Upload
virginia-bradford
View
214
Download
1
Tags:
Embed Size (px)
Citation preview
Fluorescent proteins
Excitation and Emission Wavelengths of AFP proteins
Strategy of FRET between CFP and YFP
Biochemical Modulation of AFP fluorescence
The General Design of FRET-based Fluorescent Probes
Some early indicators for second messenger
Using fluorescence protein to imaging signaling transduction
Biological Questions on GPCR
• Dimer/oligomerization
• Ligand induced Conformational change
• G protein coupling
• Second messenger signaling
cAMP/PKA, PKC, DAG, IP3, Ca2+
Scaffold protein/signal complex
Biological Questions on GPCR
• Dimer/oligomerization– Homo/hetreo dimer– Constitutive vesus ligand induced dimer– Dimer in receptor maturation in ER– New agonism– Functional diversity and Signaling integration
Biological Questions on GPCR
• Ligand induced Conformational change– Structural functional study– Agonism/antagonism– Receptor activation
Biological Questions on GPCR
G protein coupling
*Real time activation
*Screen model for drug discovery
*Signaling/function co-relation.
*Single cell study, instead of biochemical assay of GTPS binding
Biological Questions on GPCR
Second messenger signaling
cAMP/PKA, PKC, DAG, IP3, Ca2+
• Spatial and temporal resolution
• Localized signaling properties, specificity and efficiency.
• Specific function
Restricted cAMP signaling in cardiac myocytes
cAMP indicator
Promiscuous G protein Coupling by GPCR
Go-CFP resistant to PTX,
Cell pretreated with PTX
CFP 430+12.5nM 465+20nMYFP 500+10nM 535+15nMFRET 430+12.5NM, 535+15nM
Control
CFP 430+12.5nM 465+20nMYFP 500+10nM 535+15nMFRET 430+12.5NM, 535+15nM
CFP 430+12.5nM 465+20nMYFP 500+10nM 535+15nMFRET 430+12.5NM, 535+15nM
514nM laser line bleach YFP about 70%-90%Concomitant CFP bleach about 7-10%
Figure 6, Quantitative Analyses of FRET
a2A-YFP + Go-CFP
a2A-YFP + Gs-CFP
Donor(Go):acceptor(receptor) at 1:1, a proportion of receptor is not coupled to G proteinIncreaing the ration leads to higher degree of precoupling. In physiological conditions, G proteins are in significant excess compared to receptor, thus a large majority of receptor are precoupled.