1. Structure of GPCRs and G proteins
Goal of the lecture:
Understanding the structural basis of how a
GPCR activates a G protein

2. Heterotrimeric G protein Pathway
Clapham Nature Jan 25;379(6563):

3. Ribbon Diagram of Rhodopsin Structure
Palczewski et al, Science Aug 4;289(5480):

4. Two dimensional Representation of Rhodopsin
Palczewski et al, Science Aug 4;289(5480):

5. The environment of 11-cis retinal chromophore
Palczewski et al, Science Aug 4;289(5480):

6. Salient features of Rhodopsin Structure
Organization of the extracellular region serves as the basis seven-helix bundle arrangement
11-cis retinal holds transmembrane regions in the inactive conformation by interacting with key residues that participate in intra-helical interactions

7. Activation of Rhodopsin
Requirement of rigid-body motion of
transmembrane helices for light activation of
rhodopsin.
Farrens et al, Science Nov 1;274(5288):

8. Design of the Experiment
Mutate all Cys to Ser
Bring back Cys of interest
Construct double
Cys mutants
Keep Cys at 139 (helix 3)
constant
Vary 2nd Cys from
in helix 6
Put spin label on the Cys
EPR spectroscopy
Farrens et al, Science Nov 1;274(5288):

9. EPR spectra of inactive (dark state) shown as red trace
and activated (meta-rhodopsin II)
shown as yellow trace
to study interactions between loops
3 and 6
Farrens et al, Science Nov 1;274(5288):

10. Results from EPR Spectroscopy
Dark State: Distance between Cys at 139
and Cys at = Å
After illumination increases in distances
23-25 Å
Conclusion: Helices 3 and 6 move apart from each other after activation

11. Biochemical Verification of EPR predicted
movement of helices
Cross link with disulfide reagent, cut with V8 protease
Run SDS-PAGE
If cross linked 1 band without DTT; 2 bands with DTT
Farrens et al, Science Nov 1;274(5288):

12. Crosslinking of helices 3 and 6 blocks the ability of Rhodopsin to activate Transducin
Fluorescence assay
to measure GTPgS
binding to transducin
Farrens et al, Science Nov 1;274(5288):

13. Conclusions Helix 6 moves with respect to Helix 3
Movement is required for
activation of transducin
Helix 6 movement causes
cytoplasmic loop3 to move
Cytoplasmic loop3 is involved
in coupling to transducin
Farrens et al, Science Nov 1;274(5288):

14. G protein structure
Lambright et al, Nature Jan 25;379(6563):

15. Space filling model of Ga interacts with Gbg
Lambright et al, Nature Jan 25;379(6563):

16. The Gb interface that interacts with Ga contains key residues required for interaction with effectors
Lambright et al, Nature Jan 25;379(6563):

17. G protein residues involved in regulation of effectors
                                                                                                                                                                                                                                      
Space filling model of Gbg. Gb is white and Gg is pink.
The green region is the area of Gb covered by Ga in the heterotrimer
The smaller regions marked by colored dashed lines identify residues involved in interactions with various effectors. Each color corresponds to an effector
Ford et al, Science May 22;280(5367):

18. In the heterotrimer the switch II region of Ga is contact with Gb
Wall et al, Cell Dec 15;83(6):

19. Changes in the conformation of Ga in the GDP vs GTP bound forms and interactions with Gb
GTPgS-Ga Red
GDP-Ga Blue
Wall et al, Cell Dec 15;83(6):

20. The Switch II region of Ga has different conformation in the GDP and GTP bound states
GTPgS
GDP
Wall et al, Cell Dec 15;83(6):

21. The heterotrimeric G protein interacts with the membrane and receptor
Lambright et al, Nature Jan 25;379(6563):

22. A structural cartoon of G protein interaction with receptor
Hamm J Biol Chem Jan 9;273(2):

23. Evolving view of receptors GPCRs exist as dimers
Park et al, Biochemistry Dec 21;43(50):

24. Atomic Force Microscopy Picture of
mouse rod-outer segment disc membrane
Fotiadis et al, Nature Jan 9;421(6919):

25. Organization of the cytoplasmic surface of rhodopsin dimers are clearly visible
                                                                                          
Fotiadis et al, Nature Jan 9;421(6919):

26. Model of Rhodopsin Dimer Here phosphorylated Rhodopsin is shown
binding to arrestin
(This would be the
Desensitized state)
Park et al, Biochemistry Dec 21;43(50):

27. Model of rhodopsin dimer binding to one molecule of transducin
Park et al, Biochemistry Dec 21;43(50):

28. Receptor Dimer Activation of G proteins
Park et al, Biochemistry Dec 21;43(50): A movie of this molecule is available from