Structural studies of GPCRs

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An overview of the history of structural studies of G protein-coupled receptors, written by Prof. Tony Harmar prior to his retirement from the University of Edinburgh and as chair of the Guide to PHARMACOLOGY.

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<ul><li>1.Structural studies of GPCRs Tony Harmar University of Edinburgh </li></ul> <p>2. The first membrane protein that was structurally characterised, by RichardHenderson (left) and Nigel Unwin (right) in 1975, was bacteriorhodopsin, a light-harvesting membrane protein from the archaean Halobacterium halobium that acts as a light-driven proton pump and is the only protein constituent of the purple membrane, a two-dimensional crystal lattice naturally present as part of the plasma membrane of the bacterium. Structure of the first membrane protein 3. Using electron diffraction, Henderson &amp; Unwin showed that the protein contains seven alpha- helices that enclose an all- trans retinal chromophore that undergoes an isomerisation process upon light absorption that results in the translocation of a proton from the cytoplasmic side to the extracellular side of the membrane.They commented, almost prophetically The purple membrane thus seems to provide a simple example of an 'intrinsic' membrane protein, a class of structure to which many molecular pumps and channels must belong. We would not be surprised if the simple arrangement of helices found here also occurs in some of these other intrinsic membrane Bacteriorhodopsin -the first 7TM protein 4. The amino acid sequence of bacteriorhodopsin was first published, almost simultaneously, by the groups of Yuri Ovchinnikov in 1978 and Nobel Laureate Har Gobind Khorana in1979. Each study represented a tour de force of protein chemistry. Amino acid sequence of bacteriorhodopsin 5. The first depiction of the 7TM topology of bacteriorhodopsin, from Ovchinnikov. 6. 1983: Complete amino acid sequence of bovine rhodopsin determined by the laboratories of Ovchinnikov (Russia) and Hargrave (USA. Amino acid sequence of the first GPCR 7. 1983:cloning of cDNA and gene encoding bovine rhodopsin by Jeremy Nathans (left) and David Hogness (right). Using a citation classic technique for homology screening devised by Hogness, they later identified three related visual pigment genes(red-, green- and blue-sensitive opsins) First cDNA and gene sequences 8. 1986:Cloning of b2 adrenoceptor the first non- sensory GPCR 9. 1986:Cloning of b2 adrenoceptor the first non- sensory GPCR 10. Cloning the b2 adrenoceptor Receptor from hamster lung solubilised in detergent and purified by affinity chromatography on alprenolol-sepharose Progress of purification monitored by binding of [125I]-cyanopindolol Attempts to obtain amino acid sequence of the intact protein failed Purified protein was subjected to chemical cleavage with cyanogen bromide (CNBr), which cleaves proteins after every methionine residue Cyanogen bromide fragments were purified by HPLC and sequenced 11. Cloning the b2 adrenoceptor by HPLC and their amino acid sequences determined. 12. Cloning the b2 adrenoceptor . 13. 1988:the first "orphan" GPCR G-21 was a genomic clone with homology to the b2AR: at first its endogenous ligand was unknown, i.e. it encoded an orphan GPCR Nature 335: 358-360 (1988) Nature 335: 358-360 (1988) 14. 1988:5-HT1A receptor deorphanised When expressed in cell lines and studied in a radioligand binding assay, G-21 exhibited the pharmacology of the 5- HT1A receptor 15. 1987:cDNA sequence encoding the the NK2 receptor was reported by the group of Shigetada Nakanishi using an ingenious expression cloning strategy 1987:Expression cloning of the NK2 receptor, the first peptide GPCR 16. 1987:pools of mRNA transcripts from bovine stomach cDNAwere injected into Xenopus oocytes and tested for electrophysiological responses to neurokinin A. Pools were progressively subdivided until Cloning the NK2 receptor by expression in Xenopus oocytes 17. 1991:The cDNA sequence was also cloned by Nakanishis group via screening of RNA transcripts in Xenopus oocytes. Picture shows mRNA distribution in hippocampus by in situ hybridisation 1991:Expression cloning of the metabotropic glutamate receptor mGlu1, the first GPCR from Class C 18. 1991:The secretin receptor was cloned by the laboratory of Shigekazu Nagata by expression in COS cells 1991:Expression cloning of the secretin receptor, the first Class B GPCR 19. 1991:Crystal structure of rhodopsin determined by Krzysztof Palczewski and colleagues (click to play movie) 1991:Crystal structure of rhodopsin 20. 1991:Whole genome sequencing prompted searches for the full mammalian complement of GPCRs and phylogenetic analysis 2003- Datamining 21. 2007:the first high-resolution structure of a GPCR. Crystal structure was determined by the labs of Brian Kobilka and Ray Stevens. Science cover caption readsStructure of the human 2-adrenergic receptor (red) embedded in a lipid membrane and bound to a diffusible ligand (green), with cholesterol (yellow) between the two receptor molecules. A cartoon of the lipidic cubic phase used for crystallization of the receptor is shown in the background 2007:Crystal structure of the b2 adrenoceptor 22. Activated human 2 adrenergic receptor (in blue ) in a complex with a heterotrimeric G protein (3 subunits:reddish to orange-brown) and hormone (gold), resolution 3.2. The boundaries of the membrane in which the GPCR sits are represented in light green. From Proteopedia(click to play movie). 23. Activated human 2 adrenergic receptor (in blue ) in a complex with a heterotrimeric G protein (3 subunits:reddish to orange-brown) and hormone (gold), resolution 3.2. The boundaries of the membrane in which the GPCR sits are represented in light green. From Proteopedia(click to play movie). 24. Awarded to Robert Lefkowitz (left)and Brian Kobilka (right) "for studies of G-protein-coupled receptors" 2012:Nobel Prize in Chemistry </p>