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

1986:Cloning of b2 adrenoceptor – the first non-sensory GPCR

1986:Cloning of b2 adrenoceptor – the first non-sensory GPCR

Cloning the b2 adrenoceptor

• Receptor from hamster lung solubilised in detergent and purified byaffinity 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

Cloning the b2 adrenoceptor

Cyanogen bromide fragments of the purified b2 AR were isolated by HPLC and their amino acid sequences determined.

Cloning the b2 adrenoceptor

Hamster genomic and cDNA libraries were screened with synthetic oligonucleotides complementary to peptide fragments of the receptor.

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”

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

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

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 a single responsive clone was identified

Cloning the NK2 receptor by expression in Xenopus oocytes

1991:The cDNA sequence was also cloned by Nakanishi’s 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

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

1991:Crystal structure of rhodopsin determined by Krzysztof Palczewski and colleagues (click to play movie)

1991:Crystal structure of rhodopsin

1991:Whole genome sequencing prompted searches for the full mammalian complement of GPCRs and phylogenetic analysis

2003- Datamining

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 reads”Structure of the human β2-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

Activated human β2 adrenergic receptor (in blue ) in a complex with a heterotrimeric G protein (3 subunits:reddish to orange-brown) and hormone (gold), resolution 3.2Å. The boundaries of the membrane in which the GPCR sits are represented in light green. From Proteopedia(click to play movie).

Activated human β2 adrenergic receptor (in blue ) in a complex with a heterotrimeric G protein (3 subunits:reddish to orange-brown) and hormone (gold), resolution 3.2Å. The boundaries of the membrane in which the GPCR sits are represented in light green. From Proteopedia(click to play movie).

Awarded to Robert Lefkowitz (left)and Brian Kobilka (right) "for studies of G-protein-coupled receptors"

2012:Nobel Prize in Chemistry