation, TRH receptors are phosphorylated at multiple Ser/Thr residues in the cytoplasmic tail. Phosphorylation catalyzed by GPCR kinase 2 (GRK2) takes place rapidly, reaching a maximum within seconds. Arrestins bind to two phosphorylated regions, but only arrestin bound to the proximal region causes desensitization and internalization. Phosphorylation at Thr365 is critical for these responses. TRH receptors internalize in clathrin-coated vesicles with bound arrestin. Following endocytosis, vesicles containing phosphorylated TRH receptors soon merge with rab5-positive vesicles. Over approximately 20 min these form larger endosomes rich in rab4 and rab5, early sorting endosomes. After TRH is removed from the medium, dephosphorylated receptors start to accumulate in rab4-positive, rab5-negative recycling endosomes. The mechanisms responsible for sorting dephosphorylated receptors to recycling endosomes are unknown. TRH receptors from internal pools help repopulate the plasma membrane. Dephosphorylation of TRH receptors begins when TRH is removed from the medium regardless of receptor localization, although dephosphorylation is fastest when the receptor is on the plasma membrane. Protein phosphatase 1 is involved in dephosphorylation but the details of how the enzyme is targeted to the receptor remain obscure. It is likely that future studies will identify biased ligands for the TRH receptor, novel arrestin-dependent signaling pathways, mechanisms responsible for targeting kinases and phosphatases to the receptor, and principles governing receptor trafficking.Frontiers in Neuroscience 12/2012; 6:180. DOI:10.3389/fnins.2012.00180 · 3.70 Impact Factor
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ABSTRACT: The thyroid hormone (TH) system is involved in several important physiological processes, including regulation of energy metabolism, growth and differentiation, development and maintenance of brain function, thermo-regulation, osmo-regulation, and axis of regulation of other endocrine systems, sexual behaviour and fertility, cardiovascular function. Therefore, concern about TH disruption (THD) has resulted in strategies being developed to identify THD chemicals (THDCs). Information on potential of chemicals causing THD is typically derived from animal studies. For the majority of chemicals, however, this information is either limited or unavailable. It is also unlikely that animal experiments will be performed for all THD relevant chemicals in the near future for ethical, financial and practical reasons. In addition, typical animal experiments often do not provide information on the mechanism of action of THDC, making it harder to extrapolate results across species. Relevant effects may not be identified in animal studies when the effects are delayed, life stage specific, not assessed by the experimental paradigm (e.g., behaviour) or only occur when an organism has to adapt to environmental factors by modulating TH levels. Therefore, in vitro and in silico alternatives to identify THDC and quantify their potency are needed. THDC have many potential mechanisms of action, including altered hormone production, transport, metabolism, receptor activation and disruption of several feed-back mechanisms. In vitro assays are available for many of these endpoints, and the application of modern '-omics' technologies, applicable for in vivo studies can help to reveal relevant and possibly new endpoints for inclusion in a targeted THDC in vitro test battery. Within the framework of the ASAT initiative (Assuring Safety without Animal Testing), an international group consisting of experts in the areas of thyroid endocrinology, toxicology of endocrine disruption, neurotoxicology, high-throughput screening, computational biology, and regulatory affairs has reviewed the state of science for (1) known mechanisms for THD plus examples of THDC; (2) in vitro THD tests currently available or under development related to these mechanisms; and (3) in silico methods for estimating the blood levels of THDC. Based on this scientific review, the panel has recommended a battery of test methods to be able to classify chemicals as of less or high concern for further hazard and risk assessment for THD. In addition, research gaps and needs are identified to be able to optimize and validate the targeted THD in vitro test battery for a mechanism-based strategy for a decision to opt out or to proceed with further testing for THD.Toxicology in Vitro 02/2013; 27(4). DOI:10.1016/j.tiv.2013.02.012 · 3.21 Impact Factor
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ABSTRACT: Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca(2+) mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg(9)-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.Neurochemical Research 08/2013; 39(6). DOI:10.1007/s11064-013-1126-9 · 2.55 Impact Factor