scholarly journals A novel luminescence-based β-arrestin membrane recruitment assay for unmodified GPCRs

2020 ◽  
Author(s):  
Maria Hauge Pedersen ◽  
Jennifer Pham ◽  
Helena Mancebo ◽  
Asuka Inoue ◽  
Jonathan A. Javitch
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Membrane Recruitment ◽  
Terminal Fragment ◽  
Downstream Effectors ◽  
Agonist Concentration ◽  
Membrane Tether

AbstractG protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, G protein-independent signaling via the arrestin pathway has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either the G protein or arrestin-signaling pathways. Currently available screening techniques that measure arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure arrestin recruitment to any unmodified receptor.NanoLuc, an engineered luciferase from ophlorus gracilirostris (deep sea shrimp), is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. Here, we have identified a novel split site and have fused the N-terminal fragment to a membrane tether and the C-terminal fragment to the N-terminus of either β-arrestin 1 or 2. Upon receptor activation, arrestin is recruited to the plasma membrane in an agonist concentration-dependent manner and the two NanoLuc fragments complement to reconstitute functional luciferase, which allows quantification of recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification. The split NanoLuc arrestin recruitment assay has promise as a new generic assay for measuring arrestin recruitment to diverse GPCR types in heterologous or native cells.

Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK involves multiple signaling pathways

2002 ◽  
Vol 283 (5) ◽  
pp. C1351-C1364 ◽  
Author(s):  
Hong Wang ◽  
Joachim J. Ubl ◽  
Rolf Stricker ◽  
Georg Reiser
Keyword(s):  
G Protein ◽  
Egf Receptor ◽  
Receptor Activation ◽  
Small Degree ◽  
Dependent Manner ◽  
Heparin Binding ◽  
Protease Activated Receptors ◽  
Concentration Dependent Manner ◽  
Erk Activation ◽  
Rat Astrocytes

Protease-activated receptors (PARs), newly identified members of G protein-coupled receptors, are widely distributed in the brain. Thrombin evokes multiple cellular responses in a large variety of cells by activating PAR-1, -3, and -4. In cultured rat astrocytes we investigated the signaling pathway of thrombin- and PAR-activating peptide (PAR-AP)-induced cell proliferation. Our results show that PAR activation stimulates proliferation of astrocytes through the ERK pathway. Thrombin stimulates ERK1/2 phosphorylation in a time- and concentration-dependent manner. This effect can be fully mimicked by a specific PAR-1-AP but only to a small degree by PAR-3-AP and PAR-4-AP. PAR-2-AP can induce a moderate ERK1/2 activation as well. Thrombin-stimulated ERK1/2 activation is mainly mediated by PAR-1 via two branches: 1) the PTX-sensitive G protein/(βγ-subunits)-phosphatidylinositol 3-kinase branch, and 2) the Gq-PLC-(InsP3receptor)/Ca2+-PKC pathway. Thrombin- or PAR-1-AP-induced ERK activation is partially blocked by a selective EGF receptor inhibitor, AG1478. Nevertheless, transphosphorylation of EGF receptor is unlikely for ERK1/2 activation and is certainly not involved in PAR-1-induced proliferation. The metalloproteinase mechanism involving transactivation of the EGF receptor by released heparin-binding EGF was excluded. EGF receptor activation was detected by the receptor autophosphorylation site, tyrosine 1068. Our data suggest that thrombin-induced mitogenic action in astrocytes occurs independently of EGF receptor transphosphorylation.

CSIG-07. ACTIVATION OF THE ADHESION G PROTEIN-COUPLED RECEPTOR GPR133 BY ANTIBODIES AGAINST THE N-TERMINUS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi34-vi34
Author(s):  
Gabriele Stephan ◽  
Joshua Frenster ◽  
Niklas Ravn-Boess ◽  
Devin Bready ◽  
Jordan Wilcox ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Receptor Activation ◽  
Cell Culture Supernatant ◽  
Dependent Manner ◽  
G Protein Coupled Receptor ◽  
Terminal Fragment ◽  
C Terminus ◽  
N Terminus ◽  
G Protein Coupled

Abstract We recently demonstrated that GPR133 (ADGRD1), a member of the adhesion G protein-coupled receptor (aGPCR) family, is necessary for growth of glioblastoma (GBM) and is de novo expressed in GBM relative to normal brain tissue. We therefore postulate that GPR133 represents a novel target in GBM, which merits development of therapeutics. Like most aGPCRs, GPR133 is characterized by an intracellular C-terminus, 7 plasma membrane-spanning α-helices and a large extracellular N-terminus. The N-terminus possesses a conserved GPCR autoproteolysis-inducing (GAIN) domain that catalyzes cleavage at a GPCR proteolysis site (GPS), resulting in a C-terminal fragment (CTF) and an N-terminal fragment (NTF). We showed that dissociation of the cleaved NTF and CTF at the plasma membrane increases canonical signaling of GPR133, which is mediated by coupling to Gs and increase in cytosolic cAMP. Toward characterizing the effect of biologics on GPR133 function, we overexpressed wild-type or mutant forms of GPR133 in HEK293T cells and patient-derived GBM cells lines. Treatment of these cells with antibodies specifically targeting the NTF of GPR133 increased receptor activation in a dose-dependent manner. No effects were elicited with an antibody against the receptor’s intracellular C-terminus. Interestingly, cells overexpressing a cleavage-deficient mutant GPR133 (H543R) did not respond to antibody stimulation, suggesting that the effect is cleavage-dependent. Following antibody treatment, co-purification of the GPR133 NTF and the N-terminal antibody from the cell culture supernatant indicated the formation of antibody-NTF complexes. Analysis of these complexes suggested that antibody binding stimulated the dissociation of the NTF from the CTF. However, the increased flexibility of the GAIN domain and NTF after cleavage, independently of dissociation, may also endow the receptor with responsiveness to the effects of the antibodies. These data constitute a proof-of-concept paradigm of modulation of GPR133 function with antibodies. This work provides rationale for pursuing development of biologics targeting GPR133 in GBM.

Tachykinins induce gelatinase production by guinea pig alveolar macrophages: involvement of NK2 receptors

1995 ◽  
Vol 269 (5) ◽  
pp. L631-L636 ◽  
Author(s):  
M. P. D'Ortho ◽  
P. H. Jarreau ◽  
C. Delacourt ◽  
S. Pezet ◽  
C. Lafuma ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Alveolar Macrophages ◽  
Culture Media ◽  
Receptor Activation ◽  
Neurokinin A ◽  
Dependent Manner ◽  
Maximum Increase ◽  
Concentration Dependent Manner ◽  
Gelatinase Activity ◽  
Terminal Fragment

To determine whether tachykinins induce gelatinase production by guinea pig alveolar macrophages (AM), and to characterize the mechanism involved, we incubated AM with substance P (SP), neurokinin A (NKA), or the NH2-terminal fragment of SP, SP(1-7). The effects of increasing concentrations of selective NK1 and NK2 agonists on tachykinin-induced gelatinase production were also evaluated, as were the effects of a selective NK2 antagonist. Gelatinase activity in conditioned culture media (CCM) was assessed by zymography and quantified by image analysis. SP increased 92-kDa gelatinase activity in CCM of AM in a concentration-dependent manner, with a maximum increase at 10(-4) M. NKA, the NH2-terminal fragment of SP, and an NK1-selective agonist had no effect. In contrast, a selective NK2 agonist induced a concentration-dependent increase in gelatinase activity. The increase in this activity induced by SP and the selective NK2 agonist was inhibited by a selective NK2 antagonist. We conclude that SP induces gelatinase production by AM through NK2 receptor activation. The release of gelatinase may constitute one mechanism through which SP contributes to the epithelial lesions observed in bronchial hyperreactivity and asthma.

scholarly journals Calcium-sensitivity of inositol 1,4,5-trisphosphate metabolism in exocrine cells from the avian salt gland

1992 ◽  
Vol 282 (3) ◽  
pp. 703-710 ◽  
Author(s):  
J P Hildebrandt ◽  
T J Shuttleworth
Keyword(s):  
Substrate Concentration ◽  
Inositol Phosphates ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Salt Glands ◽  
Concentration Dependent Manner ◽  
Intact Cells ◽  
Exocrine Cells ◽  
Tissue Homogenates

The generation of inositol phosphates upon muscarinic-receptor activation was studied in [3H]inositol-loaded exocrine cells from the nasal salt glands of the duck Anas platyrhynchos, and the metabolism of different inositol phosphates in vitro was studied in tissue homogenates, with particular reference to the possible interaction of changes in intracellular [Ca2+] ([Ca2+]i) with the metabolic processes. In intact cells, there was a rapid (within 15 s) generation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, followed by an accumulation of their breakdown products, Ins(1,3,4)P3 and inositol bis- and monophosphates. Ca(2+)-sensitivity of the Ins(1,4,5)P3 3-kinase was demonstrated in tissue homogenates, with the rate of phosphorylation increasing 2-fold at free Ca2+ concentrations greater than 1 microM. However, addition of calmodulin or the presence of the calmodulin inhibitor W-7 (up to 100 microM) had no effect. 3-Kinase activity increased proportionally with the initial Ins(1,4,5)P3 concentration up to 1 microM, but a 10-fold higher substrate concentration produced only a doubling in the phosphorylation rate. Ins(1,3,4,5)P4 was dephosphorylated to Ins(1,3,4)P3, which accumulated in the homogenate assays as well as in intact cells. Depending on its concentration, Ins(1,3,4)P3 was phosphorylated [in part to Ins(1,3,4,6)P4] or dephosphorylated. To investigate the Ca(2+)-sensitivity of the 3-kinase in intact cells, excess quin2 was used to buffer the receptor-mediated transient changes in [Ca2+]i in [3H]inositol-loaded cells. These experiments revealed that increasing [Ca2+]i from less than 100 to approx. 400 nM (i.e. within the physiological range) has no effect on the partitioning of Ins(1,4,5)P3 metabolism (phosphorylation versus dephosphorylation) and on the accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. This indicates that activation of the 3-kinase by physiologically relevant Ca2+ concentrations may not play a major role in the generation of Ins(1,3,4,5)P4 signals upon receptor activation in these cells. The latter are mainly achieved by the receptor-mediated increase in Ins(1,4,5)P3 in the cell and its phosphorylation by the 3-kinase in a substrate-concentration-dependent manner.

Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery

1993 ◽  
Vol 13 (1) ◽  
pp. 399-407
Author(s):  
I J McEwan ◽  
A P Wright ◽  
K Dahlman-Wright ◽  
J Carlstedt-Duke ◽  
J A Gustafsson
Keyword(s):  
Glucocorticoid Receptor ◽  
Protein Interactions ◽  
Core Promoter ◽  
Specific Interaction ◽  
Direct Interaction ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Concentration Dependent Manner

We have used a yeast (Saccharomyces cerevisiae) cell free transcription system to study protein-protein interactions involving the tau 1 transactivation domain of the human glucocorticoid receptor that are important for transcriptional transactivation by the receptor. Purified tau 1 specifically inhibited transcription from a basal promoter derived from the CYC1 gene and from the adenovirus 2 major late core promoter in a concentration-dependent manner. This inhibition or squelching was correlated with the transactivation activity of tau 1. Recombinant yeast TATA-binding protein (yTFIID), although active in vitro, did not specifically reverse the inhibitory effect of tau 1. In addition, no specific interaction between tau 1 and yTFIID could be shown in vitro by affinity chromatography. Taken together, these results indicate that the tau 1 transactivation domain of the human glucocorticoid receptor interacts directly with the general transcriptional apparatus through some target protein(s) that is distinct from the TATA-binding factor. Furthermore, this assay can be used to identify interacting factors, since after phosphocellulose chromatography of a whole-cell yeast extract, a fraction that contained an activity which selectively counteracted the squelching effect of tau 1 was found.

Regulation of Na(+)-3HCO3- cotransport in rabbit proximal convoluted tubule via adenosine A1 receptor

1993 ◽  
Vol 265 (4) ◽  
pp. F511-F519 ◽  
Author(s):  
M. Takeda ◽  
K. Yoshitomi ◽  
M. Imai
Keyword(s):  
Basolateral Membrane ◽  
Receptor Activation ◽  
Proximal Convoluted Tubule ◽  
Adenosine A1 Receptor ◽  
Intracellular Camp ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Adenosine A1 ◽  
A1 Receptor

We investigated the role of adenosine A1-receptor in the regulation of basolateral Na(+)-3HCO3- cotransporter in the rabbit proximal convoluted tubule (PCT) microperfused in vitro by monitoring basolateral membrane potential and intracellular pH. FK-453, a highly specific A1 antagonist, inhibited basolateral HCO3- conductance in a concentration-dependent manner (10(-10)-10(-5) M). Other A1 antagonists, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) at 10(-5) M and theophylline at 10(-3) M, also had similar effects. N6-cyclohexyladenosine (CHA) at 10(-7) M attenuated the effect of low concentration (10(-8) M) of FK-453. Either enhancement of the degradation of adenosine by 0.1 U/ml adenosine deaminase (ADA) or inhibition of adenosine release from the cells by 10(-6) M S-(4-nitrobenzyl)-6-thioinosine (NBTI) mimicked the effects of A1 antagonists. These observations suggest that endogenous adenosine is released from PCT cells and stimulates Na(+)-3HCO3- cotransporter. Both 10(-4) M 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and 10(-6) M forskolin also inhibited basolateral HCO3- conductance. Both 10(-6) M FK-453 and 10(-4) M CPT-cAMP decreased the initial rate as well as the magnitude of intracellular acidification induced by reduction of peritubular HCO3- concentration from 25 to 0 mM. Neither 10(-6) M FK-453 nor 10(-7) M CHA changed intracellular Ca2+ concentration as measured by fura-2 fluorescence. These results indicate that adenosine might stimulate HCO3- exit across the basolateral membrane through Na(+)-3HCO3- cotransporter by decreasing intracellular cAMP via A1-receptor activation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Presynaptic GABAB Receptors Regulate Retinohypothalamic Tract Synaptic Transmission by Inhibiting Voltage-Gated Ca2+ Channels

2006 ◽  
Vol 95 (6) ◽  
pp. 3727-3741 ◽  
Author(s):  
Mykhaylo G. Moldavan ◽  
Robert P. Irwin ◽  
Charles N. Allen
Keyword(s):  
Glutamate Release ◽  
Receptor Activation ◽  
Gabab Receptors ◽  
Channel Blockers ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Epsc Amplitude ◽  
Receptor Modulation ◽  
Joint Application ◽  
Retinohypothalamic Tract

Presynaptic GABAB receptor activation inhibits glutamate release from retinohypothalamic tract (RHT) terminals in the suprachiasmatic nucleus (SCN). Voltage-clamp whole cell recordings from rat SCN neurons and optical recordings of Ca2+-sensitive fluorescent probes within RHT terminals were used to examine GABAB-receptor modulation of RHT transmission. Baclofen inhibited evoked excitatory postsynaptic currents (EPSCs) in a concentration-dependent manner equally during the day and night. Blockers of N-, P/Q-, T-, and R-type voltage-dependent Ca2+ channels, but not L-type, reduced the EPSC amplitude by 66, 36, 32, and 18% of control, respectively. Joint application of multiple Ca2+ channel blockers inhibited the EPSCs less than that predicted, consistent with a model in which multiple Ca2+ channels overlap in the regulation of transmitter release. Presynaptic inhibition of EPSCs by baclofen was occluded by ω-conotoxin GVIA (≤72%), mibefradil (≤52%), and ω-agatoxin TK (≤15%), but not by SNX-482 or nimodipine. Baclofen reduced both evoked presynaptic Ca2+ influx and resting Ca2+ concentration in RHT terminals. Tertiapin did not alter the evoked EPSC and baclofen-induced inhibition, indicating that baclofen does not inhibit glutamate release by activation of Kir3 channels. Neither Ba2+ nor high extracellular K+ modified the baclofen-induced inhibition. 4-Aminopyridine (4-AP) significantly increased the EPSC amplitude and the charge transfer, and dramatically reduced the baclofen effect. These data indicate that baclofen inhibits glutamate release from RHT terminals by blocking N-, T-, and P/Q-type Ca2+ channels, and possibly by activation of 4-AP–sensitive K+ channels, but not by inhibition of R- and L-type Ca2+ channels or by Kir3 channel activation.

Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells

1997 ◽  
Vol 273 (1) ◽  
pp. F129-F135 ◽  
Author(s):  
J. M. Arthur ◽  
G. P. Collinsworth ◽  
T. W. Gettys ◽  
L. D. Quarles ◽  
J. R. Raymond
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cellular Activation ◽  
Protein Activation ◽  
A Cell ◽  
Cation Sensing

Extracellular cations such as Ca2+ stimulate a G protein-coupled, cation-sensing receptor (CaR). We used microphysiometry to determine whether an extracellular cation-sensing mechanism exists in Madin-Darby canine kidney (MDCK) cells. The CaR agonists Ca2+ and Gd3+ caused cellular activation in a concentration-dependent manner. mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PCR) using nested CaR-specific primers, identification of an appropriately located restriction site, and sequencing of the subcloned fragment obtained by PCR. G protein activation was evaluated using the GTP photoaffinity label [alpha-32P]GTP azidoanalide (AA-GTP). After stimulation with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for Gq/11 alpha and Gi alpha family members. Gd3+ increased incorporation of AA-GTP into Gq/11 alpha precipitates by 146 +/- 48% and into G alpha i-2 and G alpha i-3 to a lesser extent but not into G alpha i-1. Direct effects of Gd3+ on the G proteins were ruled out using partially purified mammalian G proteins expressed in Escherichia coli or Sf9 cells. We conclude that MDCK cells possess a cell-surface CaR that activates Gq/11 alpha, G alpha i-2, and G alpha i-3 but not G alpha i-1.

Effects of estrogen on cerebral blood flow and pial microvasculature in rabbits

2000 ◽  
Vol 279 (3) ◽  
pp. H1208-H1214 ◽  
Author(s):  
M. T. Littleton-Kearney ◽  
D. M. Agnew ◽  
R. J. Traystman ◽  
P. D. Hurn
Keyword(s):  
Blood Flow ◽  
Estrogen Receptor ◽  
Cerebral Blood Flow ◽  
Receptor Activation ◽  
Receptor Subtypes ◽  
Dependent Manner ◽  
Pial Arteries ◽  
Concentration Dependent Manner ◽  
Cranial Window ◽  
Pial Arterioles

We tested the hypothesis that intracarotid estrogen infusion increases cerebral blood flow (CBF) in a concentration-dependent manner and direct application of estrogen on pial arterioles yields estrogen receptor-mediated vasodilation. Rabbits of both genders were infused with estrogen via a branch of the carotid artery. Estrogen doses of 20 or 0.05 μg · ml−1 · min−1 were used to achieve supraphysiological or physiological plasma estrogen levels, respectively. CBF and cerebral vascular resistance were determined at baseline, during the infusion, and 60-min postinfusion, and effects on pial diameter were assessed via a cranial window. Pial arteriolar response to estrogen alone and to estrogen after administration of tamoxifen (10−7), an antiestrogen drug that binds to both known estrogen receptor subtypes, was tested. No gender differences were observed; therefore, data were combined for both males and females. Systemic estrogen infusion did not increase regional CBF. Estradiol dilated pial arteries only at concentrations ranging from 10−4–10−7 M ( P ≤ 0.05). Pretreatment with tamoxifen alone had no effect on arteriolar diameter but inhibited estrogen-induced vasodilation ( P < 0.001). Our data suggest that estrogen does not increase CBF under steady-state conditions in rabbits. In the pial circulation, topically applied estradiol at micromolar concentrations dilates vessels. The onset is rapid and dependent on estrogen receptor activation.

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