1. Signal Transducing Complexes
Ted Wensel, Baylor College of Medicine

2. Why are complexes associated with G protein-coupled receptors the most important complexes for human health ?
They are the most common class of drug targets
They will continue to be the most attractive drug targets

3. Targets of Top-Selling Prescription Drugs

5. Structure of a G protein-coupled receptor
Carbohydrate chains
Extracellular domain
Palczewski et al., July, 2000, Science
Transmembrane Helices
Bound ligand
Membrane Phospholipids
Cytoplasmic face: binds G proteins, kinases, arrestins, etc.
Palmitoylated cysteine residues
More post-translational modifications: disulfides; phosphorylation, proteolysis

6. Multi-protein complexes of Vertebrate Phototransduction
Rhod-
opsin
Dimer ? Multimer ? Ga
Gb1 Pg
Rhod.
kinase
Arrestin Gg
PDE Pa Pb
Gb5
RGS9 Pg
R9AP
Dimer ?
565 kDa
Also: PKCa, Cam

7. RGS domain of RGS9
Fragment of
effector, PDE6g
G protein a subunit

8. Gabg-membrane complexGb Ga Gg

9. Non-catalytic cGMP-binding GAF-domains
Catalytic domains
Isoprenylated C-termini C
Visual Effector
PDE6abgg
cGMP phospho-diesterse C

10. PDE6 bound to vesicles.
PDE6 + activated G protein bound to vesicles.

11. GTPase assay verifies all are functional
Vesicles reconstituted with 8 polypeptides: Rhodopsin, R9AP, Gat, Gbgt, RGS9-1, Gb5, and PDEg.
GTPase assay verifies all are functional
100
1000
10000
1.0
2.0
3.0
4.0
5.0 8 80
800
[R9AP]
[Vesicle]
Fold-enhancement

12. PDZ-Domain Scaffolds for GPCR Signaling Complexes
565 80 40
74 (inaD)
80 (PKC)
125
Total:
964 X 4 ? X 2 ?
PDZ-Domain Scaffolds for GPCR Signaling Complexes
Hall & Lefkowitz, 2002

13. MS raw file analyzed by BioworksBrowser against NR database

14. PDZ-Domain Scaffolds for GPCR Signaling Complexes
565 80 40
74 (inaD)
80 (PKC)
125
Total:
964 X 4 ? X 2 ?
PDZ-Domain Scaffolds for GPCR Signaling Complexes
Hall & Lefkowitz, 2002

15. TRP channel purified from yeast via 1-D4 epitope tag TRPV1 (side)
TRPV1 (bottom)
K+ channel
Vera Moiseenkova

16. Kvap voltage –gated K+ channel
Yeast TRPC
YVC1 (bottom)
Yeast TRPC
YVC1 (side)
Kvap voltage –gated K+ channel
Vera Moiseenkova

17. Photoreceptor disk rims
Plasma membrane
Disk rims
disks
cytoplasm
ABCR
Trans-porter
lumen
Guanylate cyclase
Peripherin/rds tetramers
Rim complex connects to cGMP gated channels and Na/Ca exchanger in plasma membrane
1.4 mm

19. Summary
GPCR are the most common receptors, and the pathways they regulate are the most important drug targets.
They function in large multi-protein complexes, usually at the plasma membrane.
Despite some success in crystallography and EM of small pieces of these complexes, full-size complex structures remain a challenge.
Visual systems of Drosophila and mammals present tractable systems- all components of mammalian system can be purified or expressed and reconstituted in quantities sufficient for EM.
Outer segments, connecting cilia, and disks can be isolated for cro-EM tomography.