gpcr structures 041213

Structural studies of GPCRsTony Harmar

University of Edinburgh

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

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”

Bacteriorhodopsin -the first 7TM protein

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

The first depiction of the 7TM topology of bacteriorhodopsin, from Ovchinnikov.

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

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

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

1986:Cloning of β2 adrenoceptor – the first non-sensory GPCR

1986:Cloning of β2 adrenoceptor – the first non-sensory GPCR

Cloning the Cloning the ββ 22 adrenoceptor adrenoceptor

• Receptor from hamster lung solubilised in detergent and purified byReceptor from hamster lung solubilised in detergent and purified byaffinity chromatography on alprenolol-sepharoseaffinity chromatography on alprenolol-sepharose

• Progress of purification monitored by binding of [Progress of purification monitored by binding of [125125I]-cyanopindololI]-cyanopindolol

• Attempts to obtain amino acid sequence of the intact protein failedAttempts to obtain amino acid sequence of the intact protein failed

• Purified protein was Purified protein was subjected to chemical cleavage with cyanogen to chemical cleavage with cyanogen bromide (CNBr), which cleaves proteins after every methionine bromide (CNBr), which cleaves proteins after every methionine residueresidue

• Cyanogen bromide fragments were purified by HPLC and Cyanogen bromide fragments were purified by HPLC and sequenced sequenced

Cloning the Cloning the ββ 22 adrenoceptor adrenoceptor

Cloning the Cloning the ββ 22 adrenoceptor adrenoceptor

1988:the first "orphan" GPCR

G-21 was a genomic clone with homology to the β2AR: at first its endogenous ligand was unknown, i.e. it encoded

an “orphan” GPCRNature 335: 358-360 (1988)

Nature 335: 358-360 (1988)

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