structure of gpcrs and g proteins goal of the lecture: understanding the structural basis of how a...

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