CHARACTERIZATION OF NOVEL G PROTEIN - T-Space

110 Pages·2000·5.27 MB·English

CHARACTERIZATION OF NOVEL G PROTEIN-COUPLED
RECEPTOR GENES AND THE NOVEL LIGAND APELIN
by
Dennis K. Lee
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Graduate Department of Pharmacology
University of Toronto
O Copyright by Dennis K. Lee 1999
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Characterization of Novel G Protein-Coupleci Receptor Genes
and the Novel Ligand Apelin
Dennis K. Lee, MeSc. 1999
Department of Pharmacology
University of Toronto
G protein-coupled receptors (GPCRs) make up the large family of integral
membrane bound proteins which mediate the signaling of extracellular stimuli to
intracelMar responses via activation of heterotrimeric G proteins and their subsequent
interaction with effector proteins. GPCRs have been implicated in a number of
physiologicd Functions including behaviour. homeostasis, cognition, appetite, and drug
addiction. This thesis describes the molecular characterization of the human and rat
genes encoding apelin, the cognate ligand for the APJ receptor. Human and rat DNA
sequences encoding apelin were retrieved by a search of the GenbanP databases, their
full length sequences cloned and used as probes for expression analyses. Comparative
sequence and tissue distribution analyses revealed both apelin and the APJ receptor to
resemble the angiotensin II peptide and the angiotensin Il receptors, suggesting roles in
similar physiological systems. In addition, a degenerate PCR strategy, database
searching and the patent literature revealed DNA sequences encoding novel GPCRs,
namely the thyrotropin-releasing hormone receptor TRH-R2,o rphan GPCRs GPR54,
GPR57, GPR58, GPR6 1, GPR62 and a pseudogene, vGPR57. With the initiai discovery
of each of these sequences, full length GPCR-encoding sequences were detennined and
used for mRNA distribution analyses by northern blot and in situ hybridization. In
addition, novel GPCR-encoding genes were localized to chromosomes by fluorescence
in situ h ybridization (FISH) and expressed for pharmacological characterization.
ACKNOWLEDGEMENTS
1 would Iike to thank my supervisors, Dr. Brian F. O'Dowd and Dr. Susan R.
George, for their support and guidance and for the opportunity to pursue my degree under
their direction. 1 would also like to express my gratitude to Tuan Nguyen for his work on
cloning rat apelin cDNA, TRH-R2, GPR54 and yGPR57, Regina Cheng for her work on
the northern blot and in situ hybridization anaIyses, and Yang Liu for her work on
isolating and sequencing the GPCR search clones- I would also thank Dr- Adriano
Marchese, Marek Sawzdargo, Ziedong Xie, Teresa Fan and the members of the O'Dowd
and George lab, both past and present, for their generous and amiable counsel, technical
support and our collaborative efforts at work and play. In addition, 1 wish to thank Brett
Clayton and acknowledge his excellent work on the three-dimensional schematic of the
GPR54 receptor. Finally, 1 would Iike to thank my family and friends for their love and
support, in hopes that 1 have returned both in kind-
PUBLICATIONS
This thesis contains work that has been published in or submitted to the scientific
literature:
A ~ e ahnd APJ
Lee, D.K., Cheng, Ra,N guyen, T., Fan, T., Kariyawasam, A.P., Liu, Y., Osmond, D.H.,
George, S.R., and O'Dowd, B.F. "Charactenzation of apelin, the ligand for the APJ
receptor", J. Neurochem. In press.
TRH-R2
O'Dowd, B.F., Lee, D.K., Huang, W., Nguyen, T.. Cheng, R., Liu, Y., Wang, B.,
Gershengorn, M C , and George, S.R. "TRH-R2 exhibits simiiztr binding and acute
signaling but distinct replation and anatomic distribution compared to TRH-RI". Mol.
Endocrin. In press.
GPR54
Lee, D.K.. Nguyen, T., O'Neill, G.P., Cheng, R., Liu, Y., Howard, A. D., Coulombe, N.,
Tan, C.P., Tang-Nguyen, A.-T., George, S.R., and O'Dowd, B. F. "Discovery of a
receptor related to the galanin receptors". FEBS Lett. (1999) 446, 103- 107.
vGPR57. GPR57. GPR58
Lee, D.K., Lynch, KR., Nguyen, T., Xie, Z., Cheng, R., Saldivia, V.R., Liu, Y., Liu.
I.S.C.. Heng, H.H.Q.,S eeman, P., George, S.R., O'Dowd, B.F. and Marchese. A.
"Cloning and characterization of additional members of the G protein-coupled receptor
farnily". Submitted.
OTHER PUBLICATIONS
Lee, D.K., Nguyen, T., Porter, C.A., Cheng, R., George, S.R., and O'Dowd, B.F. 'TWO
related G protein-coupled recepton: The distribution of GPR7 in rat brain and the
absence of GPR8 in redents". Brain Res. Mol. Brain Res- (1999) 71,96403.
Sawzdargo, M., Nguyen, T., Lee, D.K., Lynch, KR., Cheng, R.. Heng, H.H.Q., George,
S.R.. and O'Dowd, B.F. "Identification and cloning of three novel human G protein-
coupled receptor genes GPR52, yGPR53 and GPR55: GPR55 is extensively expressed
in human brain". Brain Res. Mol. Brain Res. (1999) 64, 193-198.
TABLE OF CONTENTS
PAGE
TITLE PAGE I
ABSTRACT II
ACKNOWLEDGEMENTS III
PUBLICATIONS IV
v
TABLE OF CONTENTS
LIST OF ABBREVIATIONS IX
LIST OF TABLES X
LIST OF FIGURES XI
1.0 INTRODUCTION
Overview of Introduction 1
The GPCR family 2
Agonists for GPCRs 5
Discovery of GPCR Genes by Molecular Cloning 6
Discovery of GPCR Genes by Database Searches 13
Reverse Pharmacology: Assigning Ligands to Novel GPCRs 16
Researc h Objectives 22
2.0 MATERIALS AND METHODS
2.1 Materials
2.1.1 Chernical Reagents
2.1.2 Enzymes
2.1.3 Isotopes and Ligands
2.1.4 Oiigonucieotides
2.1.5 Kits
2.1.6 CeU lines, Plamnids and DNA Libraries
2.2 Methods
Computationd DNA and Protein Sequence Andysis
PCR: Polymerase Chain Reaction
GenbankTMD atabase Searches
Subcloning of PCR Products
DNA Minipreparation, Restriction Digestion and
Electrophoresis
DNA Sequencing
DNA Probe Extraction and Radiolabelhg by Nick
Translation
Genomic and cDNA Library Screening
Bacteriophage DNA Preparation
2.2.10 Southern Blotting
2.2.11 Northern Blotting
2.2.12 In Situ Hybridization
2.2.13 Chromosomal localization
2.2.14 Creation of an Intronless GPR58 Receptor Gene
Expression Construct
2.2.15 Maxi DNA Preparation
2.2.16 Calcium Phosphate TC811Sfection
2.2.17 Membrane Preparation and Binding Studies
3.0 RESULTS
Determination of the Human Apelin Genomic Structure
and Cloning of Rat Apeiin cDNA
mRNA Tissue Distribution of Apelin and the APJ Receptor
Discovery and Cloning of the TRH-R2 Receptor Gene
mRNA Tissue Distribution of the TRH-R2 Receptor
Discovery and Cloning of the GPR54 Receptor
mRNA Tissue Distribution of the GPR54 Receptor
Discovery and Cloning of the GPR57 and GPR58 Receptor
Genes and a Pseudogene F R 5 7
GPR57 and GPRSS Expression
Attempted Pharmacological Characterization of the
GPR58 Receptor
Chromosomal Locaîization of the GPR58 Receptor Genes and 74
Pseudogene yGPR57
Discovery and Cloning of the GPR61 and GPR62 Receptor 74
Genes
mRNA Tissue Distribution of the GPR61 Receptor 79
4.0 DISCUSSION
4.1 Apelin: Characterization of the Endogenous Peptide 80
Ligand for the APJ Receptor
4.2 TRH-R2: Discovery and Characterization of a Second 82
GPCR for Thyrotropin-Releasing Hormone
4.3 GPR54: Discovery and Characterization of a NoveI GPCR 85
related to the Gala& Receptors
4.4 GPR57 and GPR58: Discovery of a Novel Subfamily of 86
GPCRs
4.5 GPR61 and GPR62: Discovery of a Novel S u b f d yo f 88
GPCRs
4.6 Conclusions 89
5.0 REFERENCES 91
LIST OF ABBREVIATIONS
ATP adenosine triphosphate
BLAST basic local aiignment search tool
base pairs
bp
cDNA complementary DNA
CHO chinese hamster ovary
DAPI 4' '6'-diamidino-2-pheny lindole
DNA deoxyribonucleic acid
EST expressed sequence tag
FISH fluorescence in situ hybridization
GPCR(s) G protein-coupled receptor(s)
G protein guanine nucleotide regulatory protein
GU(s) G protein-coupled receptor kinase
GSS genomic survey sequences
hr hours
HTGS high throughput genomic sequence
MAGE Integrated Molecular Analysis of Gene Expression
IPTG isopropylthio-B-D-galactosidase
kb kilobase pairs
LB Luria-Bertani media
min minutes
mRNA messenger RNA
NCB 1 Nationai Center for Biotechnology Information
nr non-redundant
oGPCR orphan G protein-coupled receptor
ORFW open reading frame(s)
PCR polymerase chain reaction
PKA CAMP-dependentp rotein kinase A
PKC protein kinase C
RNA ribonucleic acid
P m revolutions per minute
SDS sodium dodecyl sulfate
sec seconds
STS sequence tagged sites
TM transmembrane domain
T m thyrotropin-releasing hormone
UTR(s ) untranslated region(s)
x-gal 5-bromo-4-chloro-3-indolyl-B-D-galactosidase