1. Fluorescent proteins

2. Excitation and Emission Wavelengths of AFP proteins

3. Strategy of FRET between CFP and YFP

4. Biochemical Modulation of AFP fluorescence

5. The General Design of FRET-based Fluorescent Probes

6. Some early indicators for second messenger

7. Using fluorescence protein to imaging signaling transduction

8. 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

12. 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

17. Biological Questions on GPCR Ligand induced Conformational change –Structural functional study –Agonism/antagonism –Receptor activation

21. 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 GTP  S binding

23. 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

24. Restricted cAMP signaling in cardiac myocytes cAMP indicator

25. Promiscuous G protein Coupling by GPCR

27. Go-CFP resistant to PTX, Cell pretreated with PTX

28. CFP 430+12.5nM 465+20nM YFP 500+10nM 535+15nM FRET 430+12.5NM, 535+15nM

29. Control CFP 430+12.5nM 465+20nM YFP 500+10nM 535+15nM FRET 430+12.5NM, 535+15nM

30. CFP 430+12.5nM 465+20nM YFP 500+10nM 535+15nM FRET 430+12.5NM, 535+15nM

32. 514nM laser line bleach YFP about 70%-90% Concomitant CFP bleach about 7-10%

34. 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 protein Increaing 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.