scholarly journals Anti-HIV Agent Trichosanthin Enhances the Capabilities of Chemokines to Stimulate Chemotaxis and G Protein Activation, and This Is Mediated through Interaction of Trichosanthin and Chemokine Receptors

1999 ◽  
Vol 190 (1) ◽  
pp. 101-112 ◽  
Author(s):  
Jian Zhao ◽  
Li-Hong Ben ◽  
Ya-Lan Wu ◽  
Wei Hu ◽  
Kun Ling ◽  
...  
Keyword(s):  
Hiv Infection ◽  
G Protein ◽  
Chemokine Receptors ◽  
Hek293 Cells ◽  
Chinese Medicinal Herb ◽  
Protein Activation ◽  
G Protein Activation ◽  
Trichosanthes Kirilowii ◽  
Stromal Cell Derived Factor ◽  
Specific Association

Trichosanthin (TCS), an active protein component isolated from a traditional Chinese medicinal herb Trichosanthes kirilowii, has been shown to inhibit HIV infection and has been applied in clinical treatment of AIDS. The recent development that chemokines and chemokine receptors play important roles in HIV infection led us to investigate the possible functional interaction of TCS with chemokines and their receptors. This study demonstrated that TCS greatly enhanced both RANTES (regulated upon activation, normal T cell expressed and secreted)– and stromal cell–derived factor (SDF)-1α–stimulated chemotaxis (EC50 ≅ 1 nM) in leukocytes (THP-1, Jurkat, and peripheral blood lymphocyte cells) and activation of pertussis toxin–sensitive G proteins (EC50 ≅ 20 nM). TCS also significantly augmented chemokine-stimulated activation of chemokine receptors CCR5 and CXCR4 as well as CCR1, CCR2B, CCR3, and CCR4 transiently expressed in HEK293 cells. A mutant TCS with 4,000-fold lower ribosome-inactivating activity showed similar augmentation activity as wild-type TCS. Moreover, flow cytometry demonstrated that the specific association of TCS to the cell membranes required the presence of chemokine receptors, and laser confocal microscopy reveals that TCS was colocalized with chemokine receptors on the membranes. The results from TCS-Sepharose pull-down and TCS and chemokine receptor coimmunoprecipitation and cross-linking experiments demonstrated association of TCS with CCR5. Thus, our data clearly demonstrated that TCS synergizes activities of chemokines to stimulate chemotaxis and G protein activation, and the effects of TCS are likely to be mediated through its interaction with chemokine receptors.

scholarly journals Analysis of agonist function at fusion proteins between the IP prostanoid receptor and cognate, unnatural and chimaeric G-proteins

1999 ◽  
Vol 342 (2) ◽  
pp. 457-463 ◽  
Author(s):  
Chee Wai FONG ◽  
Graeme MILLIGAN
Keyword(s):  
Fusion Protein ◽  
G Protein ◽  
G Proteins ◽  
Fusion Proteins ◽  
Hek293 Cells ◽  
Gtpase Activity ◽  
Prostanoid Receptor ◽  
Protein Activation ◽  
G Protein Activation ◽  
Stimulation Of

Direct measures of G-protein activation based on guanine nucleotide exchange and hydrolysis are frequently impossible to monitor for receptors which interact predominantly with Gsα. An isolated FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-epitope-tagged human IP prostanoid receptor and fusion proteins generated between this form of the receptor and the α subunits of its cognate G-protein Gs, Gi1, a G-protein which it fails to activate in co-expression studies, and a chimaeric Gi1-Gs6 (a form of Gi1 in which the C-terminal six amino acids were replaced with the equivalent sequence of Gs) were stably expressed in HEK293 cells. These were detected by [3H]ligand-binding studies and by immunoblotting with both an anti-FLAG antibody and with appropriate antisera to the G-proteins. Each construct displayed similar affinity to bind the agonist iloprost. Iloprost stimulated adenylate cyclase activity in clones expressing both IP prostanoid receptor and the IP prostanoid receptor-Gsα fusion protein, and both constructs were shown to interact with and activate endogenously expressed Gsα. Addition of iloprost to membranes of cells expressing the isolated receptor resulted in a small stimulation of high-affinity GTPase activity. Iloprost produced no stimulation of GTPase activity which could be attributed to the IP prostanoid receptor-Gi1α fusion. However, the fusion proteins containing either Gsα or Gi1-Gs6α produced substantially greater stimulation of GTPase activity than the isolated IP prostanoid receptor. Treatment of cells expressing the IP prostanoid receptor-Gi1-Gs6α fusion protein with a combination of cholera and pertussis toxins allowed direct measurement of agonist activation of the receptor-linked G-protein. Normalization of such results for levels of expression of the IP prostanoid receptor constructs demonstrated a 5-fold higher stimulation of GTPase activity when using the Gsα-containing fusion protein and a 9-fold improvement when using the fusion protein containing Gi1-Gs6α to detect G-protein activation compared with expression of the isolated receptor.

scholarly journals Heterogeneity of G-protein activation by the calcium-sensing receptor

2021 ◽  
Author(s):  
Hasnat Ali Abid ◽  
Asuka Inoue ◽  
Caroline M. Gorvin
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Receptor Activation ◽  
Assay System ◽  
Hek293 Cells ◽  
Calcium Sensing Receptor ◽  
Protein Activation ◽  
Calcium Sensing ◽  
G Protein Activation ◽  
Protein Dissociation

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that plays a fundamental role in extracellular calcium (Ca2+e) homeostasis by regulating parathyroid hormone release and urinary calcium excretion. The CaSR has been described to activate all four G-protein subfamilies (Gαq/11, Gαi/o, Gα12/13, Gαs), and mutations in the receptor that cause hyper/hypocalcaemia, have been described to bias receptor signalling. However, many of these studies are based on measurements of second messenger proteins or gene transcription that occurs many steps downstream of receptor activation and can represent convergence points of several signalling pathways. Therefore, to assess CaSR-mediated G-protein activation directly, we took advantage of a recently described NanoBiT G-protein dissociation assay system. Our studies, performed in HEK293 cells stably expressing CaSR, demonstrate that Ca2+e stimulation activates all Gαq/11 family and several Gαi/o family proteins, although Gαz was not activated. CaSR stimulated dissociation of Gα12/13 and Gαs from Gβ-subunits, but this occurred at a slower rate than that of other Gα-subunits. Investigation of cDNA expression of G-proteins in three tissues abundantly expressing CaSR, the parathyroids, kidneys and pancreas, showed Gα11, Gαz, Gαi1 and Gα13 genes were highly expressed in parathyroid tissue, indicating CaSR most likely activates Gα11 and Gαi1 in parathyroids. In kidney and pancreas, the majority of G-proteins were similarly expressed, suggesting CaSR may activate multiple G-proteins in these cells. Thus, these studies validate a single assay system that can be used to robustly assess CaSR variants and biased signalling and could be utilised in the development of new pharmacological compounds targeting CaSR.

Opioid-Activated Postsynaptic, Inward Rectifying Potassium Currents in Whole Cell Recordings in Substantia Gelatinosa Neurons

1998 ◽  
Vol 80 (6) ◽  
pp. 2954-2962 ◽  
Author(s):  
S. P. Schneider ◽  
W. A. Eckert ◽  
A. R. Light
Keyword(s):  
Membrane Potential ◽  
G Protein ◽  
Potassium Currents ◽  
Substantia Gelatinosa ◽  
Opioid Agonist ◽  
Membrane Hyperpolarization ◽  
Whole Cell ◽  
Protein Activation ◽  
G Protein Activation ◽  
Whole Cell Recordings

Schneider, S. P., W. A. Eckert III, and A. R. Light. Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons. J. Neurophysiol. 80: 2954–2962, 1998. Using tight-seal, whole cell recordings from isolated transverse slices of hamster and rat spinal cord, we investigated the effects of the μ-opioid agonist (d-Ala2, N-Me-Phe4,Gly5-ol)-enkephalin (DAMGO) on the membrane potential and conductance of substantia gelatinosa (SG) neurons. We observed that bath application of 1–5 μM DAMGO caused a robust and repeatable hyperpolarization in membrane potential ( V m) and decrease in neuronal input resistance ( R N) in 60% (27/45) of hamster neurons and 39% (9/23) of rat neurons, but significantly only when ATP (2 mM) and guanosine 5′-triphosphate (GTP; 100 μM) were included in the patch pipette internal solution. An ED50 of 50 nM was observed for the hyperpolarization in rat SG neurons. Because G-protein mediation of opioid effects has been shown in other systems, we tested if the nucleotide requirement for opioid hyperpolarization in SG neurons was due to G-protein activation. GTP was replaced with the nonhydrolyzable GTP analogue guanosine-5′- O-(3-thiotriphosphate) (GTP-γ-S; 100 μM), which enabled DAMGO to activate a nonreversible membrane hyperpolarization. Further, intracellular application of guanosine-5′- O-(2-thiodiphosphate) (GDP-β-S; 500 μM), which blocks G-protein activation, abolished the effects of DAMGO. We conclude that spinal SG neurons are particularly susceptible to dialysis of GTP by whole cell recording techniques. Moreover, the depletion of GTP leads to the inactivation of G-proteins that mediate μ-opioid activation of an inward-rectifying, potassium conductance in these neurons. These results explain the discrepancy between the opioid-activated hyperpolarization in SG neurons observed in previous sharp electrode experiments and the more recent failures to observe these effects with whole cell patch techniques.

scholarly journals β-Amyloid Peptide-induced Apoptosis Regulated by a Novel Protein Containing a G Protein Activation Module

2001 ◽  
Vol 276 (22) ◽  
pp. 18748-18756 ◽  
Author(s):  
Eileen M. Kajkowski ◽  
C. Frederick Lo ◽  
Xiaoping Ning ◽  
Stephen Walker ◽  
Heidi J. Sofia ◽  
...  
Keyword(s):  
G Protein ◽  
Amyloid Peptide ◽  
Protein Activation ◽  
G Protein Activation ◽  
Β Amyloid ◽  
Β Amyloid Peptide ◽  
Induced Apoptosis ◽  
Novel Protein

Pregnancy enhances G protein activation and nitric oxide release from uterine arteries

2001 ◽  
Vol 280 (5) ◽  
pp. H2069-H2075 ◽  
Author(s):  
L. P. Thompson ◽  
C. P. Weiner
Keyword(s):  
Nitric Oxide ◽  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Nitric Oxide Synthase Inhibitor ◽  
Protein Activation ◽  
Nitric Oxide Release ◽  
G Protein Activation ◽  
Hormonal Modulation ◽  
Synthase Inhibitor

We hypothesized that pregnancy modulates receptor-mediated responses of the uterine artery (UA) by altering G protein activation or coupling. Relaxation and contraction to NaF (0.5–11.5 mM), acetylcholine (10−9–10−5 M), and bradykinin (10−12–3 × 10−5 M) were measured in isolated UA of pregnant and nonpregnant guinea pigs. Responses were measured in the presence and absence of either cholera toxin (2 μg/ml) or pertussis toxin (Gαs and Gαiinhibitors, respectively). NaF relaxation was endothelium dependent and nitro-l-arginine sensitive (a nitric oxide synthase inhibitor). Relaxation to NaF, acetylcholine, and bradykinin were potentiated by pregnancy. Cholera but not pertussis toxin increased relaxation to acetylcholine and bradykinin in UA from nonpregnant animals, had no effect in UA from pregnant animals, and abolished the pregnancy-induced differences in acetylcholine relaxation. Cholera toxin potentiated the bradykinin-induced contraction of UA of both pregnant and nonpregnant animals, whereas pertussis toxin inhibited contraction of UA from pregnant animals only. Therefore, pregnancy may enhance agonist-stimulated endothelium-dependent relaxation and bradykinin-induced contraction of UA by inhibiting GTPase activity or enhancing Gαs but not Gαi activation in pregnant animals. Thus the diverse effects of pregnancy on UA responsiveness may result from hormonal modulation of G proteins coupled to their specific receptors.

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