Download GFP PLCδ PH domain – membrane to cytoplasm redistribution assay

GFP PLCδ
δ PH domain –
membrane to cytoplasm redistribution assay
Phospholipase C plays a
key role in signal transduction
processes for many cell
surface receptors
Generates DAG and IP3 as
second messengers
GFP is fused to the Pleckstrin
Homology (PH) domain of
PLC. This binds to
phosphatidylinositol lipids
(specifically PIP2). This
fusion protein translocates
like native PLC
Generally applicable to all areas
because many agonists that
activate signal transduction
pathways stimulate PLC delta
GFP-PLCδ
δ PH domain
membrane to cytoplasm redistribution assay
Agonist,
5-10 sec
Un-stimulated cell:
majority of GFP-PLCδ localized on
inner surface of plasma membrane.
20-40 sec
Stimulated cell:
GFP-PLCδ redistributes transiently
to cytoplasmic space,
Post-stimulation cell:
GFP-PLCδ re-accumilates at
plasma membrane
GFP-PLCδ PH domain
Translocation index
- (% D peak)
ATP Dose Response curve
40
30
20
10
0
-7
-6
-5
-4
ATP conc. (M)
Image acquisition & analysis:
IN Cell Analyzer 3000
-3
GFP Translocation Assays
Assay
Response pathways
MAPKAP-k2
Rac-1
PLCδ PH domain
2XFYVE domain
(domain from
Hrs)
Example Therapeutic Areas
Stress response pathways
Inflammation, neuronal growth and
differentiation, pain, CNS disorders,
ischemia, seizures, skeletal muscle
regulation
Cytokinesis, phagocytosis, pinocytosis,
axon outgrowth, morphogenesis, cellcell contacts, cell polarity,
transformation, adhesion, migration
Colon and breast cancer, cardiac
hypertrophy, myofibrillogenesis, proinflammatory signalling, leukemia,
rheumatoid arthritis, progression to
AIDS
PI(4,5)P2 signalling pathways,
adrenoreceptor function, cytoskeletal
organization, responses to purinergic
agonists, mechanical stress pathways
Cardiac diseases/ hypertrophy,
cardiac injury, Alzheimer’s disease,
bipolar disorders
Growth factor (RTK) and cytokine
signalling pathways, PI(3)P levels
(class III PI3Kinase sensor)
Identification of class-specific PI3K
inhibitors for development of cancer
treatments, immuno-suppressants and
anti-inflammatory drugs
(Slide 1 of 2)
GFP Translocation Assays
Response pathways
Assay
Akt1
Cell survival, proliferation, apoptosis,
insulin response pathways
Example Therapeutic Areas
Cancer (breast, prostate, ovarian,
gastric), tumor progression, drug and
radiation resistance in cancer therapy,
invasion, angiogenesis, diabetes
TGF-beta signalling, growth,
differentiation, cell survival, regulation
of cell proliferation and migration,
elaboration of extracellular matrix
Oncogenesis, tumor progression,
cardiac hypertension, embryonic
development, tissue repair, immune
function
STAT3
Acute-phase response pathways,
cytokine signalling, leptin signalling
Acute-phase responses, oncogenesis
(hematologic, breast, head, neck,
prostate cancers), obesity
NFAT-c1
Immune responses (IL2, IL4, TNFα
gene expression), prostaglandin
signalling
Immunosuppression, muscle growth
SMAD2
(Slide 2 of 2)