Adhesion-GPCRs – GPCR proteolytic site GTP – Guanosine

Adhesion-GPCRs are a mysterious sub-family of G protein-coupled receptors with a
distinctive structure. The large N’ terminal fragment (NTF), composed of multiple adhesive
domains, associates non-covalently with the seven transmembrane domain and intracellular
C’ terminal. This heterodimeric conformation arises from post-translational autocatalytic
cleavage at the GPCR proteolytic site, a motif located within a highly conserved domain
upstream of the transmembrane domain.

Mutated Adhesion-GPCRs are implicated in numerous diseases including Usher’s
syndrome, and bilateral frontoparietal polymicrogyria, yet remain largely uncharacterised.
Despite being recently proven to function as classical GPCRs, a definitive mechanism of
signal transduction remains ambiguous. Studies indicate that NTF removal exposes a
tethered agonist which then interacts with the TM domain, causing receptor activation.
However the process by which fragment removal occurs in vivo is unclear. Using atomic
force microscopy we aim to define the force required to cause NTF removal and determine
its physiological reproducibility in the instance of ligand binding, providing a paradigm for
signalling activation. Additionally solving the crystal structure of the extracellular domain of
these receptors will provide insight into how this receptor family interacts with external
stimuli, and provide a basis for future drug design via high-throughput molecule screening.

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List of Abbreviations

BAI1 – Brain angiogenesis inhibitor 1
BGH – Bovine growth hormone
CL1 – CIRL/Latrophilin 1
CTF – C’ terminal fragment

DMEM – Dulbecco’s modified eagle medium
EGF-TM7 – Epidermal-growth factor seven transmembrane
EO-PCR – Extension overlap polymerase chain reaction
GAIN – GPCR autoproteolytic inducing
GDP – Guanosine diphosphate
GPCR – G protein-coupled receptor
GPS – GPCR proteolytic site
GTP – Guanosine triphosphate
HEK – Human embryonic kidney
His-tag – Histidine tag
NTF – N’ terminal fragment
Opti-MEM – Reduced serum minimal essential medium
PKD – Polycystic kidney disease
PC – Polycystin protein
SDS – Sodium dodecyl sulphate
SDS-PAGE – SDS-polyacrylamide gel electrophoresis
TM7 – Seven transmembrane
VLGR1 – Very large G-protein coupled receptor 1

Acknowledgements

I’d like to thank my supervisor Martin Stacey and co-supervisor Chi Trinh for continuing to
impart their knowledge on to me and for support throughout the project. I would also like to
thank Tom for his help with my many obvious questions. This project is co-supervised and
co-funded by GlaxoSmithKline.

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1. Introduction
1.1 G Protein-Coupled Receptors
As the largest receptor gene family in the human genome with over 800 members, the
transmembrane signal transducing G protein-coupled receptors (GPCRs) play critical roles
in the nearly all conceivable physiological processes; ranging from the sensing of photons
and odorants to metabolic homeostasis and migration of leukocytes. Based upon the
proposed GRAFS classification system (Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2,
Secretin), GPCRs are grouped in to five distinct phylogenetic subfamilies(1). Although all
members share a characteristic seven transmembrane (TM7) topology, GPCRs exhibit little
sequence homology. Their diverse sequences are reflected by a large repertoire of ligands
which include monoamines, neurotransmitters, chemokines, and other naturally occurring
molecules such as calcium ions (2).

Despite their diversity the majority of GPCRs are believed to signal though a similar
mechanism; that is the transduction of external stimuli into intracellular secondary
messengers via associated heterotrimeric G protein complexes (3). These complexes
consist of ?, ? and ? subunits that prior to GPCR-ligand binding, exist in an inactive GDP
bound state. However upon ligand binding conformational changes through the seven
transmembrane helices of the receptor facilitate the GDP-GTP exchange within the ?-
subunit resulting in the dissociation from the ?? complex (4). The ? and ?? subunits are
then able to regulate various effectors leading to the production of downstream secondary
messengers that can cause rapid changes in cellular phenotype, or the activation of various
transcription factors and concomitant alterations in gene expression. Specificity of signalling
is mediated in part by the regulated expression of numerous types of ? and ?? subunits
which engage different effectors and through the regulation of their activity. Their activity is
controlled by numerous protein families including, G protein exchange factors (GEFs) and
Regulators of G-protein Signalling, (RGS) which enhance ?-subunit GDP-GTP exchange
and GTPase activity, activating and deactivating G protein signalling, respectively. Further
regulation of signalling is mediated via receptor desensitisation by GPCR kinases (GRKs)
and arrestin proteins, which again possess regulated and restricted expression profiles.

GPCRs are expressed in most physiological systems, influencing gene transcription, cell
survival, and growth and motility in the nervous, endocrine, immune, cardiovascular and
reproductive systems. Both inherited and acquired mutations of GPCRs have been
documented for numerous human diseases, including hyperparathyroidism, in which the
parathyroid glands produce excess parathyroid hormone; retinitis pigmentosa, a
degenerative eye disease; and various forms of cancer. Being active in a variety of systems

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makes the GPCR superfamily desirable drug targets. Currently, drugs targeted to GPCRs
account for approximately 40% of those licensed, and they remain a large research focus in
the pharmaceutical industry (5). However, only a small number of receptors are
therapeutically exploited and much remains to be understood about the activity and targeting
of non-rhodopsin classes of GPCRs. .

As with other transmembrane proteins, GPCRs are notoriously difficult to crystallise.
However in recent years, an increasing number of GPCRs in the rhodopsin class have been
solved, providing mechanistic insights into the conformational changes within the TM7
domain upon ligand interaction with the extracellular domain. The structural and signalling
knowledge has allowed for the development of high throughput compound screens and
identification of potential small molecule inhibitors for therapeutic use. It has also enabled
the identification of residues, implicated in known disease states associated with GPCRs.

Figure 1. A schematic representation of the downstream effects of ligand binding the
extracellular domain of GPCRs. Ligand interaction causes conformational changes of the TM7
domain, activating the receptor and allowing the interaction with the ?-subunit of the heterotrimeric G-
protein. This results in the activation of the G-protein’s GTPase activity, facilitating the cycling of GDP
to GTP, and the separation of the ?- and ??-subunits.

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1.2 Adhesion-GPCRs

Adhesion-GPCRs form the second largest subgroup of GPCRs in humans (6) behind the
extensive Rhodopsin family and consist of 33 proteins (7) which can be further divided into 9
groups (Figure 2). Like the others in the superfamily, Adhesion-GPCRs are broadly
expressed across all human tissues, however individual members or subgroups may be
restricted to tissues/cell types including gastrointestinal and reproductive tracts, the central
nervous system, and the immune system. Although certain members have been detailed in
fundamental processes, the group currently remain poorly understood and under
researched. The majority of members remain orphaned with no known binding partners.

The adhesion class are distinguishable from other GPCRs by their hybrid molecular
structure. The extracellular region is often unusually large, composed of an array of modular
protein domains known to facilitate cellular migration and protein-protein interactions (Figure
2) including epidermal growth factor (EGF)-like repeats, immunoglobulin (Ig) domains,
leucine rich repeats (LRRs), and cadherin repeats (8). Further to this, glycosylation sites are
present which are hypothesised to facilitate interactions with extracellular ligands and
therefore mediate changes in response to the external environment (9). Adhesion-GPCRs
share the characteristic GPCR TM7 domain, and an intracellular C-terminal which,
demonstrated by increasing evidence, provides the interface at which the receptor interacts
with and activates effector G-proteins (10)(11).