Signal Transducing Complexes Ted Wensel, Baylor College of Medicine

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

Targets of Top-Selling Prescription Drugs

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

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

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

Gabg-membrane complex Gb Ga Gg

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

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

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

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

MS raw file analyzed by BioworksBrowser against NR database

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

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

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

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

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.