A constitutively active mutant thyrotropin-releasing hormone receptor is chronically down-regulated in pituitary cells: evidence using chlordiazepoxide as a negative antagonist.

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to ␤-arrestin after agonist exposure. To define the importance of receptor phosphorylation and ␤-arrestin binding in desensitization, and to determine whether ␤-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking ␤-arrestin-1 and/or ␤-arrestin-2. Apparent affinity for [ 3 H]MeTRH was increased 8-fold in cells expressing ␤-arrestins, including a ␤-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of ␤-arrestins, but receptors remained primarily on the plasma membrane without ␤-arrestin. ␤-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous ␤-arrestins reduced TRH-stimulated inositol phosphate production by 48% (␤-arrestin-1), 71% (␤-arrestin-2), and 84% (␤-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by ␤-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without ␤-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking G␣ q and G␣ 11 or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that ␤-arrestin binding is critical for desensitization and internalization.The type 1 thyrotropin-releasing hormone (TRH) 2 receptor is a seven-transmembrane helix protein that regulates thyroid stimulating hormone and prolactin release from the anterior pituitary. Upon exposure to agonist, the TRH receptor stimulates phospholipase C␤ through coupling to the GTP-binding proteins G␣ q or G␣ 11 , leading to the formation of inositol 1,4,5-trisphosphate (IP 3 ) and the subsequent release of Ca 2ϩ from the endoplasmic reticulum. This signaling pathway ultimately leads to the release of thyroid stimulating hormone and is essential for proper thyroid function and targeted deletion of the type 1 TRH receptor results in hypothyroidism (1).When activated, most G protein-coupled receptors (GPCRs) undergo phosphorylation, which is followed by receptor interaction with ␤-arrestins and desensitization and endocytosis (2). Phosphorylation is carried out by second messenger-activated kinases, such as protein kinase C, or by G protein-coupled receptor kinases (GRKs). Less is known about dephosphorylation, but it is believed to be a crucial step in the resensit...

Section: Discussionsupporting

confidence: 71%

β-Arrestin Mediates Desensitization and Internalization but Does Not Affect Dephosphorylation of the Thyrotropin-releasing Hormone Receptor

Jones

Hinkle

2005

“…The findings presented here, together with our previous findings concerning a constitutively active TRH-R mutant (11,14,23), strongly suggest that constitutively active receptors cause heterologous desensitization either in Xenopus oocytes or in mammalian cells. Considering the existence of pathologies resulting from mutations that cause constitutive activity and the findings that many wild type receptors exhibit some degree of constitutive activity, these novel desensitization phenomena should be further investigated.…”

Section: Fig 9 Expression Of Kshv-gpcrs Depletes Oocyte Calcium Poosupporting

confidence: 82%

“…Indeed, there is a direct correlation between the degree of heterologous desensitization caused by KSHV-GPCR (Fig. 1B) and the C335Stop mutant TRH-R (14) in Xenopus oocytes and the level of constitutive signaling activity observed in mammalian cells (2,23).…”

Section: Fig 9 Expression Of Kshv-gpcrs Depletes Oocyte Calcium Poomentioning

confidence: 93%

Constitutive Signaling by Kaposi's Sarcoma-associated Herpesvirus G-protein-coupled Receptor Desensitizes Calcium Mobilization by Other Receptors

Lupu-Meiri

Silver

Simons

et al. 2001

We coexpressed Kaposi's sarcoma-associated herpesvirus G protein-coupled receptors (KSHV-GPCRs) with thyrotropin-releasing hormone (TRH) receptors or m1-muscarinic-cholinergic receptors in Xenopus oocytes and in mammalian cells. In oocytes, KSHV-GPCR expression resulted in pronounced (81%) inhibition (heterologous desensitization) of Ca 2؉ -activated chloride current responses to TRH and acetylcholine. 45 Ca 2؉ efflux. In KSHV-GPCR-expressing oocytes, responses to microinjected inositol 1,4,5-trisphosphate were inhibited by 74%, and this effect was partially reversed by interferon-␥-inducible protein 10. Treatment with thapsigargin suggested that the pool of calcium available for mobilization by TRH was decreased in oocytes coexpressing KSHV-GPCRs. These results suggest that constitutive signaling by KSHV-GPCR causes heterologous desensitization of responses mediated by other receptors, which signal via the phosphoinositide/calcium pathway, which is caused by depletion of intracellular calcium pools. KSHV-GPCR,1 the gene product of ORF74 in the KSHV (human herpesvirus 8) genome, is a GPCR homologous to human chemokine CXC receptor 2 (1, 2). Our group demonstrated that rodent fibroblasts expressing KSHV-GPCRs formed tumors in mice (3). Recently, transgenic mice in which the KSHV-GPCR gene was regulated by the human CD2 enhancer/promoter and locus control region were shown to develop angioproliferative lesions that resembled the lesions of KS (4). These reports make the mechanism of action of KSHV-GPCR interesting in terms of tumorigenesis. We reported that KSHV-GPCR exhibited marked constitutive, agonist-independent activity in mammalian cells (2). We also demonstrated that the constitutive activity of the viral GPCR could be inhibited by IP-10, acting as an inverse agonist (5). These results were later confirmed by Rosenkilde et al. (6). Last, we showed that signaling by KSHV-GPCR could be inhibited by GPCR-specific protein kinases and suggested that this may represent a form of homologous desensitization of this viral receptor (7). However, the effect of the constitutive activity of KSHV-GPCR on signaling by other receptors (heterologous desensitization) has not been previously investigated.To investigate the effect of constitutive signaling by KSHV-GPCR on other receptors that utilize the phosphoinositide/ calcium signaling pathway, we studied the behavior of this receptor in Xenopus oocytes and in mammalian cells. Our results show that KSHV-GPCR causes marked desensitization of responses mediated by TRH-Rs and m1-Rs by inhibiting InsP 3 -stimulated mobilization of Ca 2ϩ . EXPERIMENTAL PROCEDURESXenopus laevis Oocytes-Defolliculated oocytes were obtained from mature Xenopus females, essentially as previously described (8). In vitro transcribed cRNAs for wild type TRH-Rs, m1-Rs (1 ng/oocyte), gastrin-releasing peptide (GRP) receptors (0.23 ng/oocyte), and KSHVGPCRs (1-3 ng/oocyte) were injected 48 h before the assay as described previously (9 -11).Electrophysiology-Two-electrode voltage clamp measurements wer...

“…Non-peptide inverse agonists have recently been identified for two other types of peptide receptors, i.e. the thyrotropin-releasing hormone and the AT 1A angiotensin II receptors (21,22). Like (R)-L-740,093, each of these non-peptide inverse agonists appears to have no intrinsic activity at the respective wild type receptors and was therefore originally classified as an antagonist.…”

Section: Resultsmentioning

confidence: 99%

Minor Modifications of a Cholecystokinin-B/Gastrin Receptor Non-peptide Antagonist Confer a Broad Spectrum of Functional Properties

Beinborn

Quinn

Kopin

1998

The development of non-peptide agonists for peptide hormone receptors would markedly expand the treatment options for a large number of diseases. However, difficulty in identifying non-peptide molecules which possess intrinsic activity has been a major obstacle in achieving this goal. At present, most of the known nonpeptide ligands for peptide hormone receptors appear in standard functional assays to be antagonists. Here, we report that a constitutively active mutant of the human cholecystokinin-B/gastrin receptor, Leu 325 3 Glu, offers the potential to detect even trace agonist activity of ligands which, at the wild type receptor isoform, appear to lack efficacy. The enhanced functional sensitivity of the mutant receptor enabled us to detect intrinsic activity of L-365,260, an established non-peptide antagonist for the cholecystokinin-B/gastrin receptor. Extending from this observation, we were able to demonstrate that minor structural modifications could convert L-365,260 into either: (i) an agonist or (ii) an inverse agonist (attenuates ligand-independent signaling). The ability to confer functional activity to small non-peptide ligands suggests that the properties of endogenous peptide hormones can be mimicked, and even extended, by considerably less complex molecules.