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.