Introduction

G protein-coupled receptors (GPCRs) are integral membrane-spanning proteins with an extracellular N-terminal domain, 7-a-helical membrane-spanning domains connected by intracellular and extracellular loops, and, finally, an intracellular C-terminal domain. GPCRs are important signal transduction proteins, and study of their function is usually carried out in native tissue or in recombinant cellular systems. A true understanding of GPCR function at a molecular level requires high-resolution three-dimensional (3D) structures of the different functional conformations across the superfamily of receptors. High-resolution crystal structures are also required to enable the range of structure- based drug design methodologies, which are routinely applied to soluble proteins to be used on GPCRs.

Despite a huge effort over many years, progress in obtaining GPCR structures, as well as other membrane proteins, has been painstakingly slow. To date, atomic structures have been obtained for 178 transmembrane proteins using X-ray crystallography (http://blanco.biomol.uci.edu/Membrane_Proteins_xtal. html). The majority of these structures are obtained from bacteria and archae-bacteria, which do not have GPCRs, only the ancestral protein bacteriorho-dopsin. In total, atomic structures have been solved for only four different GPCRs (Table 14.1), three of which have been published in 2008. There are several ways to obtain 3D structures of proteins: X-ray crystallography,

GPCR Molecular Pharmacology and Drug Targeting: Shifting Paradigms and New Directions,

Edited by Annette Gilchrist

Copyright © 2010 John Wiley & Sons, Inc.

TABLE 14.1 List of GPCR Crystal Structures

Rhodopsin: bovine ROS

2.8Â

1F88

[1]

Rhodopsin: bovine ROS

2.6Â

1L9H

[32]

Rhodopsin: bovine ROS

2.65 Â

1GZM

[33]

Rhodopsin: bovine ROS

2.2Â

1U19

[35]

Rhodopsin: recombinant bovine rhodopsin mutant,

3.4Â

2J4Y

[36]

N2C/D282C

Rhodopsin, photoactivated: bovine ROS

3.8-4.15 Â

2I37

[43]

Opsin: bovine ROS

2.9Â

3 CAP

[202]

Rhodopsin: squid

2.5 Â

2Z73

[38]

Rhodopsin: squid

3.7Â

2ZIY

[37]

Turkey ß1AR (StaR engineered for stability)

2.7 Â

2VT4

[4]

Complex with cyanopindolol

3.4/3.7 Â

Human ß2AR Fab5 complex

2R4R

[3]

Complex with carazolol

2.4 Â

2R4S

Engineered human ß2AR

2RH1

[2]

T4L replaces third ICL. Complex with carazolol

2.8 Â

Engineered human ß2AR

3D4S

[9]

T4L replaces third ICL. E122W stability mutation

Complex with timolol

2.6 Â

Human A2a adenosine receptor, in complex with a

3EML

[5]

high-affinity subtype-selective antagonist ZM241385

high-affinity subtype-selective antagonist ZM241385

TABLE 14.2 Hurdles Preventing Crystallization of GPCRs and How They Are Being Overcome

Crystallization Problem with Solution Reference

Step GPCRs

TABLE 14.2 Hurdles Preventing Crystallization of GPCRs and How They Are Being Overcome

Crystallization Problem with Solution Reference

Step GPCRs

Protein

Low expression in

High - level recombinant

expression

native tissues

expression systems

Protein

Instability

Engineering stability

[9,135,136,

purification

Low yield of functional protein

203]

Detergent

Instability in

Engineering stability

[9, 135, 136,

solubilization

detergent

Lipid bicelles Lipidic cubic phase

203-205]

Crystallization

Structural heterogeneity

E. coli expression Mutagenesis of sites for PTM

Conformational

Ligand binding

[135, 136]

heterogeneity

Conformational stabilization

Low polar surface

Antibody complex

[2, 3, 164]

area

T4L fusion

Small crystals

Synchrotron microfocus beam

TABLE 14.3 Comparative Heterologous Expression Levels of the Human P2AR

Expression

Strain

N-Terminus

C-Terminus

Expression

Minimal Level of

Scalability

Reference

Host

Level (pmol/mg)

Expression Required (pmol/mg) [133,137]

E. coli

XLl-blue

MalE

His 6

3

13

High

[138]

Yeast

P. pastoris

a-factor signal His6

6

5

High

[139]

Yeast

S. cerevisiae

Ste2

115

5

High

[129]

Insect

Sf9

73

26

Medium

[95]

Insect

Sf9 stable

26

Medium

[140]

Mammalian

Stable HEK239

1D4 epitope

220

26

Low

[141]

Cell Free

E. coli S30 extract

TrxA

His 6

150|ig/mLa

N/A

Low

[115]

'Expression levels in |.ig/mL of CF expression reaction mixture.

'Expression levels in |.ig/mL of CF expression reaction mixture.

electron cryomicroscopy (cryo-EM), and nuclear magnetic resonance (NMR) spectroscopy. X-ray crystallography using synchrotron radiation sources is the most powerful method for obtaining high-resolution structures of GPCRs, and this chapter will focus exclusively on this area. However, many of the methodological improvements described will also benefit EM and NMR studies.

Until very recently, the only GPCR that had been crystallized was the photopigment from retinal rod cells—rhodopsin. This receptor is unique in terms of its natural abundance within rod membranes and its stability in detergent solutions. The structure, which was first obtained in 2000 [1], has proved extremely valuable as a template for homology modeling of other Family A receptors despite the fact that the homology is less than 30%. The use of rhodopsin as a basis for modeling Family B and Family C, however, is questionable, due to the lack of sequence homology across these families.

The last 2 years have seen several major breakthroughs in obtaining highresolution structures of GPCRs, which have resulted in the publication of two structures of the p2-adrenergic receptor (p2AR) [2, 3], the structure of the closely related p1-adrenergic receptor (p1AR) [4] and the adenosine A2a receptor [5]. The new approaches, which are now being applied in the field of GPCR crystallography (Table 14.2), provide hope that the next decade will see a wealth of new GPCR structures that will transform our understanding of GPCR function and guide novel drug discovery.

In this chapter, we discuss the technical challenges that have hindered the production of stable functional receptor protein for structural studies. We will review the history and impact of the structures of rhodopsin, and we will describe the new developments that have resulted in the recent p - adrenergic (pAR) and adenosine receptor structures along with the applicability of these approaches to other GPCRs. We will also consider the new insights obtained from the new structures in terms of understanding ligand binding and receptor function, and we conclude by discussing likely future development in this rapidly growing area of GPCR biology.

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