scholarly journals Stimulation of Proton Sensing G-Protein Coupled Receptors Prevent pH Induced Fibroblast Activation

2020 ◽  
Author(s):  
A.R. Rackow ◽  
R.S. Clough ◽  
G. Zapas ◽  
R.M. Kottmann
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Fibroblast Activation ◽  
G Protein Coupled ◽  
Stimulation Of

scholarly journals The metalloprotease Kuzbanian (ADAM10) mediates the transactivation of EGF receptor by G protein–coupled receptors

2002 ◽  
Vol 158 (2) ◽  
pp. 221-226 ◽  
Author(s):  
Yibing Yan ◽  
Kyoko Shirakabe ◽  
Zena Werb
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Egf Receptor ◽  
Critical Role ◽  
G Protein Coupled Receptors ◽  
Heparin Binding ◽  
Egf Receptors ◽  
Gpcr Activation ◽  
G Protein Coupled ◽  
Stimulation Of

Communication between different signaling pathways enables cells to coordinate the responses to diverse environmental signals. Activation of the transmembrane growth factor precursors plays a critical role in this communication and often involves metalloprotease-mediated proteolysis. Stimulation of G protein–coupled receptors (GPCR) transactivates the EGF receptors (EGFRs), which occurs via a metalloprotease-dependent cleavage of heparin-binding EGF (HB-EGF). However, the metalloprotease mediating the transactivation remains elusive. We show that the integral membrane metalloprotease Kuzbanian (KUZ; ADAM10), which controls Notch signaling in Drosophila, stimulates GPCR transactivation of EGFR. Upon stimulation of the bombesin receptors, KUZ increases the docking and activation of adaptors Src homology 2 domain–containing protein and Gab1 on the EGFR, and activation of Ras and Erk. In contrast, transfection of a protease domain–deleted KUZ, or blocking endogenous KUZ by morpholino antisense oligonucleotides, suppresses the transactivation. The effect of KUZ on shedding of HB-EGF and consequent transactivation of the EGFR depends on its metalloprotease activity. GPCR activation enhances the association of KUZ and its substrate HB-EGF with tetraspanin CD9. Thus, KUZ regulates the relay between the GPCR and EGFR signaling pathways.

Functional Screening of G Protein—Coupled Receptors by Measuring Intracellular Calcium with a Fluorescence Microplate Reader

2002 ◽  
Vol 7 (3) ◽  
pp. 233-246 ◽  
Author(s):  
Matthias U. Kassack ◽  
Barbara Höfgen ◽  
Jochen Lehmann ◽  
Niels Eckstein ◽  
J. Mark Quillan ◽  
...  
Keyword(s):  
G Protein ◽  
Screening Method ◽  
G Protein Coupled Receptors ◽  
Functional Screening ◽  
Binding Studies ◽  
Fluorescence Measurements ◽  
Agonist Stimulation ◽  
Microplate Reader ◽  
G Protein Coupled ◽  
Stimulation Of

Ligand binding studies reveal information about affinity to G protein—coupled receptors (GPCRs) rather than functional properties. Increase in intracellular Ca2+ appears to represent a universal second messenger signal for a majority of recombinant GPCRs. Here, we exploit Ca2+ signaling as a fast and sensitive functional screening method for a number of GPCRs coupled to different G proteins. Ca2+ fluorescence measurements are performed using Oregon Green 488 BAPTA-1/AM and a microplate reader equipped with an injector. Buffer alone or test compounds dissolved in buffer are injected into a cell suspension, and fluorescence intensity is recorded for 30 s. Each of the GPCRs tested—Gq-coupled P2Y2, Gs-coupled dopamine D1 and D5, Gi-coupled dopamine D2L, and Gq/11-coupled muscarinic acetylcholine M1—yielded a significant rise in intracellular free [Ca2+] on agonist stimulation. Agonist stimulation was dose dependent, as shown for ATP or UTP stimulation of P2Y2 receptors (EC50 = 1 μM), SKF38393 stimulation of hD1 and hD5 (EC50 = 18.1 nM and 2.7 nM), and quinpirole at hD2L (EC50 = 6.5 nM). SCH23390 (at hD1 and hD5) and spiperone, haloperidol, and clozapine (at hD2L) competitively antagonized the Ca2+ response. Furthermore, the Ca2+ assay served to screen suramin analogs for antagonistic activity at P2Y2 receptors. Screening at dopamine receptors revealed LE300, a new lead for a dopamine receptor antagonist. Advantages of the assay include fast and simple 96- or 384-well plate format (high-throughput screening), use of a visible light-excitable fluorescent dye, applicability to a majority of GPCRs, and simultaneous analysis of distinct Ca2+ fluxes.

Regulation of adenylyl cyclase in polarized renal epithelial cells by G protein-coupled receptors

1997 ◽  
Vol 273 (6) ◽  
pp. F883-F891 ◽  
Author(s):  
Mark D. Okusa ◽  
Liping Huang ◽  
Akemi Momose-Hotokezaka ◽  
Long P. Huynh ◽  
Amy J. Mangrum
Keyword(s):  
Epithelial Cells ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Cyclase Activity ◽  
Camp Accumulation ◽  
Adenylyl Cyclase Activity ◽  
Renal Epithelial Cells ◽  
G Protein Coupled ◽  
Stimulation Of

We employed two guanine nucleotide binding protein (G protein)-coupled receptors known to be targeted to opposite domains in renal epithelial cells to test the hypothesis that the polarized receptor expression of receptors regulates the activity of the receptor’s effector molecule, adenylyl cyclase. We used LLC-PK1 cells stably transfected with cDNA encoding the α2B-adrenergic receptor (α2B-AR) or A1-adenosine receptor (A1-AdR). Immunohistochemistry and Western blot analysis confirmed the basolateral and apical expression of α2B-ARs and A1-AdRs, respectively. Adenylyl cyclase activity was assessed by measuring cAMP accumulation following the addition of forskolin (10 μM) in the presence of 3-isobutyl-1-methylxanthine to apical or basolateral chambers of confluent monolayers. A five- to sixfold increase in cAMP accumulation occurred following apical (or basolateral) stimulation of LLC-PK1 cells expressing apical (or basolateral) receptors in comparison to forskolin stimulation of corresponding domains of untransfected cells. We conclude 1) adenylyl cyclase activity is present at or near the apical and basolateral domains of LLC-PK1 cells, and 2) factors that regulate the polarized expression of inhibitory G protein-coupled receptors may also regulate local adenylyl cyclase activity.

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