Towards the Structures of the Parathyroid Hormone Receptors

G protein-coupled receptors (GPCRs) are a superfamily of membrane proteins that
transduce extracellular signals to invoke physiological responses. Class B GPCRs are
regulated by peptide hormones and are characterised by a seven transmembrane
domain, a 120-150 residue extracellular domain (ECD), and an intracellular C-terminus.
The parathyroid hormone 1/2 receptors (PTH1R/PTH2R) belong to this family and, despite
being closely related, have different physiological roles: PTH1R is crucial in bone metabolism,
whereas PTH2R function is significantly less well defined.

The original aims of this research were to solve the structures of these two receptors.
This involved creating various receptor constructs that would be suitable candidates
for downstream structural trials. Insect cell expressed receptors were quantified
through fluorescent intensity densitometry analysis and a potential candidate for
further structural studies was found in the form of a mini Gs protein bound to an
apocytochrome, b562RIL (BRIL) fusion protein following a FLAG purification.
However, a constitutively active receptor, created by tethering PTH(1-14) to BRIL-PTH1R
and verified through LANCE® cAMP assays, was unable to form this complex.
PTH2R proved a major challenge, so attempts to solve the isolated ECD were made
instead. This was pursued using Escherichia coli expressed protein in various
oxidising environments, though due to low protein yields and poor stability it was
impossible to obtain a candidate for further trials.

Finally, the novel integral membrane protein stability selector (IMPROvER) program
was used to create a thermostable PTH1R. Through fluorescent based thermostability
assays, 40% of 20 mutations were stabilising with a maximum increase of 3.4 ± 1.1 °C.
Combinations of these mutations resulted in a construct with an improved stability of
10 ± 0.76 °C. This research gives a strong foundation for future structural work
regarding PTH1R and has highlighted that IMPROvER is a valid alternative to more
laborious methods such as alanine scanning.