Detection of phospholipase C-β 2 activation by G-protein subunits

1998 ◽  
Author(s):  
Suzanne Scarlata ◽  
Loren Runnels ◽  
Mario Rebecchi
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Protein Subunits

Dexamethasone increases G alpha q-11 expression and hormone-stimulated phospholipase C activity in UMR-106-01 cells.

1997 ◽  
Vol 273 (3) ◽  
pp. E528 ◽  
Author(s):  
J Mitchell ◽  
A Bansal
Keyword(s):  
Adenylyl Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Beta Subunits ◽  
Adenylyl Cyclase Activity ◽  
Protein Subunits ◽  
G Alpha Q ◽  
Umr 106 ◽  
G Protein Coupled

Glucocorticoids regulate responsiveness of many cells to hormones that bind to G protein-coupled receptors. We examined the effect of glucocorticoids on parathyroid hormone (PTH) activation of two G protein-activated signal transduction pathways, phospholipase C (PLC) and adenylyl cyclase, in osteosarcoma UMR-106-01 cells. Dexamethasone (100 nM) increased PTH-stimulated and NaF-stimulated PLC activity by > 100% over 4 days (223 +/- 8 and 293 +/- 8.2% of control after 4 days for PTH and NaF-stimulated activity, respectively). The increase in PTH-stimulated adenylyl cyclase response in the same cells was more modest (162 +/- 5.4 and 171 +/- 6.8% of control after 4 days for PTH and NaF-stimulated activity, respectively). PTH activation of PLC was blocked by antiserums to G alpha q-11 and activation of adenylyl cyclase by G alpha s antiserums. Quantification of these G protein subunits in control and dexamethasone-treated cells showed a 78% increase in G alpha q-11 (from 18.1 +/- 1.2 to 32.2 +/- 1.5 pmol/mg), whereas G alpha s was increased only 34% (from 6.2 +/- 0.5 to 8.2 +/- 0.3 pmol/mg) and G beta-subunits were increased 40% (from 54 +/- 2.3 to 75.2 +/- 3.8 pmol/mg). These results suggest that glucocorticoids are more potent regulators of PLC activity than adenylyl cyclase activity in UMR cells, and this is mediated, at least in part, by differential increases in G alpha q-11 proteins.

scholarly journals Role of Dynamic Interactions in Effective Signal Transfer for Gβ Stimulation of Phospholipase C-β2

2002 ◽  
Vol 277 (51) ◽  
pp. 49707-49715 ◽  
Author(s):  
Elizabeth Buck ◽  
Peter Schatz ◽  
Suzanne Scarlata ◽  
Ravi Iyengar
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Binding Affinity ◽  
Protein Interactions ◽  
Signal Transfer ◽  
Dynamic Interactions ◽  
Protein Subunits ◽  
Functional Studies ◽  
Effective Signal ◽  
Stimulation Of

Heterotrimeric G protein subunits regulate their effectors by protein-protein interactions. The regions involved in these direct interactions have either signal transfer or general binding functions (Buck, E., Li, J., Chen, Y., Weng, G., Scarlata, S., and Iyengar, R. (1999) Science 283, 1332–1335). Although key determinants of signal transfer regions for G protein subunits have been identified, the mechanisms of signal transfer are not fully understood. We have used a combinatorial peptide approach to analyze one Gβ region, Gβ86–105, involved in signal transfer to the effector phospholipase C (PLC)-β2 to gain a more mechanistic understanding of Gβ/PLC-β2 signaling. Binding and functional studies with the combinatorial peptides on interaction with and stimulation/inhibition of phospholipase Cβ2 indicate that binding affinity can be resolved from EC50for functional effects, such that peptides that have wild type binding affinities have 15- to 20-fold lower EC50values. Although more potent, these peptides display a much lower extent of maximal stimulation. These peptides synergize with Gβγ or peptides encoding the second Gβ42–54 signal transfer region in maximally stimulating phospholipase C-β2. Other combinatorial peptides from the Gβ86–105 region that bind to PLC-β2 by themselves submaximally stimulate and extensively inhibit Gβγ stimulation of PLC-β2. The intrinsic stimulation function can be attributed to Arg-96 and Ser-97, the synergy function to Trp-99, and the binding affinity to Thr-87, Val-90, Pro-94, Arg-96, Ser-97, and Val-100. These results indicate that, even within signal transfer regions, residues involved in binding can be resolved from those involved in signal transfer and that signal transfer is likely to be achieved through dynamic rather than steady-state interactions.

scholarly journals Signalling through the leukotriene B4 receptor involves both αi and α16, but not αq or α11 G-protein subunits

1998 ◽  
Vol 335 (1) ◽  
pp. 15-18 ◽  
Author(s):  
Rémi GAUDREAU ◽  
Christian Le GOUILL ◽  
Salim MÉTAOUI ◽  
Stéphane LEMIRE ◽  
Jana STANKOVÀ ◽  
...  
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Inositol Phosphates ◽  
Leukotriene B4 ◽  
The Other ◽  
Protein Subunits ◽  
Other Hand ◽  
Leukotriene B4 Receptor

COS-7 cells transfected with the leukotriene (LT) B4 receptor (BLTR) cDNA were unable to produce LTB4-induced inositol phosphates (IPs) in spite of the presence of endogenous Gαi, Gαq and Gα11 proteins. Co-transfection of BLTR with Gα16, however, resulted in high levels of IP production, which were 17-, 10- and 6-fold higher than with co-transfected Gα11, Gαq and Gα14, respectively. Co-transfection of BLTR with phospholipase C (PLC) β2, on the other hand, resulted in efficient IP production and co-transfection of BLTR with both Gα16 and PLCβ2 resulted in a greater than additive response.

Functional characterization of phosphoinositide-specific phospholipase C-beta 1 and -beta 3 in intestinal smooth muscle

1995 ◽  
Vol 269 (4) ◽  
pp. C969-C978 ◽  
Author(s):  
K. S. Murthy ◽  
G. M. Makhlouf
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Longitudinal Muscle ◽  
Functional Characterization ◽  
Circular Muscle ◽  
Muscle Cells ◽  
Protein Subunits ◽  
G Alpha Q ◽  
Hydrolysis Of

Soluble and membrane phosphoinositide-specific phospholipases obtained separately from dispersed circular and longitudinal intestinal muscle cells were characterized for substrate specificity and G protein dependence using selective antibodies to various isoforms of phospholipase C (PLC) and G protein subunits. Western blot analysis disclosed the presence of the main PLC isozymes, PLC-gamma 1, PLC-delta 1, and PLC-beta 1. Soluble PLC from circular and longitudinal muscle was stimulated by guanosine 5'-O-(3-thiophosphate) and inhibited by PLC-beta 1 antibody (80-90%) and PLC-beta 3 antibody (approximately 25%) but not by G protein antibodies. Membrane PLC from circular and longitudinal muscle was stimulated by cholecystokinin octapeptide (CCK-8) and inhibited selectively by PLC-beta 1 antibody (85%), PLC-beta 3 antibody (15%), and G alpha q/11 antibody (90%). CCK-8-induced contraction in permeabilized circular muscle cells was also selectively inhibited by PLC-beta 1 antibody (76%), PLC-beta 3 antibody (24%), and G alpha q/11 antibody (86%). The combined effects of PLC-beta 1 and PLC-beta 3 antibodies on PLC activity and muscle contraction were additive, causing complete inhibition. Soluble and membrane PLC from circular and longitudinal muscle were immunologically similar but functionally different. The enzymes from circular muscle preferentially hydrolyzed endogenous and exogenous phosphatidylinositol 4,5-biphosphate (PIP2), confirming previous findings of preferential hydrolysis of PIP2 in dispersed intestinal circular muscle cells

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