Protease-activated receptor 2 (PAR2) is a G-protein coupled receptor that is unusual in being activated by proteases. Enzymes such as trypsin, tryptase and factor VIIa cleave the extracellular N-terminal domain of PAR2 at a specific site to expose a new N-terminal “tethered ligand”. This tethered ligand can self-bind to PAR2, stimulating downstream intracellular signalling and functions. Synthetic peptides and small molecules have been designed as activators (agonists) or inhibitors (antagonists) of PAR2 by mimicking or modifying tethered ligand sequences. PAR2 is important in a variety of physiological functions and diseases, including inflammatory conditions, cancers, obesity, and central nervous system disorders. However, there is no effective, potent agonist or antagonist for use in vivo. This thesis aims to characterize cell signalling and functional profiles of new PAR2 agonists/antagonists, and facilitate a better understanding of the roles of PAR2 in physiology and disease. Chapter 1 succinctly reviews PAR2 discovery, structure, activation, signalling and functions as well as known agonists and antagonists. It systemically summarizes PAR2 activation and signalling, including canonical, non-canonical and biased activation, PAR2 involvement in a variety of physiological functions and diseases, and proteases, agonists, and antagonists known to date to modulate PAR2. Chapter 2 investigates ligand structures for potent and selective PAR2 activation, towards a better understanding of biased signalling and functions. Structure-activity relationships for ligands that act on PAR2 are reported for two different signalling pathways, calcium and ERK1/2 signalling, leading to a comparison of three PAR2 biased agonists. One calcium-biased agonist DF253 (2f-LAAAAI-NH2) induced PAR2-mediated calcium release without ERK1/2 phosphorylation in CHO cells transfected with hPAR2 (CHO-hPAR2 cells). Compound AY77 (Isox-Cha-Chg-NH2) was a more potent calcium-biased agonist, but did stimulate weak ERK1/2 activation. A third compound AY254 (Isox-Cha-Chg-A-R-NH2) was biased towards ERK1/2 signalling, triggering more ERK1/2 phosphorylation than calcium activation. This signalling bias translated into different functions in human colorectal carcinoma cells (HT-29), where AY254 but not DF253 or AY77 induced three different ERK1/2 mediated functions. These findings suggest that biased ligands can be used as novel tools for selectively regulating PAR2 signalling functions. Chapter 3 characterizes properties of a potent PAR2 antagonist, the imidazopyridazine I-191, which inhibited multiple PAR2-dependent signalling functions in cancer cells. I-191 was shown for the first time to bind to PAR2, as deduced by competition with a europium-labelled PAR2 agonist on CHO-hPAR2 cells. It also inhibited PAR2-mediated Ca2+release, ERK1/2 phosphorylation, RhoA activation and cAMP accumulation. Importantly, I-191 did not show any agonist activity over a wide concentration range. This antagonist was a negative allosteric modulator of agonist 2f-LIGRL-NH2in Ca2+assays, acting in an insurmountable and non-competitive manner. It also potently attenuated PAR2-mediated cytokine expression and secretion, cell apoptosis and migration in human colon (HT-29) and breast (MDA-MB-231) cancer cells. These results indicate that I-191 is a potent PAR2 antagonist that inhibits multiple PAR2-induced signalling pathways and functions in cancer cells. Chapter 4 explores signalling profiles of four new compounds (AZ3451, AZ8838, AZ2623, AZ0107). All four compounds were found to bind to PAR2 and were all antagonists of Ca2+and ERK signalling. The benzimidazole, AZ3451 and AZ2623, were more potent than imidazoles, AZ0107 and AZ8838, in antagonizing calcium release and ERK1/2 phosphorylation induced by PAR2 agonist, 2f-LIGRL-NH2. AZ0107 and AZ8838 were weak inhibitors of trypsin-induced PAR2 activation, but AZ3451 and AZ2623 did not inhibit. While AZ3451 and AZ8838 were negative allosteric modulators that acted in a non-competitive and insurmountable manner, their signalling patterns were dependent on the agonist used and the signalling pathway measured. AZ3451 and AZ8838 showed little cooperativity in calcium and ERK1/2 signalling, whereas there was a positive cooperativity between AZ3451 and I-191 in both pathways, suggesting a potential for combination therapy. This chapter provides a better pharmacological understanding of PAR2 allosterism. Chapter 5 probes the role and mechanism of action of PAR2 in ovarian cancer cells. PAR2 is overexpressed in human ovarian tumour tissues and cancer cell lines. A PAR2 protease (trypsin), a synthetic peptide (2f-LIGRL-NH2) and an antagonist (I-191) were used to investigate PAR2 in OV90 cells representative of high-grade serous ovarian cancer versus KOC7C cells from a clear cell ovarian cancer. PAR2 was found to stimulate calcium and ERK1/2 signalling in these cells. Specifically, PAR2-mediated ERK1/2 signalling promoted expression of transcription factor genes (FOS, MYC, STAT3) and COX-2 (PTGS2), and consequently stimulated PGE2 secretion. The PAR2-MEK-ERK1/2-transcription factors-COX-2-PGE2 signalling axis operated through transactivating epidermal growth factor receptor (EGFR) via β-arrestins/Src/MMPs, leading to ovarian cancer cell migration and invasion in vitro. Chapter 6 summarizes key findings from this thesis and provides suggestions for future work. The thesis contributes to a better understanding of PAR2 signalling and functions, identified new PAR2 biased agonists, allosteric antagonists, and new roles for PAR2 in cancer. It provides a platform for future development of PAR2 based drugs and further understanding of PAR2 functions in physiology and disease.