structure of gpcrs and g proteins goal of the lecture: understanding the structural basis of how a...
TRANSCRIPT
Structure of GPCRs and G proteins
Goal of the lecture:
Understanding the structural basis of how a GPCR activates a G protein
Heterotrimeric G protein Pathway
Clapham Nature. 1996 Jan 25;379(6563):297-299.
Ribbon Diagram of Rhodopsin Structure
Palczewski et al, Science. 2000 Aug 4;289(5480):739-745.
Two dimensional Representation of Rhodopsin
Palczewski et al, Science. 2000 Aug 4;289(5480):739-745.
The environment of 11-cis retinal chromophore
Palczewski et al, Science. 2000 Aug 4;289(5480):739-745.
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
Activation of Rhodopsin
Requirement of rigid-body motion of
transmembrane helices for light activation of
rhodopsin.
Farrens et al, Science. 1996 Nov 1;274(5288):768-770.
Design of the Experiment
Mutate all Cys to Ser
Bring back Cys of interest
Construct doubleCys mutants
Keep Cys at 139 (helix 3)constant Vary 2nd Cys from 247-252in helix 6
Put spin label on the Cys
EPR spectroscopy
Farrens et al, Science. 1996 Nov 1;274(5288):768-770.
EPR spectra of inactive (dark state)shown as red traceand activated (meta-rhodopsin II)shown as yellow traceto study interactions between loops3 and 6
Farrens et al, Science. 1996 Nov 1;274(5288):768-770.
Results from EPR Spectroscopy
Dark State: Distance between Cys at 139
and Cys at 248-251 = 12-14 Å
After illumination increases in distances
23-25 Å
Conclusion: Helices 3 and 6 move apart from each other after activation
Biochemical Verification of EPR predicted movement of helices
Cross link with disulfide reagent, cut with V8 proteaseRun SDS-PAGEIf cross linked 1 band without DTT; 2 bands with DTT
Farrens et al, Science. 1996 Nov 1;274(5288):768-770.
Crosslinking of helices 3 and 6 blocks the ability of Rhodopsin to activate Transducin
Fluorescence assayto measure GTPS binding to transducin
Farrens et al, Science. 1996 Nov 1;274(5288):768-770.
Conclusions
Helix 6 moves with respect to Helix 3
Movement is required for activation of transducin
Helix 6 movement causescytoplasmic loop3 to move
Cytoplasmic loop3 is involved in coupling to transducin
Farrens et al, Science. 1996 Nov 1;274(5288):768-770.
G protein structure
Lambright et al, Nature. 1996 Jan 25;379(6563):311-319.
Space filling model of G interacts with G
Lambright et al, Nature. 1996 Jan 25;379(6563):311-319.
The G interface that interacts with G contains key residues required for
interaction with effectors
Lambright et al, Nature. 1996 Jan 25;379(6563):311-319.
G protein residues involved in regulation of effectors
Ford et al, Science. 1998 May 22;280(5367):1271-1274.
Space filling model of G. Gis white and G is pink.The green region is the area of G covered by Gin the heterotrimer
The smaller regions marked by colored dashed lines identify residues involved in interactions with various effectors. Each color corresponds to an effector
In the heterotrimer the switch II region of G is contact with G
Wall et al, Cell. 1995 Dec 15;83(6):1047-1058.
G
GTPS-G Red GDP-G Blue
Changes in the conformation of G in the GDP vs GTP bound forms and interactions with G
Wall et al, Cell. 1995 Dec 15;83(6):1047-1058.
The Switch II region of G has different conformation in the GDP and GTP bound states
GTPS
GDP
Wall et al, Cell. 1995 Dec 15;83(6):1047-1058.
The heterotrimeric G protein interacts with the membrane and receptor
Lambright et al, Nature. 1996 Jan 25;379(6563):311-319.
A structural cartoon of G protein interaction with receptor
Hamm J Biol Chem. 1998 Jan 9;273(2):669-672.
Evolving view of receptors GPCRs exist as dimers
Park et al, Biochemistry. 2004 Dec 21;43(50):15643-15656.
Atomic Force Microscopy Picture of mouse rod-outer segment disc membrane
Fotiadis et al, Nature. 2003 Jan 9;421(6919):127-128.
Organization of the cytoplasmic surface of rhodopsin dimers are clearly visible
Fotiadis et al, Nature. 2003 Jan 9;421(6919):127-128.
Model of RhodopsinDimer
Here phosphorylatedRhodopsin is shown binding to arrestin(This would be the Desensitized state)
Park et al, Biochemistry. 2004 Dec 21;43(50):15643-15656.
Model of rhodopsin dimer binding to one molecule of transducin
Park et al, Biochemistry. 2004 Dec 21;43(50):15643-15656.
Receptor Dimer Activation of G proteins
Park et al, Biochemistry. 2004 Dec 21;43(50):15643-15656. A movie of this molecule is available from http://stke.sciencemag.org/cgi/content/full/sigtrans;2005/276/tr10/DC1