The C-terminal of the α1b-adreneroceptor is a key determinant for its structure integrity and biological functions

2021 ◽  
Author(s):  
Ying Liu ◽  
Yu-Ting Shao ◽  
Richard Ward ◽  
Li Ma ◽  
Hao-Xin Gui ◽  
...  
Keyword(s):  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Biological Functions ◽  
Protein Activation ◽  
G Protein Activation ◽  
Signaling Function ◽  
Signal Changes ◽  
C 30 ◽  
C 50

Abstract The C-terminal of GPCRs is now recognized as being important for G protein activation and signaling function. To detect the role of C-terminal tail in receptor activation, we used the α1b-AR, which has a long C-terminal of 164 amino acids. We constructed the intramolecular FRET sensors, in which the C-terminal was truncated to 10 (∆C-10), 20 (∆C-20), 30 (∆C-30), 50 (∆C-50), 70 (∆C-70) or 90 (∆C-90). The truncated mutants of ∆C-10, ∆C-20 or ∆C-30 cannot induce FRET signal changes and downstream ERK1/2 phosphorylation. However, the truncated mutants of ∆C-50, ∆C-70 or ∆C-90 induce significant FRET signal changes and downstream ERK1/2 phosphorylation, especially ∆C-90. This is particularly true in the case of the ∆C-90, ∆C-70, or ∆C-50 which retained the potential phosphorylation sites (Ser401, Ser404, Ser408 or Ser410). The ∆C-90 showed an increase in agonist-induced FRET signal changes and ERK1/2 phosphorylation in PKC- or endocytosis-dependent and EGFR-, src- or β-arrestin 2-independent.

scholarly journals Mutations in the ‘DRY’ motif of the CB1 cannabinoid receptor result in biased receptor variants

2014 ◽  
Vol 54 (1) ◽  
pp. 75-89 ◽  
Author(s):  
Pál Gyombolai ◽  
András D Tóth ◽  
Dániel Tímár ◽  
Gábor Turu ◽  
László Hunyady
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Double Mutant ◽  
Cannabinoid Receptor ◽  
Camp Accumulation ◽  
Protein Activation ◽  
Cb1 Cannabinoid Receptor ◽  
G Protein Activation ◽  
Dry Motif

The role of the highly conserved ‘DRY’ motif in the signaling of the CB1cannabinoid receptor (CB1R) was investigated by inducing single-, double-, and triple-alanine mutations into this site of the receptor. We found that the CB1R-R3.50A mutant displays a partial decrease in its ability to activate heterotrimeric Goproteins (∼80% of WT CB1R (CB1R-WT)). Moreover, this mutant showed an enhanced basal β-arrestin2 (β-arr2) recruitment. More strikingly, the double-mutant CB1R-D3.49A/R3.50A was biased toward β-arrs, as it gained a robustly increased β-arr1 and β-arr2 recruitment ability compared with the WT receptor, while its G-protein activation was decreased. In contrast, the double-mutant CB1R-R3.50A/Y3.51A proved to be G-protein-biased, as it was practically unable to recruit β-arrs in response to agonist stimulus, while still activating G-proteins, although at a reduced level (∼70% of CB1R-WT). Agonist-induced ERK1/2 activation of the CB1R mutants showed a good correlation with their β-arr recruitment ability but not with their G-protein activation or inhibition of cAMP accumulation. Our results suggest that G-protein activation and β-arr binding of the CB1R are mediated by distinct receptor conformations, and the conserved ‘DRY’ motif plays different roles in the stabilization of these conformations, thus mediating both G-protein- and β-arr-mediated functions of CB1R.

scholarly journals NADPH Oxidase Mediates Membrane Androgen Receptor–Induced Neurodegeneration

Endocrinology ◽  
2019 ◽  
Vol 160 (4) ◽  
pp. 947-963 ◽  
Author(s):  
Mavis A A Tenkorang ◽  
Phong Duong ◽  
Rebecca L Cunningham
Keyword(s):  
Androgen Receptor ◽  
Nadph Oxidase ◽  
Neuronal Cell ◽  
Cell Loss ◽  
Receptor Activation ◽  
Reduced Form ◽  
Negative Effects ◽  
Protein Activation ◽  
Trisphosphate Receptor

Abstract Oxidative stress (OS) is a common characteristic of several neurodegenerative disorders, including Parkinson disease (PD). PD is more prevalent in men than in women, indicating the possible involvement of androgens. Androgens can have either neuroprotective or neurodamaging effects, depending on the presence of OS. Specifically, in an OS environment, androgens via a membrane-associated androgen receptor (mAR) exacerbate OS-induced damage. To investigate the role of androgens on OS signaling and neurodegeneration, the effects of testosterone and androgen receptor activation on the major OS signaling cascades, the reduced form of NAD phosphate (NADPH) oxidase (NOX)1 and NOX2 and the Gαq/inositol trisphosphate receptor (InsP3R), were examined. To create an OS environment, an immortalized neuronal cell line was exposed to H2O2 prior to cell-permeable/cell-impermeable androgens. Different inhibitors were used to examine the role of G proteins, mAR, InsP3R, and NOX1/2 on OS generation and cell viability. Both testosterone and DHT/3-O-carboxymethyloxime (DHT)–BSA increased H2O2-induced OS and cell death, indicating the involvement of an mAR. Furthermore, classical AR antagonists did not block testosterone’s negative effects in an OS environment. Because there are no known antagonists specific for mARs, an AR protein degrader, ASC-J9, was used to block mAR action. ASC-J9 blocked testosterone’s negative effects. To determine OS-related signaling mediated by mAR, this study examined NOX1, NOX2, Gαq. NOX1, NOX2, and the Gαq complex with mAR. Only NOX inhibition blocked testosterone-induced cell loss and OS. No effects of blocking either Gαq or G protein activation were observed on testosterone’s negative effects. These results indicate that androgen-induced OS is via the mAR–NOX complex and not the mAR–Gαq complex.

Role of G proteins in stimulation of Na-H exchange by cell shrinkage

1992 ◽  
Vol 262 (2) ◽  
pp. C533-C536 ◽  
Author(s):  
B. A. Davis ◽  
E. M. Hogan ◽  
W. F. Boron
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Transport Processes ◽  
Cell Shrinkage ◽  
Protein Activation ◽  
Kinase C ◽  
G Protein Activation ◽  
Barnacle Muscle Fibers ◽  
Stimulation Of

Many cells respond to shrinkage by stimulating specific ion transport processes (e.g., Na-H exchange). However, it is not known how the cell senses this volume change, nor how this signal is transduced to an ion transporter. We have studied the activation of Na-H exchange in internally dialyzed barnacle muscle fibers, measuring intracellular pH (pHi) with glass microelectrodes. When cells are dialyzed to a pHi of approximately 7.2, Na-H exchange is active only in shrunken cells. We found that the shrinkage-induced stimulation of Na-H exchange, elicited by increasing medium osmolality from 975 to 1,600 mosmol/kgH2O, is inhibited approximately 72% by including in the dialysis fluid 1 mM guanosine 5'-O-(2-thiodiphosphate). The latter is an antagonist of G protein activation. Even in unshrunken cells, Na-H exchange is activated by dialyzing the cell with 1 mM guanosine 5'-O-(3-thiotriphosphate), which causes the prolonged activation of G proteins. Activation of Na-H exchange is also elicited in unshrunken cells by injecting cholera toxin, which activates certain G proteins. Neither exposing cells to 100 nM phorbol 12-myristate 13-acetate nor dialyzing them with a solution containing 20 microM adenosine 3',5'-cyclic monophosphate (cAMP) (or 50 microM dibutyryl cAMP) plus 0.5 mM 3-isobutyl-1-methylxanthine substantially stimulates the exchanger. Thus our data suggest that a G protein plays a key role in the transduction of the shrinkage signal to the Na-H exchanger via a pathway that involves neither protein kinase C nor cAMP.

scholarly journals Agonist-induced formation of unproductive receptor-G12complexes

2020 ◽  
Vol 117 (35) ◽  
pp. 21723-21730
Author(s):  
Najeah Okashah ◽  
Shane C. Wright ◽  
Kouki Kawakami ◽  
Signe Mathiasen ◽  
Joris Zhou ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Receptor Internalization ◽  
Protein Association ◽  
Protein Activation ◽  
Protein Receptor ◽  
G Protein Activation

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2receptors (V2R) associate with both Gsand G12heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gscomplexes, V2R-G12complexes are not destabilized by guanine nucleotides and do not promote G12activation. Activating V2R does not lead to signaling responses downstream of G12activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12heterotrimers. Overexpressing G12inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12that are insensitive to nucleotides, suggesting that unproductive GPCR-G12complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

Role of Carboxylmethylation in Chemoattractant Receptor-Stimulated G Protein Activation and Functional Responses

1994 ◽  
Vol 200 (3) ◽  
pp. 1604-1614 ◽  
Author(s):  
E.D. Lederer ◽  
A.A. Jacobs ◽  
J.L. Hoffman ◽  
G.B. Harding ◽  
J.D. Robishaw ◽  
...  
Keyword(s):  
G Protein ◽  
Functional Responses ◽  
Protein Activation ◽  
G Protein Activation

scholarly journals C-terminal threonines and serines play distinct roles in the desensitization of rhodopsin, a G protein-coupled receptor

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Anthony W Azevedo ◽  
Thuy Doan ◽  
Hormoz Moaven ◽  
Iza Sokal ◽  
Faiza Baameur ◽  
...  
Keyword(s):  
G Protein ◽  
Single Photon ◽  
Low Noise ◽  
Tight Control ◽  
G Protein Coupled Receptor ◽  
Rod Photoreceptors ◽  
Protein Activation ◽  
G Protein Activation ◽  
G Protein Coupled

Rod photoreceptors generate measurable responses to single-photon activation of individual molecules of the G protein-coupled receptor (GPCR), rhodopsin. Timely rhodopsin desensitization depends on phosphorylation and arrestin binding, which quenches G protein activation. Rhodopsin phosphorylation has been measured biochemically at C-terminal serine residues, suggesting that these residues are critical for producing fast, low-noise responses. The role of native threonine residues is unclear. We compared single-photon responses from rhodopsin lacking native serine or threonine phosphorylation sites. Contrary to expectation, serine-only rhodopsin generated prolonged step-like single-photon responses that terminated abruptly and randomly, whereas threonine-only rhodopsin generated responses that were only modestly slower than normal. We show that the step-like responses of serine-only rhodopsin reflect slow and stochastic arrestin binding. Thus, threonine sites play a privileged role in promoting timely arrestin binding and rhodopsin desensitization. Similar coordination of phosphorylation and arrestin binding may more generally permit tight control of the duration of GPCR activity.

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