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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.