To study phosphorylation of the endogenous type I thyrotropin-releasing hormone receptor in the anterior pituitary, we generated phosphosite-specific polyclonal antibodies. The major phosphorylation site of receptor endogenously expressed in pituitary GH3 cells was Thr(365) in the receptor tail; distal sites were more phosphorylated in some heterologous models. beta-Arrestin 2 reduced thyrotropin-releasing hormone (TRH)-stimulated inositol phosphate production and accelerated internalization of the wild type receptor but not receptor mutants where the critical phosphosites were mutated to Ala. Phosphorylation peaked within seconds and was maximal at 100 nm TRH. Based on dominant negative kinase and small interfering RNA approaches, phosphorylation was mediated primarily by G protein-coupled receptor kinase 2. Phosphorylated receptor, visualized by immunofluorescence microscopy, was initially at the plasma membrane, and over 5-30 min it moved to intracellular vesicles in GH3 cells. Dephosphorylation was rapid (t((1/2)) approximately 1 min) if agonist was removed while receptor was at the surface. Dephosphorylation was slower (t((1/2)) approximately 4 min) if agonist was withdrawn after receptor had internalized. After agonist removal and dephosphorylation, a second pulse of agonist caused extensive rephosphorylation, particularly if most receptor was still on the plasma membrane. Phosphorylated receptor staining was visible in prolactin- and thyrotropin-producing cells in rat pituitary tissue from untreated rats and much stronger in tissue from animals injected with TRH. Our results show that the TRH receptor can rapidly cycle between a phosphorylated and nonphosphorylated state in response to changing agonist concentrations and that phosphorylation can be used as an indicator of receptor activity in vivo.
"* denotes statistical significance at í µí± < 0.0025 when compared to 100% control using Student's í µí±¡-test. was not sensitive enough to capture the receptors being internalized so we decided to utilize a modified cell-surface ELISA  to measure agonist-mediated internalization of an N-terminal VSVg-tagged MCHR1. The VSVg-tag allowed us to utilize a high-affinity antibody for this assay. "
[Show abstract][Hide abstract] ABSTRACT: The regulation of appetite is complex, though our understanding of the process is improving. The potential role for the melanin-concentrating hormone (MCH) signaling pathway in the treatment of obesity is being explored by many. It was hypothesized that internalization of MCH receptors would act to potently desensitize cells to MCH. Despite potent desensitization of ERK signaling by MCH in BHK-570 cells, we were unable to observe MCH-mediated internalization of MCH receptor 1 (MCHR1) by fluorescence microscopy. A more quantitative approach using a cell-based ELISA indicated only 15% of receptors internalized, which is much lower than that reported in the literature. When β-arrestins were overexpressed in our system, removal of receptors from the cell surface was facilitated and signaling to a leptin promoter was diminished, suggesting that internalization of MCHR1 is sensitive to cellular β-arrestin levels. A dominant-negative GRK construct completely inhibited loss of receptors from the cell surface in response to MCH, suggesting that the internalization observed is phosphorylation-dependent. Since desensitization of MCH-mediated ERK signaling did not correlate with significant loss of MCHR1 from the cell surface, we hypothesize that in this model system regulation of MCH signaling may be the result of segregation of receptors from signaling components at the plasma membrane.
International Journal of Endocrinology 11/2013; 2013:143052. DOI:10.1155/2013/143052 · 1.95 Impact Factor
"Arrestin interactions with TRH receptors have been monitored by a variety of approaches including translocation of GFP-arrestin (Groarke et al., 1999, 2001; Oakley et al., 1999, 2000; Yu and Hinkle, 1999; Smith et al., 2001; Hanyaloglu et al., 2002), co-precipitation of arrestin and receptor (Jones et al., 2007), effect of arrestin on agonist affinity (Jones and Hinkle, 2005, 2008), and BRET (Kroeger et al., 2001; Hanyaloglu et al., 2002). GFP-arrestin is diffusely localized in the cytoplasm of cells expressing TRH receptors. "
[Show abstract][Hide abstract] 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 · 3.66 Impact Factor
"Cells were plated in 24 well dishes and transfected the next day with 250 ng total DNA/well (100–200 ng of receptor DNA and 50–150 ng MRAP). The next day, the density of receptors on the plasma membrane was measured by an ELISA that has been described(Jones et al., 2007). In brief, live cells were incubated with mouse monoclonal anti-HA antibody diluted 1:5000 for 30–60 min and then washed extensively and fixed with 2% paraformaldehyde. "
[Show abstract][Hide abstract] ABSTRACT: The melanocortin 2 (MC(2)) receptor differs from other melanocortin family members in its pharmacological profile and reliance on an accessory protein, MC(2) receptor accessory protein (MRAP), for surface expression and signal transduction. To identify features of the MC(2) receptor responsible for these characteristics, we created chimeras between MC(2) and MC(4) receptors and expressed these in CHO cells, where MRAP is essential for trafficking and signaling by MC(2) but not MC(4) receptors. Replacing the first transmembrane segment of the MC(2) receptor with the corresponding region from the MC(4) receptor allowed some surface expression in the absence of an accessory protein, while ACTH-induced cAMP production remained entirely MRAP-dependent. On the other hand, replacing the last two transmembrane domains, third extracellular loop and C-terminal tail of the MC(4) receptor with the corresponding regions from the MC(2) receptor resulted in MRAP-dependent signaling. Surprisingly, replacing the second and third transmembrane domains and the intervening first extracellular loop of MC(2) receptors with MC(4) sequences generated a chimera (2C2) that responded to both adrenocorticotropic hormone (ACTH) and to the potent MSH analog 4-norleucine-7-d-phenylalanine-α-melanocyte stimulating hormone (NDP-α-MSH), which does not activate native MC(2) receptors. The 2C2 chimeric receptor was able to respond to NDP-α-MSH without MRAP, but MRAP shifted the EC50 value for NDP-α-MSH to the left and caused constitutive activity. These results identify the first transmembrane domain as important for surface expression and regions from the second to third transmembrane segments of the MC(2) receptor as important for MRAP dependent-signal transduction and ligand specificity.
European journal of pharmacology 06/2011; 660(1):94-102. DOI:10.1016/j.ejphar.2010.10.113 · 2.53 Impact Factor
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