The document summarizes key developments in the structural characterization of G protein-coupled receptors (GPCRs), beginning with the 1975 structure of bacteriorhodopsin determined by Henderson and Unwin, showing its 7 transmembrane alpha helical structure. It then discusses important milestones such as the 1983 cloning of bovine rhodopsin, the first GPCR cDNA; the 1986 cloning of the beta-2 adrenergic receptor, the first non-sensory GPCR; the 1988 cloning of the 5-HT1A receptor, the first "orphan" GPCR to be deorphanized; and the 1991 crystal structure of rhodopsin, the first GPCR structure. The document concludes with the

1. Structural studies of GPCRs
Tony Harmar
University of Edinburgh

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

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. 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. 1986:Cloning of β2 adrenoceptor – the first nonsensory GPCR

9. 1986:Cloning of β2 adrenoceptor – the first nonsensory GPCR

10. 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

11. Cloning the β 2 adrenoceptor

12. Cloning the β 2 adrenoceptor

13. 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)

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 5HT1A receptor

15. 1987:Expression cloning of the NK2 receptor,
the first peptide GPCR
1987:cDNA sequence encoding the the NK2 receptor was
reported by the group of Shigetada Nakanishi using an
ingenious expression cloning strategy

16. Cloning the NK 2 receptor by expression in
Xenopus oocytes
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

17. 1991:Expression cloning of the metabotropic
glutamate receptor mGlu1, the first GPCR from
Class C
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

18. 1991:Expression cloning of the secretin receptor,
the first Class B GPCR
1991:The secretin receptor was cloned by the laboratory of
Shigekazu Nagata by expression in COS cells

19. 1991:Crystal structure of rhodopsin
1991:Crystal structure of rhodopsin determined by Krzysztof Palczewski and
colleagues (click to play movie)

20. 2003- Datamining
1991:Whole genome sequencing prompted searches for the full
mammalian complement of GPCRs and phylogenetic analysis

21. 2007:Crystal structure of the β2 adrenoceptor
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”

22. 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).

23. 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).

24. 2012:Nobel Prize in Chemistry
Awarded to Robert Lefkowitz (left)and Brian Kobilka (right) "for studies of
G-protein-coupled receptors"