Gpcr structures 041213

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Gpcr structures 041213

  1. 1. Structural studies of GPCRs Tony Harmar University of Edinburgh
  2. 2. Structure of the first membrane protein 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 lightdriven 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.
  3. 3. Bacteriorhodopsin -the first 7TM protein Using electron diffraction, Henderson & 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 proteins”
  4. 4. Amino acid sequence of bacteriorhodopsin 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.
  5. 5. The first depiction of the 7TM topology of bacteriorhodopsin, from Ovchinnikov.
  6. 6. Amino acid sequence of the first GPCR 1983: Complete amino acid sequence of bovine rhodopsin determined by the laboratories of Ovchinnikov (Russia) and Hargrave (USA.
  7. 7. First cDNA and gene sequences 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
  8. 8. Amino acid sequence of the first GPCR 1983: Complete amino acid sequence of bovine rhodopsin determined by the laboratories of Ovchinnikov (Russia) and Hargrave (USA).
  9. 9. 1986:Cloning of β2 adrenoceptor – the first nonsensory GPCR
  10. 10. 1986:Cloning of β2 adrenoceptor – the first nonsensory GPCR
  11. 11. Cloning the β 2 adrenoceptor • Receptor from hamster lung solubilised in detergent and purified by affinity chromatography on alprenolol-sepharose • Progress of purification monitored by binding of [ 125 I]-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
  12. 12. Cloning the β 2 adrenoceptor
  13. 13. Cloning the β 2 adrenoceptor
  14. 14. 1988:the first "orphan" GPCR Nature 335: 358-360 (1988) G-21 was a genomic clone with homology to the β2AR: at first its endogenous ligand was unknown, i.e. it encoded an “orphan” GPCR Nature 335: 358-360 (1988)
  15. 15. 1988:5-HT1A receptor “deorphanised” When expressed in cell lines and studied in a radioligand binding assay, G-21 exhibited the pharmacology of the 5HT1A receptor

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