Mammalian reproduction is driven by gonadotrophin -releasing hormone (GnRH) - a
decapeptide released from hypothalamic neurones into the pituitary portal blood
vessels. The aim of this thesis was to study structure- function relationships of the Gprotein coupled GnRH receptor (GnRH -R), and has focused on the identification of
key amino acids involved in GnRH ligand- receptor interactions as well as the role of
putative disulphide bridge formation within the receptor itself. In addition, the role
of disulphide bridge formation has also been explored in another G- protein coupled
receptor (GPCR), the thyrotrophin- releasing hormone receptor (TRH -R).
Comparative sequence analysis and computer molecular modelling approaches were
used to target potentially important amino acids for site -directed mutagenesis study.
Wild -type and receptor mutants were then expressed in mammalian cells, and
receptor binding, expression, and activational properties compared between
constructs.
The majority of GPCRs contain two conserved extracellular Cys residues which
have been postulated to form a covalently linked disulphide bridge structure. In the
GnRH -R, these Cys residues are positioned at Cys 114 and Cys 195 in the first and
second extracellular loops respectively. In addition, the GnRH -R contains two non - conserved Cys residues at Cys 14 in the amino terminus and Cys 199 in the second
extracellular loop. Substitution of these Cys residues to serine resulted in a loss of
ligand binding. A comparative study in the TRH -R, substituting the conserved
extracellular Cys residues, Cys98 and Cys 179, to either serine or alanine, confirmed
these findings. This data suggests that extracellular Cys residues, through putative
disulphide bridge formation, may maintain the tertiary extracellular structure of the
receptor and therefore facilitate ligand- receptor binding. Further studies, using
chemical modifying reagents, have indicated that Cys residues with free sulfhydryl
groups may also be important in TRH-R binding.
The GnRH -R despite its structural homology to other GPCRs exhibits some unique
features. These differences include the interchange of a highly conserved Asp and
Asn residue in the transmembrane (TM) domains. Individual substitutions of Asn87
(in TM II) to Asp87 and Asp318 (in TM VII) to Asn318, revealed that Asn87 is
important for GnRH agonist and antagonist binding whereas Asp318 is important for
receptor activation. To investigate if the function as well as the position of these
amino acids were transposed, a double mutation substituting both residues
simultaneously was generated. However, this mutant receptor showed only a small
degree of GnRH agonist binding, indicating that the functional role of these specific
residues is not interchangeable.
Amongst GPCRs, the GnRH -R is particularly suitable for three dimensional
molecular modelling and computer aided simulations because of its short
extracellular and intracellular domains. Using this approach, it has been possible to
predict putative amino acids involved in ligand- receptor interactions. During this
study, GnRH molecular models have evolved from a template predicted by the
Baldwin model to a series of energy minimised computer generated three
dimensional structures. To simulate GnRH ligand- receptor interactions, a model of
the native GnRH peptide has also been constructed. The initial Baldwin model
highlighted a series of TM located polar amino acids in TM II, TM III, TM IV and
TM VII of the GnRH -R. Both the position and nature of these amino acids rendered
them capable of interacting with the GnRH ligand. Mutations at these sites
identified two residues, His305 located at the TM VII/extracellular interface and
Asn314 in TM VII, that were potentially important for GnRH -R binding.
Further modelling studies, using GnRH ligand- receptor computer simulations,
predicted that amino acids Phe312 in TM VII and Leu83 in TM II may interact with
Trp3 and Leu7 in the GnRH ligand respectively. Substituting these amino acids to
residues of either similar, different or neutral hydropathicity, showed that a
hydrophobic amino acid was essential for GnRH -R binding at position 312 and for
receptor activation at residue 83. Altogether, 15 sites within the GnRH -R have been
experimentally modified and the information derived from site -directed mutagenesis
studies has been utilised to redefine the structure of the molecular models.
In conclusion, the formation of a putative disulphide bond between extracellular
cysteine residues in both the GnRH -R and TRH -R is important in maintaining
tertiary protein structure. In addition, amino acids located in TM II and TM VII are
essential for binding interactions between GnRH and its receptor. Analysis of
structure -function relationships, particularly using this dual biochemical and
molecular modelling approach, will greatly facilitate rational drug design. In the
light of the enormous clinical applications of GnRH and its analogues, information
regarding the mechanisms of hormone -receptor interactions will be of benefit in the
development of new and novel drugs for clinical use in reproductive medicine.
