analysis of the human tachykinin NK2 receptor and expression of the human dopamine D2 receptor
Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften
an der Universität Konstanz
Mathematisch -Naturwissenschaftliche Sektion Fachbereich Biologie
Vorgelegt von Vijay S.N. Narasimhan
Tag der mündlichen Prüfung: 14. Dezember 2009
Prüfungskommission:
Vorsitzende: Prof. Dr. I. Adamska 1. Prüfer : Prof. Dr. W. Welte
2. Prüfer: PD Dr. J.-H. Kleinschmidt
Konstanzer Online-Publikations-System (KOPS) URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-97878
URL: http://kops.ub.uni-konstanz.de/volltexte/2009/9787/
TABLE OF CONTENTS CHAPTER 1: INTRODUCTION
1.1 : INTRODUCTION TO MEMBRANE PROTEINS 1
1.1.1: Biological membranes and membrane lipids 1
1.1.2: Membrane proteins 4
1.1.3: Membrane protein folding 7
1.2 : INTRODUCTION TO THE GUANINE-NUCLEOTIDE BINDING PROTEIN COUPLED RECEPTORS (GPCRs) 9
1.3 : CLASSIFICATION OF THE GPCR FAMILIES 10
1.4 : GPCR SIGNAL TRANSDUCTION 12
1.5 : STRUCTURE OF GPCRs 13
1.5.1: Rhodopsin 13
1.5.2: β2-Adrenergic Receptor 15
1.6 : OVER-EXPRESSION OF GPCRs FOR REFOLDING AND STRUCTURAL STUDIES 17
1.6.1: Bottlenecks in the overexpression of GPCRs 17
1.6.2: Expression systems employed for the overexpression of GPCRs 19
1.7 : REFOLDING OF GPCRs 21
1.8 : THE DOPAMINE RECEPTORS 22
1.8.1: The D-1, D-2 like Receptors 22
1.9 : THE DOPAMINE 2 RECEPTOR (D2R) 23
1.9.1: Isoforms of the D2R 25
1.9.2: Ligand binding site of the D2R 25
1.10: THE TACHYKININ RECEPTORS 26
1.10.1: Types of the tachykinin receptors 27
1.11: THE TACHYKININ 2 RECEPTOR (NK2R) 29
1.11.1: The NK2R agonist and antagonist 29
1.11.2: Ligand binding site of the NK2R 31
1.12: PROJECT AIMS 32
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2.1: MATERIALS 33
2.1.1: General chemicals and reagents 33
2.1.2: Molecular biology reagents 33
2.1.3: Media 33
2.1.4: Antibiotics 34
2.1.5: Plasmid vectors 34
2.1.5.1: pcDNA3 vector: 5446 bp 34
2.1.5.2: pCR-Blunt II TOPO vector: 3159 bp 34
2.1.6: Originating constructs 36
2.1.6.1: pcDNA3 D2(L)R 36
2.1.6.2: pcDNA3 NK2R 36
2.1.7: Strains 36
2.1.7.1: TOP 10 36
2.1.7.2: XL1 Blue 36
2.2: METHODS 37
2.2.1: Propagation of cells 37
2.2.1.1: Growth and maintenance of Escherichia coli (E.coli) cells 37
2.2.2: Molecular biology techniques 37
2.2.2.1: Transformation of E.coli cells 37
2.2.2.2: Small scale preparation of the plasmid DNA from E.coli 37
2.2.2.3: Medium scale preparation of the plasmid DNA from E.coli 37
2.2.2.4: Phenol:Chloroform:Isoamyl Alcohol extraction and DNA precipitation 38
2.2.2.5: Agarose Gel Electrophoresis of the DNA 39
2.2.2.6: Agarose gel extraction of the DNA fragments 39
2.2.2.7: Restriction digestion 39
2.2.2.8: Ligation of the DNA fragments 39
2.2.2.9: Polymerase Chain Reaction (PCR) 40
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2.2.2.10: Zero Blunt II TOPO PCR cloning 40
2.2.2.11: SDS-PAGE 41
2.2.2.12: Semi-Dry Electro-Blotting 41
2.2.2.13: Immunoblotting 41
2.2.2.14: Invision In-gel staining 42
2.2.2.15: Radioligand binding assay 42
CHAPTER 3: EXPRESSION OF THE DOPAMINE 2 AND TACHYKININ 2 RECEPTORS IN ESCHERICHIA COLI(E.coli) 44
3.1: INTRODUCTION 44
3.2: MATERIALS 44
3.3: METHODS 45
3.3.1: Subcloning of the D2R and NK2R cDNA into the pET-22b vector 45
3.3.2: Transformation of the plasmid DNA into the BL21 (DE3) E.coli cells 46
3.3.3: Expression trials for the D2R and NK2R in E.coli 46
3.4: RESULTS 47
3.4.1: Expression of the D2R and NK2R in E.coli 47
CHAPTER 4: EXPRESSION OF THE DOPAMINE 2 AND TACHYKININ 2 RECEPTORS IN DROSOPHILA SCHNEIDER 2 (S2) CELLS 48
4.1: INTRODUCTION 48
4.2: MATERIALS 48
4.3: METHODS 49
4.3.1: Subcloning of the D2R and NK2R cDNA into the pAc 5.1-V5/His vector 49
4.3.2: Culture and general maintenance of the S2 cells 50
4.3.3: Calcium phosphate transfection of the plasmid DNA into the S2 cells 50
4.4: RESULTS 51
4.4.1: Expression of the D2R and NK2R in S2 cells 51
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5.1: INTRODUCTION 53
5.2: MATERIALS 54
5.3: METHODS 55
5.3.1: Subcloning of the D2R and NK2R cDNA into the pBiEx-3 vector 55
5.3.2: Culture and general maintenance of the Sf9 cells 55
5.3.3: Transfection of the plasmid DNA into Sf9 cells using Insect Gene juice reagent 56
5.4: RESULTS 57
5.4.1: Expression of the D2R in Sf9 cells – anti-D2R blot 57
5.4.1: Expression of the NK2R in Sf9 cells – anti-His blot 57
CHAPTER 6: EXPRESSION OF THE DOPAMINE 2 RECEPTOR IN PICHIA PASTORIS EXPRESSION SYSTEM 59
6.1: INTRODUCTION 59
6.2: MATERIALS 61
6.3: METHODS 62
6.3.1: Subcloning of the D2R cDNA into the pPICZ and pPICZα vector 62
6.3.2: Growth and maintenance of Pichia pastoris cells 63
6.3.3: Electroporation of the Pichia pastoris cells 63
6.3.4: Expression test in Pichia pastoris 64
6.3.5: Preparation of the Pichia pastoris membranes 64
6.3.6: Purification of the D2R using Ni-NTA spin column 64
6.3.7: Radioligand binding assay 65
6.4: RESULTS 65
6.4.1: Expression of the D2R in Pichia pastoris cells using the pPICZ vector 65
6.4.2: Expression of the D2R in Pichia pastoris cells using the pPICZα vector 66
6.4.3: Purification of the D2R using Ni-NTA spin column 66
6.4.4: Radioligand Binding studies 67 iv
CHAPTER 7: EXPRESSION OF THE DOPAMINE 2 AND TACHYKININ 2 RECEPTORS IN
CELL-FREE EXPRESSION SYSTEM 69
7.1: INTRODUCTION 69
7.2: MATERIALS 70
7.3: METHODS 71
7.3.1: The RTS E. coli Linear Template Generation 71
7.3.2: Subcloning of the Nk2R and D2R cDNA into the pIVEX2.4d vector 72
7.3.3: Expression trials using the RTS 100 E. coli HY Kit 73
7.4: RESULTS 74
7.4.1: Expression of the NK2R and D2R using the linear template generation set 74
7.4.2: Expression of the NK2R and D2R using the pIVEX2.4d vector 75
CHAPTER 8: EXPRESSION OF THE TACHYININ 2 AND DOPAMINE 2 RECEPTORS IN E.COLI WITH TRANSMEMBRANE DOMAIN OF OUTER MEMBRANE PROTEIN A AS FUSION PARTNER AND RARE CODON EXCHANGE TECHNIQUE 77
8.1: INTRODUCTION 77
8.1.1: The effect of rare codons on high level expression of heterologous protein in E.coli 78
8.2: MATERIALS 79
8.3: METHODS 80
8.3.1: Construction of the TM-OmpA-GPCR fusion plasmid 80
8.3.2: Transformation of BL21-Codonplus (DE3)-RIPL cells 83
8.3.3: Expression trials in E.coli cells 83
8.3.4: Construction of the TM-OmpA-NK2R N-terminus fragment fusion plasmid 83
8.3.5: Construction of various TM-OmpA-NK2R fragments fusion plasmid 84
8.3.6: The effect of rare codons on expression of the NK2R in E.coli 85
8.3.7: The QuikChange® Multi Site-Directed Mutagenesis 87
8.3.8: Site directed mutagenesis to exchange rare codons in the NK2R DNA 88
8.3.9: Solubilisation trials for the TM-OmpA-Nk2R inclusion bodies 90
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8.3.12: Construction of the pET-22b-NK2R plasmid 91
8.4: RESULTS 92
8.4.1: Expression trial of the Nk2R in BL21-RIPL cells 92
8.4.2: Expression of the TM-OmpA-NK2R N-terminus fragment in E.coli 93
8.4.3: Expression of the TM-OmpA-NK2R fragments in E.coli 93
8.4.4: Expression of the TM-OmpA-NK2R individual helices in E.coli 94
8.4.5: Site directed mutagenesis to exchange the rare codons in the NK2R DNA 96
8.4.6: Expression of the TM-OmpA-Nk2R in E.coli after codon replacement 97
8.4.7: Comparison of expression of the NK2R with and without fusion partner after codon replacement 99
8.4.8: Solubilisation trials of the TM-OmpA-NK2R inclusion bodies 100
8.4.9: The enterokinase cleavage of the fusion protein 101
8.4.10: His-pull down to purify the NK2R expressed in E.coli 102
CHAPTER 9: HETEROLOGOUS EXPRESSION IN E.COLI, PURIFICATION AND MASS SPECTROMETRY ANALYSIS OF HUMAN TACHYKININ NK2 RECEPTOR ABSTRACT 103
INTRODUCTION 104
MATERIALS AND METHODS 106
Materials 106
Construction of the recombinant expression vector 106
Exchange of the rare codons present in the hNK2R for expression in E.coli 107
Expression of the MBP-hNK2R in E.coli 110
Analysis of the expression of the MBP-hNK2R fusion protein in E.coli 111
Purification of the MBP-hNK2R fusion protein 111
Cleavage of the MBP-hNK2R fusion protein and isolation of the hNK2R 112 vi
In-gel digestion and mass spectroscopic analysis of the purified hNK2R 113
Radioligand binding assay to determine the functional activity of the hNK2R 113
Circular dichroism spectra of the purified hNK2R 114
RESULTS 115
Expression of the hNK2R as a fusion protein in E.coli 115
Purification of the hNK2R fusion protein 118
Cleavage of the MBP-hNK2R fusion protein and isolation of the hNK2R 119
Mass spectroscopic analysis of the purified hNK2R 120
Analysis of the activity and the secondary structure of the purified hNK2R 122
DISCUSSION 123
REFERENCES 126
SUMMARY 128
ZUSAMMENFASSUNG 133
REFERNCES 138
LIST OF PUBLICATIONS 149
ACKNOWLEDGEMENTS 150
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AC adenylyl cyclase
AcMNPV Autographa california multiple embedded nuclear polyhedrosis virus AOX1 alcohol Oxidase 1
BMGY buffered glycerol-complex medium BMMY buffered methanol-complex medium BSA bovine serum albumin
cAMP adenosine 3’,5’-cyclic monophosphate CD circular dichroism
CE crude extract CF cell-free
CMC critical micellar concentration CPM count per minute
D2 (L)R Dopamine 2 receptor – long form E. coli Escherichia coli
EDTA ethylenediaminetetraacetic acid FBS fetal bovine serum
GPCR G Protein Coupled Receptors hMOR human µ-opioid receptor
IPTG isopropyl-1-thio-β-D-galactopyranoside kDa kilo Dalton
LB Agar Luria-Bertani agar
LDAO N-lauroyl-N,N-dimethylammonium-N-oxide
LMPG 1-Myristoyl-2-Hydroxy-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (sodium salt) LTB4 Leukotriene B4 receptor
MBP maltose binding protein of E. coli Mdeg millidegrees
Ni-NTA Nickel-Nitrilotriacetic acid NK2R Tachykinin 2 Receptor NKA Neurokinin A
NKB Neurokinin B
OmpA outer membrane protein A from Escherichia coli viii
ORF open reading frame
PMSF phenylmethylsulfonyl fluoride RTS rapid translation system S2 cells Drosophila Schneider 2 cells SDS sodium dodecyl sulfate
SDS-PAGE SDS-polyacylamide gel electrophoresis Sf9 cells Spodoptera frugiperda cells
SP Substance P T4L T4 lysozyme TM transmembrane
TM-OmpA transmembrane domain of OmpA Tris tris(hydroxymethyl)aminomethane YPD yeast extract, peptone and dextrose
YPDS yeast extract, peptone, dextrose and Sorbitol β2AR β2-adrenergic receptor
λ wavelength
ix
Hereby I declare that all the experiments in this thesis are carried out by me and all the chapters of the thesis are written by me under the direct supervision of PD Dr. Jörg Kleinschmidt.
The exception to the above is:
The plasmids pcDNA3-D2(L)R and pcDNA3-NK2R were provided by our collaborator, Prof. John B.C. Findlay, University of Leeds, United Kingdom.
Vijay S.N. Narasimhan Konstanz, 09.2009
x
Genetic code and amino acids
2ND position 1st position
5’ end U C A G
3RD position 3’ end
U
Phe Ser Tyr Cys U
Phe Ser Tyr Cys C
Leu Ser STOP STOP A
Leu Ser STOP Trp G
C
Leu Pro His Arg U
Leu Pro His Arg C
Leu Pro Gln Arg A
Leu Pro Gln Arg G
A
Ile Thr Asn Ser U
Ile Thr Asn Ser C
Ile Thr Lys Arg A
Met Thr Lys Arg G
G
Val Ala Asp Gly U
Val Ala Asp Gly C
Val Ala Glu Gly A
Val Ala Glu Gly G
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xii
A Ala Alanine
C Cys Cysteine
D Asp Aspartic acid
E Glu Glutamic acid
F Phe Phenylalanine
G Gly Glycine
H His Histidine
I Ile lsoleucine
K Lys Lysine
L Leu Leucine
M Met Methionine
N Asn Asparagine
P Pro Proline
Q Gln Glutamine
R Arg Arginine
S Ser Serine
T Thr Threonine
V Val Valine
W Trp Tryptophan
Y Tyr Tyrosine
Introduction Chapter 1: Introduction
1.1: Introduction to Membrane Proteins
1.1.1: Biological membranes and membrane lipids
The cell is the structural and functional unit of all known living organisms. It is the smallest unit of an organism that is classified as living, and is often called the building block of life. The eukaryotic cell is highly organized into many functional units or organelles. The eukaryotic cells include a variety of membrane-bound structures, collectively referred to as the internal membrane system or endomembrane system. The endomembrane system compartmentalizes the cell for various different but interrelated cellular functions.
The cell/plasma membranes are the structuring elements of the cell and act as an interface between the cellular machinery inside the cell and the environment outside the cell. The plasma membrane is permeable to specific molecules and allows nutrients and other essential elements to enter the cell and waste materials to leave the cell. Small molecules, such as oxygen, carbon dioxide and water can pass freely across the membrane, but the passage of larger molecules, such as amino acids and sugars, are carefully regulated. The membranes allow selective receptivity and signal transduction by providing transmembrane receptors that bind the signaling molecules. In essence, membranes are essential for the integrity and function of the cell (Bruce Alberts May 2002).
The fluid-mosaic model (Singer and Nicolson 1972) encompasses our initial understanding of the membrane structure. The essential feature of the fluid-mosaic model is that biological membranes are considered to be quasi fluid structures in which the lipids and integral proteins are arranged in a mosaic manner. The Singer-Nicolson model has been refined over the years. Donald Engelman in 2005 suggested that membranes are more mosaic than fluid. According to this model, membranes are patchy with segregated regions of structure and function. The that lipid regions vary in thickness and composition, and that
1
crowding and ectodomains limit exposure of lipid to the adjacent aqueous regions (Engelman 2005) The figure 1.1 shows the Singer-Nicolson model of membrane and the amended version by Donald Engelman.
Figure 1.1: General models for the membrane structure
a: The Singer–Nicholson ‘fluid mosaic model’; b: An amended and updated version by Donald Engelman.
The plasma membrane that surrounds eukaryotic cells is a dynamic structure composed of two layers of lipid molecules interspersed with proteins. The lipids are amphiphatic in nature, and composed of a hydrophilic, head and two hydrophobic tails.
Hydrophobic tails form the hydrophobic core of the bilayer, while the hydrophilic head forms the polar/apolar interface at the membrane surface. There are different classes of
2
Introduction lipid molecules. The major lipids of eukaryotic plasma membrane are phospholipids, glycolipids and cholesterol (Figure 1.2).
The phospholipids are major component of all cell membranes. The phosphoglycerides are the most common phospholipids in the eukaryotic cell membranes.
Phosphoglycerides contains three essential parts: a backbone of glycerol, two long-chain fatty acids and a polar head group attached to a phosphate esterified to carbon 3 of the glycerol.
The glycolipids are lipids that are attached to a short carbohydrate chain (mono- or oligosaccharides). The role of glycolipids is to serve as markers for cell to cell communication and to provide energy.
The cholesterol is an important constituent of cell membranes; it has a rigid ring system and a short branched hydrocarbon tail. Cholesterol is largely hydrophobic, but it has one polar group and a hydroxyl making it amphiphatic. The cholesterol is an essential component of mammalian cell membranes where it is required to establish proper membrane permeability and fluidity.
Cholesterol has also been implicated in the cell signaling processes, assisting in the formation of lipid rafts in the plasma membrane. It also reduces the permeability of the plasma membrane to protons and sodium ions (Haines 2001).
Figure 1.2: Major lipids of the eukaryotic plasma membrane
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1.1.2: Membrane proteins
Proteins are organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in every process within the cells. Many proteins are enzymes that catalyze biochemical reactions and are vital to metabolism of the cells.
The cell membrane contains a large number of proteins that is responsible for its various activities. The amount of protein differs between species and according to function, however the typical amount of protein in a cell membrane is 50%. The main functions of the membrane proteins include cell-cell contact, surface recognition, cytoskeleton contact, signaling, enzymatic activity, or transporting substances across the membrane (Campbell et al. 2003).
The membrane proteins are classified into intrinsic membrane proteins and extrinsic membrane proteins or peripheral membrane proteins. The extrinsic membrane proteins are associated with the lipid bilayer of a cell or organelle. They are bound to the lipid bilayer by molecular attractions (ionic, hydrogen, and/or Van der Waals bonds). These proteins may interact with other proteins or directly with the lipid bilayer. In the latter case, they are then known as amphitropic proteins. Some proteins, such as G-proteins and certain protein kinases, interact with transmembrane proteins and the lipid bilayer simultaneously (Johnson and Cornell 1999).
The intrinsic or integral membrane proteins usually extend from one side of the membrane to the other and are also called as the transmembrane proteins. Based on the structure of the transmembrane domain, integral membrane proteins are subdivided into two major classes, α-helical and β-barrel transmembrane proteins (Figure 1.3).
4
Introduction
α-helical protein β-barrel protein Bacteriorhodopsin Outer membrane Protein G
Figure 1.3: Examples of the two classes of integral membrane proteins
The β-barrel proteins are so far found only in outer membranes of gram-negative bacteria, cell wall of gram-positive bacteria and outer membranes of mitochondria and chloroplasts. The β-barrel is composed of β-sheets connected by loops that twist and coils to form a closed structure in which the first strand is hydrogen bonded to the last strand.
The β-strands in β-barrels are typically arranged in an antiparallel fashion (Wimley 2003).
All known integral membrane proteins with transmembrane β-strands form barrel structures in which at least 8 neighboring β-strands are connected by hydrogen bonds. The outer membrane proteins of bacteria form transmembrane β-barrels with even numbers of β-strands ranging from 8 to 24 (Kleinschmidt 2005)
The α-helix is a common motif in the secondary structure of proteins. In an α-helix, the polypeptide chain is coiled tightly in the fashion of a spring. The "backbone" of the peptide forms the inner part of the coil while the side chains extend outward from the coil.
The amino acids in an α-helix are arranged in a right-handed helical structure, 5.4 Å (= 0.54 nm) wide. Each amino acid corresponds to a 100° turn in the helix (i.e., the helix has 3.6 residues per turn), and a translation of 1.5 Å (= 0.15 nm) along the helical axis.
5
Most importantly, the N-H group of an amino acid forms a hydrogen bond with the C=O group of the amino acid four residues earlier; this repeated hydrogen bonding defines an α-helix (Figure 1.4). Similar structures include the 310 helix (
hydrogen bonding) and the π-helix ( hydrogen bonding) (Pauling et al. 1951).
Hydrogen Bond
5.4 Å
Figure 1.4: A typical α-helix The figure shows a typical α-helical structure (see text for details)
The helices observed in α-helical proteins can range from four to over forty residues long, but a typical helix contains about ten amino acids (about three turns). The backbone hydrogen bonds of α-helices are generally considered slightly weaker than those found in β-sheets, and are readily attacked by the ambient water molecules. However, in more hydrophobic environments such as the plasma membrane, or in the presence of co-solvents such as trifluoroethanol, oligopeptides readily adopt stable α-helical structure (Hudgins and Jarrold 1999).
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Introduction 1.1.3: Membrane protein folding
A pressing challenge in modern biology is to understand how protein sequences encode biological activity. Since the first and fundamental task common to every protein sequence is to fold into its functional state, the prediction of a protein’s native conformation from its amino acid sequence is an important hurdle to be overcome. Solving this protein-folding problem will provide researchers with the expertise to predict the structure of the protein from sequence and structural changes in response to genetic mutations. And also helps to rationally engineer safe and effective pharmaceutical agents (Stanley and Fleming 2008).
Progress in understanding the folding of membrane proteins has lagged far behind that of soluble proteins, due to several reasons. The proteins that reside in biological membranes have very different surface properties to water-soluble proteins. The membrane proteins expose the hydrophobic surfaces to the membrane interior, whilst polar and charged amino acids lie on the protein exterior that interacts with membrane lipid headgroups and the aqueous regions at either side of a membrane. The proteins are also susceptible to the lateral forces and elastic properties of their surrounding lipid bilayer (Bezrukov 2000).
In vitro folding of membrane proteins require either detergents or lipids for functional refolding of the protein. Detergent micelles have been used extensively in the refolding of Bacteriorhodopsin (BR) [(Huang et al. 1981), (Renthal et al. 1990)], Outer membrane protein A (OmpA) (Kleinschmidt et al. 1999), OmpF (Surrey et al. 1996) and OmpG (Conlan and Bayley 2003). Surrey and Jähnig showed first that OmpA can spontaneously insert and fold into phospholipid bilayers (Surrey and Jahnig 1992). The refolding of the BR was successfully demonstrated using lipids extracted from Halobacteriurn halobium (Popot et al. 1987) and lipid-detergent mixture (London and Khorana 1982).
As proposed by Popot and Engelman, folding of many, perhaps most of the α-helical membrane protein can be considered as a two stage process: insertion and folding.
In the first stage, the individual stable helices are formed across the membrane lipid bilayer. This stage can be both directed and catalysed by a translocon complex, in contrast
7
to the fold of soluble proteins, which is normally defined entirely within the sequence itself (Bowie 2005).
In the second stage, the tertiary and quaternary structures are built. In this stage the helices interact with each other to form a higher ordered structure. The second stage involves assembly and reorientation of the TM segments established in the first phase (Popot and Engelman 1990) (Figure 1.5a).
Figure 1.5a: Two stage model for folding of α-helicalmembrane protein The figure was adapted from Bowie 2005 (see text for details)
The two stage model for folding of α-helical membrane protein was revised to a three stage model by Donald et al. in 2003. This model gives consideration to ligand binding, folding of the extramembranous loops, insertion of peripheral domains and the formation of quaternary structure (Donald et al. 2003). The figure 1.5b shows the three stage model for folding of two protein fragments of bacteriorhodopsin, comprising the helices 1-2 and 3-7.
In the first stage (a), the independent transmembrane helices are formed across the lipid bilayer. In the second stage (b), the helices interact with one another to form an ordered structure. In a possible third stage, these higher order structures can facilitate partitioning of additional polypeptide regions such as coil regions or short helices and/or prosthetic groups into the membrane.
8
Introduction
Figure 1.5b: Three stage model for folding of α-helical membrane protein
1.2: Introduction to the Guanine-nucleotide binding Protein Coupled Receptors (GPCRs)
Communication between cells is a fundamental aspect for multicellular organisms.
The majority of “signal molecules” such as hormones, bioactive chemicals and ions are unable to pass through the hydrophobic bilayer. Small molecules can pass through the membrane via transport mechanisms such as ion channels and transporters. Other larger signaling molecules transduce their intracellular response via membrane bound receptors.
There are three broad categories in which these receptors fall into: intrinsic enzyme activity
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(e.g. the insulin receptor), ligand-gated ion channels (e.g. the nicotine acetylcholine receptor) and G protein-coupled receptors (GPCRs) (e.g. adrenergic receptors).
The GPCRs comprise a large protein family of transmembrane receptors that sense molecules outside the cell and activate signal transduction pathways and, ultimately, cellular responses. The GPCRs are found only in eukaryotes, including yeast, plants, choanoflagellates, and animals (King et al. 2003). The ligands that bind and activate these receptors include light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters, and vary in size from small molecules to peptides to large proteins. The GPCRs are involved in many diseases, and are also the target of around half of all modern medicinal drugs (Filmore 2004). The GPCRs are involved in a wide variety of physiological processes. Some examples of their physiological roles include:
• the visual sense: the opsins use a photoisomerization reaction to translate electromagnetic radiation into cellular signals. Example : Rhodopsin
• the sense of smell: receptors of the olfactory epithelium bind odorants (olfactory receptors) and pheromones (vomeronasal receptors)
• behavioral and mood regulation: receptors in the mammalian brain bind several different neurotransmitters like serotonin, dopamine, GABA, and glutamate
• regulation of immune system activity and inflammation: chemokine receptors bind ligands that mediate intercellular communication between cells of the immune system; receptors such as histamine receptors bind inflammatory mediators and engage target cell types in the inflammatory response
• autonomic nervous system transmission: both the sympathetic and parasympathetic nervous systems are regulated by GPCR pathways, responsible for control of many automatic functions of the body such as blood pressure, heart rate, and digestive processes
1.3: Classification of GPCR families
The members of the GPCR superfamily have been subdivided into 5 subfamilies labeled A-E according to a 20% sequence similarity within the transmembrane helices (Table 1.1). All GPCRs have seven transmembrane α-helices and upon agonist stimulation
10
Introduction undergo a conformational change leading to an intracellular response. A new family of receptors has been discovered in mammals that are not part of the superfamily of the GPCRs but are thought to be related, these are known as the “frizzled/smoothened”
receptors. These “frizzled” proteins demonstrate a new membrane topology speculated to consist of 4 α-helices and 2 short β-strands and bind to wnt proteins (molecules involved in cell to cell signaling during embryogenesis) (Kristiansen 2004).
Family Sub families Receptors
Family A (Rhodopsin/β- Adrenergic like)
I Olfactory, adenosine, melanocortin and some orphan receptors
II Biogenic and amine (muscarinic, adrenergic, serotonin, etc) receptors
III Vertebrate opsins and neuropeptide receptors IV Invertebrate opsins
V Chemokine, chemotactic, somatostatin, opioids and others
VI Melatonin receptors and others not classified
Family B (Secretin-like)
I Calcitonin, calcitonin-like and calcitonin related factor receptors
II Parathyroid and parathyroid related peptide receptors
III Glucagon, secretin receptors and others IV Latrotoxin receptors and others
Family C (Metabotrophic
glutamate-like)
I Metabotrophic glutamate receptors
II Calcium receptors
III GABA-B receptors
IV Putative pheromone receptors Family D Ste2 and Ste3 pheromone receptors
Family E cAMP receptors
Table 1.1: GPCR families 11
1.4: GPCR signal transduction
The interaction of an agonist/antagonist with a cell surface receptor can lead to transmission of information across the cell membrane. The GPCRs transduce their signal response via their interactions with a heterotrimeric G protein complex that is composed of α, β, and γ subunits. Upon agonist binding to the GPCR, conformational changes within its structure are thought to confer the nucleotide exchange of the bound Gα-GTP subunit causing the concomitant dissociation of the Gβγ sub-complex and modulation of secondary effectors/messenger systems (Strader et al. 1994). Gα only re-associates with Gβγ once the GTPase activity in Gα subunit hydrolyses the GTP nucleotide to GDP (Figure 1.6). It is believed that a receptor molecule exists in a conformational equilibrium between active and inactive biophysical states (Rubenstein 1998).
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Gβγ activates intergran/soluble effectors
Free α-GTP stimulates effector
GDP-GTP exchange and dissociation of subunits
Gβγ reunites with α-GDP α-GDP
associates with Gβγ
Agonist/Antagonist
Hydrolysis of GTP inactivates α-subunit
R R*
Effector II
Effector I
GDP α
γ β
γ β GTP α
GDP GTP γ GDP α β
γ GDP α β
Figure 1.6: The GPCR signal transduction pathway
The figure represents an overview of the signal transduction pathway of GPCRs. R represents the GPCR and R*
represents the activated GPCR. (See text for details)
Introduction 1.5: Structure of GPCRs
The GPCRs share a common membrane topology, consisting of an extracellular N-terminal segment, seven transmembrane (TM) spanning helices, connected via three extracellular loops and three cytoloops, and a C-terminal segment. A fourth cytoplasmic loop is formed when the C-terminal segment is palmitoylated at cysteine residue. Each of the seven TMs is generally composed of 20-27 amino acids. On the other hand, N-terminal segments (7-595 amino acids), loops (5-230 amino acids), and C-terminal segments (12- 359 amino acids) vary in size, an indication of their diverse structures and functions.
1.5.1: Rhodopsin
Rhodopsin’s key function is photoreception and is the basis of the visual process in animals (Ovchinnikov Yu 1982). Upon absorption of light, 11-cis retinal attached to lysine 296 in TM 7 via a protonated Schiff’s-base linkage is isomerised to all-trans retinal.
All-trans retinal is detached from rhodopsin after hydrolysis of the protonated Schiff’s-base leading to photobleaching (Pepe 1999). Since rhodopsin is expressed in large quantities and is easily extracted it is highly amenable to biochemical and structural studies (Findlay and Pappin 1986). The X-ray and spectroscopic studies reveal that almost 50% of rhodopsin is buried in the membrane and is composed of mainly helical structure (Rothschild et al.
1980).
In 2000 Palczewski et al., solved the structure of bovine rhodopsin to 2.8 Å in the inactive state (Palczewski et al. 2000) (Figure 1.7). This is the first published GPCR structure, allowing structural predictions on other GPCRs based on a more accurate map.
The structure of rhodopsin has now been resolved further to a resolution of 2.2 Å (Okada et al. 2004). The new crystalline structure completely resolves the peptide sequence and provides further details into the chromophore binding pocket (Li et al. 2004). A comparison between the original low resolution structure and the more recent X-ray structure suggests that they are in broad agreement.
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Figure1.7: Ribbon drawings of the rhodopsin.
(A) Parallel to the membrane. View into the membrane plane from the (B) cytoplasmic side, (C) intradiscal side of the membrane. The figure is adapted from Palczewski et al., 2000.
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Introduction 1.5.1: β2-Adrenergic Receptor
The adrenergic receptors belong to class A of GPCRs and play a central role in mediating the effects of catecholamine hormones. In contrast to rhodopsin, which is expressed at very high levels in photoreceptor cells and has the ligand retinal covalently bound (Khorana 1992), other GPCRs such as the β2-adrenergic receptor (β2AR) are generally expressed at low levels, bind diffusible ligands, and exhibit greater functional and structural plasticity (Kobilka and Deupi 2007). The β2AR was the first non-rhodopsin GPCR to be cloned and has been one of the most extensively studied members of this large receptor family.
However, significant evidence of sequence homology was detected between rhodopsin and the mammalian β-adrenergic receptor. This suggested the β-adrenergic receptor shares a similar 7 TM spanning helical structure to that of the rhodopsin (Dixon et al. 1986) (Dohlman et al. 1987).
In 2007 Rosenbaum et al., published the structure human β2-adrenergic receptor (β2AR) to 2.4 Å which was engineered to replace the intracellular loop 3 with T4 lysozyme (T4L) (Figure 1.8). The optimized β2AR-T4L protein was crystallized in lipidic cubic phase, and the resulting 2.4 Å resolution crystal structure reveals the interface between the receptor and the ligand carazolol, a partial inverse agonist (Rosenbaum et al. 2007)
Figure 1.8 Stereoview of the overall fold of the β2AR-T4L The receptor and T4L are colored gray and green, respectively.
Carazolol is shown in blue; the lipid molecules bound to the receptor are in yellow. Figure is adapted from Rosenbaum et al., 2007
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The final model of β2AR-T4L includes 442 amino acids. This model also includes a palmitic acid, an acetamide molecule as well as one carazolol molecule, three cholesterol molecules, two sulfate ions, and two butanediol molecules that interact with β2AR. The β2AR has a fold composed of seven transmembrane helices forming a helical bundle. The residues that make up the helices (I to VII) in β2AR are as follows: helix I, positions 29 to 60; helix II, positions 67 to 96; helix III, positions 103 to 136; helix IV, positions 147 to 171; helix V, positions 197 to 229; helix VI, positions 267 to 298; and helix VII, positions 305 to 328. The residues that make up the intra cellular loop (ICL) 3 are positions 230 to 266 (residues 231 to 262 are replaced by T4L residues 2 to 161). The helices II, V, VI, and VII each have a proline-induced kink at conserved positions along the span of the transmembrane segments. These kinks are thought to enable the structural rearrangements required for activation of the G protein effectors (Cherezov et al. 2007).
In the β2AR-T4L construct, the T4L is fused to the truncated cytoplasmic ends of helices V and VI. In the crystal structure, the T4L moiety is tilted slightly away from the center axis of β2AR drawn normal to the membrane (Figure 1.9). As a result, the interactions between T4L and β2AR are minimal, with only 400 Å2 of surface area buried between them.
Figure 1.9 Intramolecular interactions between the β2AR and T4L
T4L is fused internally into the third intracellular loop of the β2AR and maintains minimal intramolecular packing interactions by tilting away from the receptor. See text for details. The figure is adapted from Cherezov, et al, 2007
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Introduction 1.6: Over-expression of GPCRs for refolding and structural studies
G protein-coupled receptors (GPCRs) are targets for 60–70% of drugs in development today. Traditionally, the drug discovery process has relied on screening of chemical compounds to identify novel and more-efficient drug molecules. The structure- based drug design, however, provides a targeted approach but has been severely hampered by limited knowledge of high resolution structures of the GPCRs owing to the difficulties encountered in their expression, purification and crystallization (Lundstrom 2005).
GPCRs, like other membrane proteins, undergo a complex and poorly understood folding and membrane-insertion mechanism. The unique properties of a cell environment that may facilitate heterologous expression of the GPCR include the specific lipid composition of the various membrane compartments, cell-type specific chaperones and unique post-translational modifications including defined glycosylation, phosphorylation, sulphonation and other covalent modification (Mancia and Hendrickson 2007).
1.6.1: Bottlenecks in the overexpression of GPCRs
A wide variety of physiological functions are regulated by the GPCRs: examples are metabolism, neurotransmission, visual perception, and immune response. The GPCRs therefore, represent major targets for drug development. Greater than 60% of the current pharmaceutical drugs exert therapeutic effects by targeting the GPCRs, generating $50 billion in pharmaceutical sales per year. The overall market of drugs that target GPCRs has been declining since the 1980s when early success in random drug screening started waning (Saunders 2005). Interestingly, less than 10% of all known GPCRs are targeted;
therefore, much effort is placed in understanding GPCRs for future drug discovery (Wise et al. 2002). Knowledge on this important class of integral membrane proteins is hindered by lack of structural and functional data. The two available techniques for obtaining high- resolution 3D structures, NMR and X-ray crystallography, require relatively large amounts (milligrams) of purified proteins. Furthermore, obtaining such quantities of GPCRs in a functional, ligand-binding state is even more challenging.
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A major limitation in characterizing GPCRs has been the difficulty in developing high-level heterologous expression systems. Even in their native cells these proteins are expressed at low levels, with rhodopsin being the lone exception. Native bovine rhodopsin expression levels have enabled a crystal structure of the receptor in its resting state (Palczewski et al. 2000) and of the photoactivated deprotonated intermediate state recently (Salom et al. 2006) .
The recombinant production of the GPCRs is still a matter of trial and error, taking up precious time that could be spent on the inevitable challenges of extraction, purification, and crystallization trials. Why is it that heterologous expression of GPCRs remains so difficult and unpredictable? The folding of GPCR and stability when expressed in their native system is quite complex and not yet fully understood. Once these proteins are expressed they must be recognized by the ER translocon and inserted/folded correctly, and then the GPCR-containing vesicle must transverse the Golgi apparatus to fuse with the plasma membrane. Although the steps involved in insertion and folding of the GPCRs is not well understood, it has been shown that proper orientation of the extracellular loops in class 1 GPCRs are maintained by a crucial disulfide bond between the extracellular loops 1 and 2. This highly conserved feature has been shown to be critical for proper folding and cell surface localization (Fu-Yue Zeng 1999). Multiple quality control systems and molecular chaperones for correct folding and transport are likely to be necessary along the way. These factors are difficult to control in heterologous systems, and therefore less systematic trial and error approaches are often used in the expression of GPCRs. The lack of information on these proteins and the probability that they are very diverse in their individual needs has hindered any rational approach to heterologous expression (McCusker et al. 2007).
Currently, laboratories approach GPCR overexpression on a case-by-case basis by testing receptor/host combination until suitable expression is achieved. In the case where little to no expression is observed after exhaustive experimentation, that GPCR is abandoned for a different, more easily expressed GPCR. As it would be expected, eukaryotic hosts are generally better for their production. However, the same few GPCRs frequently arise as high expressing proteins in these systems, thereby neglecting ~ 90% of GPCRs for in vitro study.
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Introduction 1.6.2: Expression systems employed for the overexpression GPCRs
The GPCRs have been successfully expressed in a multitude of hosts, ranging from bacteria to mammalian cells. In this context, every available expression system has been tested (Table 1.2) (Lundstrom 2005).
Expression
system GPCR Advantages Disadvantages References Escherichia coli Adenosine A2a,
Neurotensin,
Leukotriene receptor BLT1
Fast, easy, safe, Scaleable, High
expression in inclusion bodies
Lack of post- translational modifications, membrane toxicity, low yields, fusion protein required Refolding required
(Luca et al. 2003)
(Baneres et al. 2003) Halobacterium
salinarium
Rhodopsin,yeast a-factor,
serotonin 5-HT2c
Fast,
colorimetric assay, scaleable
Cloning, transformation complicated, fusion protein required
(Turner et al. 1999) Saccharomyces
cerevisiae
Yeast a-factor, dopamine D1A
Relatively easy, scaleable
Hyper
glycosylation,
thick cell wall (David et al. 1997) Schizosaccharo
myces pombe
Dopamine D2 Relatively easy, scaleable
Non-
mammalian glycosylation
(Sander et al. 1994) Pichia pastoris β2-adrenergic
and other GPCRs
Relatively easy
High biomass
Thick cell wall
(Weiss et al. 1998) Baculovirus Neurokinin-1
β2-adrenergic
Mammalian- like
production
Slow virus
stock (Mazina et al. 1994) (Guan et al. 1992) Transient
mammalian Several GPCRs e.g. 5-HT1E
Mammalian Transfection methods to be established for each GPCR
(McAllister et al.
1992)
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Stable
mammalian Rhodopsin,
Several GPCRs Inducible Stability
problems (Reeves et al. 2002) Viral, SFV Neurokinin-1 Broad host
range, Extreme expression, Scaleable
Relative expensive , Safety issues
(Kenneth Lundström 1994)
Cell-free translation
β2-adrenergic Simple, fast Very low yields
(Ishihara et al. 2005) Table 1.2: Expression systems employed for the in vitro expression of the GPCR
In addition to E. coli, other prokaryotes such as Halobacterium salinarium and Lactococcus lactis have been used as hosts for the recombinant protein expression.
Generally, bacterial expression has been hampered by the relatively low yields of GPCRs owing to the toxic effects caused by these 7TM receptors when inserted into bacterial membranes. By contrast, when GPCRs are directed to bacterial inclusion bodies the highly expressed recombinant receptors are inactive and require refolding. The expression in E.
coli using a Maltose Binding Protein (MBP) fusion vector has proved successful for many GPCRs. In 2007 Rosenbaum et al., solved the structure of the human β2-adrenergic receptor (β2AR) to 2.4 Å. β2AR was expressed as a fusion protein with MBP in E. coli. The human neurotensin type 1 receptor (White et al. 2004), the human central CB1 cannabinoid receptors (Calandra et al. 1997) and the peripheral CB2 cannabinoid receptors (Alexei A.
Yeliseev 2005) were successfully expressed as MBP fusion proteins.
Several yeast strains viz., Saccharomyces cerevisiae. Schizosaccharomyces pombe Pichia pastoris have been investigated for the expression of various GPCRs. The expression of GPCRs has also frequently been conducted in insect cells, particularly using baculovirus vectors. The yields of the human neurokinin-1 receptor were in the milligram range with a specific binding of 60 pmol/mg protein (Mazina et al. 1994). The mammalian expression systems have also been applied for GPCRs expression, however the yields from stable cell lines were relatively modest (Hermans 2004).
Protein production by the cell-free (CF) expression techniques does not depend on cellular integrity and could therefore provide a general advantage for the synthesis of problematic membrane proteins (Klammt et al. 2006). Various GPCRs like the human
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Introduction β2-adrenergic receptor (Ishihara et al. 2005), the human melatonin 1B receptor, the human endothelin B receptor, the human and porcine vasopressin type 2 receptors and the human neuropeptide Y4 receptor were successfully expressed using the cell-free expression system (Klammt et al. 2007).
1.7: Refolding of GPCRs
Most of the biophysical studies on α-helical membrane protein folding have been carried out with bacteriorhodopsin as a model system, since it can be purified in high yields and is relatively stable in solution (Lanyi and Luecke 2001). As is the case with GPCRs, it possesses seven transmembrane α-helical segments. Although it may not be a full representative of GPCRs at the folding level, it is nevertheless a good model system to gain a better understanding of receptor folding (Klein-Seetharaman 2005).
The human leukotriene B4 (LTB4) receptor BLT1 was refolded on column by Baneres et.al in 2003. The BLT1 was expressed in E.coli as inclusion bodies, the denatured protein was refolded on a Ni-NTA column in the presence of LDAO, and the secondary structure was characterized using circular dichroism (CD) spectroscopy (Baneres et al.
2003). Similarly, the Serotonin 5-HT4 receptor was refolded in the presence of 1%
dimyristoylphosphatidylcholine (DMPC), 1% CHAPS, and 0.02% cholesteryl hemisuccinateand characterized by CD spectroscopy and ligand binding studies (Baneres et al. 2005).
The refolding of fragments containing some of the transmembrane helices of GPCRs has been studied. These fragments range from a single transmembrane domain to several transmembrane helices. As predicted by the two-stage model for membrane protein refolding (Popot and Engelman 1990), these fragments form folded domains when transferred from denaturing conditions to a milder environment. This suggests that GPCR transmembrane helices can also be considered as individual folding units. Besides their interest for a better understanding of the molecular processes involved in the receptor folding, these studies indicate that the study of receptor fragments could be an alternative to the analysis of the structural properties of the GPCRs. H.G. Khorana in 1984 demonstrated the regeneration of native bacteriorhodopsin structure from fragments in phospholipid
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vesicles (Liao et al. 1984). The transmembrane helices two and three of µ-opioid receptor was successfully refolded in lipid vesicles (Kerman and Ananthanarayanan 2005).
Similarly, synthetic peptides corresponding to the seven transmembrane helices of human adenosine A2a receptor were successfully refolded in lipid vesicles as individual helices.(Lazarova et al. 2004)
1.8: The Dopamine receptors
The dopamine is an important neurotransmitter in the brain. The dopamine is involved in a variety of functions including, motor control, emotion, cognition and endocrine regulation. The dopamine also plays a role in the peripheral nervous system controlling cardiovascular function, vascular tone, hormone secretion, catecholamine release and gastrointestinal motility (Missale et al. 1998).
The disregulation of the dopaminergic transmission system has been linked to a range of pathological conditions including, Parkinson’s disease, schizophrenia, Tourettte’s syndrome and hyperlactinemia. The dopamine receptor antagonists have been used to reduce hallucinations in patients suffering from schizophrenia, whilst agonists are used to alleviate hypokinesia in Parkinson’s disease patients.
The first evidence for the existence of the dopamine receptors in the Central Nervous System came in 1972 from biochemical studies showing that dopamine was able to stimulate adenylyl cyclase (AC). In 1978, dopamine receptors were first proposed, on the basis of pharmacological and biochemical evidence, to exist as two discrete populations, one positively coupled to AC and the other one independent of the adenosine 3’,5’-cyclic monophosphate (cAMP)-generating system (P. F. Spano 1978)
1.8.1: The D-1, D-2 like Receptors
Currently five dopamine receptors have been cloned. These distinct dopamine receptors have been characterized according to their biochemical and pharmacological properties. The receptors are divided into two classes, D-1 like and D-2 like, according to their ability to stimulate/inhibit the cAMP pathway (Kebabian and Calne 1979). Overall the
22
Introduction amino acid sequence identity between the receptors is 35%, however when split into the sub type groups this identity is increased to 75% for D-1 like and 52% for D-2 like receptors (Civelli et al. 1993b).
The D-1 like receptor family consists of the “classical” D1 (also known as D1A) receptor and the D5 (D1B) receptor. The genes coding for these receptors are intronless, a characteristic shared by most GPCRs (MacKenzie et al. 1993). The D1 and D5 receptors share an overall 62% sequence similarity and 78% between the transmembrane regions (Gingrich and Caron 1993). These receptors are likely membrane anchored by palmitoylation of a conserved cysteine residue in the long carboxy-terminus. The D-1 like receptors specifically couple to Gαs and stimulate adenylate cyclase activity resulting in an increase in cAMP (Missale et al. 1998).
The D-2 like sub type consists of D2, D3 and D4 receptors and unlike most GPCRs (rhodopsin is a notable exception) these receptor genes have introns (Gingrich and Caron 1993). The first D-2 like gene to be identified was the D2 receptor and the gene has been mapped to chromosome 11 (Civelli et al. 1993b), (Jaber et al. 1996). The D3 receptor gene has been mapped to chromosome 5 in humans (Missale et al. 1998). The D4 receptor shares overall 75% identity and overall 53% identity within the transmembrane regions to the D2 receptor. Its gene locus is at the tip of the short arm of chromosome 11 (Gelernter et al. 1992). Like the D-1 subtype counterparts the D-2 like family have a putative palmitoylation site located on the short carboxy terminus at the terminal cysteine residue.
The receptors within this sub-family inhibit the activity of adenylate cyclase although this still has to be proven for D4 (Civelli et al. 1993b).
1.9: The dopamine 2 receptor (D2R)
There has been great interest in the D2sub type as it is a major dopamine receptor in the brain and is the prime target for antiparkinsonian and antipsychotic drugs. So far, most antipsychotic drugs were developed as antagonists to the D2 receptor. This would then imply that most neuroleptics have a higher affinity to the D2 receptors in the brain (Civelli et al. 1993b). The figure 1.10 shows the membrane topography of the D2 dopamine receptor.
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Figure 1.10 Membrane topography of the human dopamine 2 receptor
Introduction 1.9.1: Isoforms of the D2R
The D2 dopamine receptor has two isoforms derived from the same gene through alternative mRNA splicing (Giros et al. 1989). The splice site occurs in the putative intracellular loop 3 involving an 87-base pair exon resulting in the addition of 29 amino acids. The resulting polypeptide chains are 415 and 444 amino acids long and termed as the D2 short (D2S) and D2 long (D2L) receptors respectively (Monsma et al. 1989b). The 29 amino acid insertion contains two potential glycosylation sites although the physiological importance is unknown since these regions are cytosolically located (Civelli et al. 1993a).
The mRNA distributions of the two forms of D2 receptor are not species or tissue specific, but D2L appears to be the major isoform (Monsma et al. 1989a).
The two isoforms of the D2 receptor share similar pharmacological profiles and both lower the intracellular levels of cAMP (Guiramand et al. 1995). However, a distinction between their intracellular responses and coupling to specific G proteins may exist. The D2L knockout mice indicated a preferential role for the D2L receptor in postsynaptic responses such as D1/D2 coupling, whilst the D2S appears to negatively regulate D1
responses in the postsynapse (Usiello et al. 2000). Furthermore, the two isoforms regulate different phosphotransmitters involved in the dopaminergic transmission (Lindgren et al.
2003). These two receptors also have preferential coupling to different G proteins, the D2L
receptor preferentially coupling to Gαi2 (Montmayeur et al. 1993).
1.9.2: Ligand binding site of the D2R
The ligand binding site of the D2 dopamine receptor is contained within a water accessible crevice formed by the seven TM segments. Using the substituted cysteine accessibility method (SCAM) with methanethiosulfonate (MTS) reagents the surface of the binding site crevice of the D2 receptor was mapped (Shi et al. 2001).
The residues in TM 1 were found to be accessible to water and were not protected by binding antagonist against MTS reagents and therefore, do not seem to contribute to the binding-site crevice. It has been suggested that Aspartate 80 in TM 2, which is highly
25
