scholarly journals Carboxyl-terminal and intracellular loop sites for CRF1 receptor phosphorylation and β-arrestin-2 recruitment: a mechanism regulating stress and anxiety responses

2007 ◽  
Vol 293 (1) ◽  
pp. R209-R222 ◽  
Author(s):  
Robert H. Oakley ◽  
J. Alberto Olivares-Reyes ◽  
Christine C. Hudson ◽  
Fabiola Flores-Vega ◽  
Frank M. Dautzenberg ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Receptor Internalization ◽  
Carboxyl Terminus ◽  
Receptor Interaction ◽  
Intracellular Loop ◽  
Membrane Expression ◽  
Receptor Phosphorylation ◽  
Third Intracellular Loop ◽  
Carboxyl Terminal ◽  
Factor Type

The primary goal was to test the hypothesis that agonist-induced corticotropin-releasing factor type 1 (CRF1) receptor phosphorylation is required for β-arrestins to translocate from cytosol to the cell membrane. We also sought to determine the relative importance to β-arrestin recruitment of motifs in the CRF1 receptor carboxyl terminus and third intracellular loop. β-Arrestin-2 translocated significantly more rapidly than β-arrestin-1 to agonist-activated membrane CRF1 receptors in multiple cell lines. Although CRF1 receptors internalized with agonist treatment, neither arrestin isoform trafficked with the receptor inside the cell, indicating that CRF1 receptor-arrestin complexes dissociate at or near the cell membrane. Both arrestin and clathrin-dependent mechanisms were involved in CRF1 receptor internalization. To investigate molecular determinants mediating the robust β-arrestin-2-CRF1 receptor interaction, mutagenesis was performed to remove potential G protein-coupled receptor kinase phosphorylation sites. Truncating the CRF1 receptor carboxyl terminus at serine-386 greatly reduced agonist-dependent phosphorylation but only partially impaired β-arrestin-2 recruitment. Removal of a serine/threonine cluster in the third intracellular loop also significantly reduced CRF1 receptor phosphorylation but did not alter β-arrestin-2 recruitment. Phosphorylation was abolished in a CRF1 receptor possessing both mutations. Surprisingly, this mutant still recruited β-arrestin-2. These mutations did not alter membrane expression or cAMP signaling of CRF1 receptors. Our data reveal the involvement of at least the following two distinct receptor regions in β-arrestin-2 recruitment: 1) a carboxyl-terminal motif in which serine/threonine residues must be phosphorylated and 2) an intracellular loop motif configured by agonist-induced changes in CRF1 receptor conformation. Deficient β-arrestin-2-CRF1 receptor interactions could contribute to the pathophysiology of affective disorders by inducing excessive CRF1 receptor signaling.

scholarly journals The Third Intracellular Loop of the Human Somatostatin Receptor 5 Is Crucial for Arrestin Binding and Receptor Internalization after Somatostatin Stimulation

2008 ◽  
Vol 22 (3) ◽  
pp. 676-688 ◽  
Author(s):  
Erika Peverelli ◽  
Giovanna Mantovani ◽  
Davide Calebiro ◽  
Andrea Doni ◽  
Sara Bondioni ◽  
...  
Keyword(s):  
Somatostatin Receptor ◽  
Hormone Secretion ◽  
Serine Phosphorylation ◽  
Receptor Internalization ◽  
Receptor Interaction ◽  
Wild Type ◽  
Intracellular Loop ◽  
Structural Domains ◽  
The Third ◽  
Third Intracellular Loop

Abstract Somatostatin (SS) is a widely distributed polypeptide that exerts inhibitory effects on hormone secretion and cell proliferation by interacting with five different receptors (SST1-SST5). β-Arrestins have been implicated in regulating SST internalization, but the structural domains mediating this effect are largely unknown. The aim of this study was to characterize the intracellular mechanisms responsible for internalization of human SST5 in the rat pituitary cell line GH3 and to identify the SST5 structural domains involved in this process. To this purpose we evaluated, by fluorescence microscopy and biochemical assay, the ability of wild-type, progressive C-terminal truncated and third cytoplasmatic loop mutants SST5-DsRed to associate with β-arrestin-enhanced green fluorescent protein and to internalize under SS28 stimulation. The truncated mutants were comparable to the wild-type receptor with respect to recruitment of β-arrestin-2 and internalization, whereas the third loop mutants R240W, S242A, and T247A showed the abolishment or reduction of arrestin association and a significant reduction of receptor internalization (14.4%, 29%, and 30.9% vs. 52.4% of wild type) and serine phosphorylation upon SS28 stimulation. Moreover, we evaluated the ability of simultaneous mutation of these three residues (R240, S242, and T247) and C-terminal truncated receptors to internalize. The progressive truncation of the C-terminal tail resulted in a progressive increased internalization (21.6%, 36.7%, and 41%, respectively) with respect to the full-length total third-loop mutant (15%). In conclusion, our results indicate the SST5 third intracellular loop as an important mediator of β-arrestin/receptor interaction and receptor internalization, whereas they suggest that residues 328–347 within the C terminus may play an inhibitory role in receptor internalization.

scholarly journals Role of the third intracellular loop of the Angiotensin II receptor subtype AT2 in ligand-receptor interaction

FEBS Letters ◽  
1999 ◽  
Vol 445 (1) ◽  
pp. 23-26 ◽  
Author(s):  
Jason Dittus ◽  
Shannon Cooper ◽  
Gerald Obermeir ◽  
Lakshmidevi Pulakat
Keyword(s):  
Angiotensin Ii ◽  
Receptor Interaction ◽  
Receptor Subtype ◽  
Angiotensin Ii Receptor ◽  
Intracellular Loop ◽  
The Third ◽  
Third Intracellular Loop

scholarly journals Association of β-Arrestin 1 with the Type 1A Angiotensin II Receptor Involves Phosphorylation of the Receptor Carboxyl Terminus and Correlates with Receptor Internalization

2001 ◽  
Vol 15 (10) ◽  
pp. 1706-1719
Author(s):  
Hongwei Qian ◽  
Luisa Pipolo ◽  
Walter G. Thomas
Keyword(s):  
Angiotensin Ii ◽  
Close Correlation ◽  
Dominant Negative ◽  
Receptor Internalization ◽  
Central Portion ◽  
Carboxyl Terminus ◽  
Angiotensin Ii Receptor ◽  
Receptor Phosphorylation ◽  
At1a Receptor ◽  
Mutant Receptors

Abstract Arrestins bind to phosphorylated G protein-coupled receptors and participate in receptor desensitization and endocytosis. Although arrestins traffic with activated type 1 (AT1A) angiotensin II (AngII) receptors, the contribution of arrestins to AT1A receptor internalization is controversial, and the physical association of arrestins with the AT1A receptor has not been established. In this study, by coimmunoprecipitating AT1A receptors and β-arrestin 1, we provide direct evidence for an association between arrestins and the AT1A receptor that was agonist- and time-dependent and contingent upon the level ofβ -arrestin 1 expression. Serial truncation of the receptor carboxyl terminus resulted in a graded loss of β-arrestin 1 association, which correlated with decreases in receptor phosphorylation. Truncation of the AT1A receptor to lysine325 prevented AngII-induced phosphorylation and β-arrestin 1 association as well as markedly inhibiting receptor internalization, indicating a close correlation between these receptor parameters. AngII-induced association was also dramatically reduced in a phosphorylation- and internalization-impaired receptor mutant in which four serine and threonine residues in the central portion of the AT1A receptor carboxyl terminus (Thr332, Ser335, Thr336, Ser338) were substituted with alanine. In contrast, substitutions in another serine/threonine-rich region (Ser346, Ser347, Ser348) and at three PKC phosphorylation sites (Ser331, Ser338, Ser348) had no effect on AngII-inducedβ -arrestin 1 association or receptor internalization. While AT1A receptor internalization could be inhibited by a dominant-negative β-arrestin 1 mutant (βarr1319–418), treatment with hyperosmotic sucrose to inhibit internalization did not abrogate the differences in arrestin association observed between the wild-type and mutant receptors, indicating that arrestin binding precedes, and is not dependent upon, receptor internalization. Interestingly, a substituted analog of AngII,[ Sar1Ile4Ile8]-AngII, which promotes robust phosphorylation of the receptor but does not activate receptor signaling, stimulated strong β-arrestin 1 association with the full-length AT1A receptor. These results identify the central portion of the AT1A receptor carboxyl terminus as the important determinant for β-arrestin 1 binding and internalization and indicate that AT1A receptor phosphorylation is crucial for β-arrestin docking.

Agonist induced receptor internalization of neuropeptide Y receptor subtypes depends on third intracellular loop and C-terminus

2008 ◽  
Vol 20 (10) ◽  
pp. 1740-1749 ◽  
Author(s):  
Ilka Böhme ◽  
Jan Stichel ◽  
Cornelia Walther ◽  
Karin Mörl ◽  
Annette G. Beck-Sickinger
Keyword(s):  
Neuropeptide Y ◽  
Receptor Internalization ◽  
Receptor Subtypes ◽  
Intracellular Loop ◽  
Neuropeptide Y Receptor ◽  
C Terminus ◽  
Third Intracellular Loop

A dileucine motif is involved in plasma membrane expression and endocytosis of rat sodium taurocholate cotransporting polypeptide (Ntcp)

2013 ◽  
Vol 305 (10) ◽  
pp. G722-G730 ◽  
Author(s):  
Claudia Stross ◽  
Stefanie Kluge ◽  
Katrin Weissenberger ◽  
Elisabeth Winands ◽  
Dieter Häussinger ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sodium Taurocholate ◽  
Dual Function ◽  
Intracellular Loop ◽  
Membrane Expression ◽  
Dileucine Motif ◽  
Plasma Membrane Expression ◽  
Third Intracellular Loop ◽  
Parenchymal Cells ◽  
Pkc Activation

The sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake transporter for bile salts into liver parenchymal cells, and PKC-mediated endocytosis was shown to regulate the number of Ntcp molecules at the plasma membrane. In this study, mechanisms of Ntcp internalization were analyzed by flow cytometry, immunofluorescence, and Western blot analyses in HepG2 cells. PKC activation induced endocytosis of Ntcp from the plasma membrane by ∼30%. Endocytosis of Ntcp was clathrin dependent and was followed by lysosomal degradation. A dileucine motif located in the third intracellular loop of Ntcp was essential for endocytosis but also for processing and plasma membrane targeting, suggesting a dual function of this motif for intracellular trafficking of Ntcp. Mutation of two of five potential phosphorylation sites surrounding the dileucine motif (Thr225 and Ser226) inhibited PKC-mediated endocytosis. In conclusion, we could identify a motif, which is critical for Ntcp plasma membrane localization. Endocytic retrieval protects hepatocytes from elevated bile salt concentrations and is of special interest, because NTCP has been identified as a receptor for the hepatitis B and D virus.

The Third Intracellular Loop and the Carboxyl Terminus of β2-Adrenergic Receptor Confer Spontaneous Activity of the Receptor

2003 ◽  
Vol 64 (5) ◽  
pp. 1048-1058 ◽  
Author(s):  
Khalid Chakir ◽  
Yang Xiang ◽  
Dongmei Yang ◽  
Sheng-Jun Zhang ◽  
Heping Cheng ◽  
...  
Keyword(s):  
Spontaneous Activity ◽  
Adrenergic Receptor ◽  
Carboxyl Terminus ◽  
Intracellular Loop ◽  
The Third ◽  
Third Intracellular Loop ◽  
Β2 Adrenergic Receptor
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