Protease-Activated Receptors (PARs) belong to an unusual family of G Protein Coupled Receptors (GPCRs). Each of the four known members is activated by its own N-terminus exposed by proteolytic cleavage and there is no other endogenous agonist known to date. PAR2 is the second member of the family and it has been implicated in wide range of pathophysiological conditions, particularly in various inflammatory diseases and cancers. In contrast, very little is known about the PAR2 receptor itself despite having been discovered more than 10 years ago. The purpose of this project was to improve our understanding of PAR2 regulation by discovering new agonists and antagonists and using them to probe the structural and functional properties of the receptor.
Chapter 1 provides a brief literature overview of the initial discovery of PAR2, what is known about the mechanism of receptor activation, information on the structures and properties of current agonists and an antagonist for PAR2, and the putative physiological roles of human PAR2. As well, it summarizes the aims of this thesis.
Chapter 2 investigates the regulation of gene expression by two different agonists of PAR2, a synthetic hexapeptide, 2f-LIGRLO-NH2, and the endogenous activator, trypsin, the idea being that genes up- or down- regulated by both agonists may more accurately profile PAR2-selective events. The effects of PAR2 activation on gene transcription in the human kidney HEK293 cell line were studied using a DNA microarray consisting of 19,000 human genes in an attempt to broadly cover the human genome and associated cell pathways with PAR2 activation. About 2,500 genes were regulated similarly by both agonists and, for genes expressed more than 5-fold, the mRNA results were further analyzed by quantitative RT-PCR techniques. PAR2 activation was shown to be associated with cellular metabolism, cell cycle, mitogen-activated protein kinase pathways, histone deacetylase and sirtuin enzymes, inflammatory cytokines and anti-complement function.
Chapter 3 described a range of molecular events surrounding the activation of the receptor. PAR2 mRNA expression was quantitated by qRT-PCR and cross-checked with an intracellular Ca2+ assay. In this way whole cell PAR2 could be correlated with cell surface expression of PAR2. Three cell lines expressing high levels of PAR2 were chosen for subsequent experiments, these being colorectal carcinoma HT29, lung carcinoma A549 and human embryonic kidney HEK293 cells. Receptor activation, internalization, desensitization and resensitization assays were carried out on these cell lines to define some key functions relevant for investigating inhibitors in subsequent chapters.
Chapter 4 reports a PAR2 mutagenesis study designed to identify the location of the binding site on PAR2 for a specific peptide agonist. A homology model of PAR2 based on bovine rhodopsin was used for docking of an agonist ligand, and the docking results were then investigated via two successive rounds of PAR2 mutagenesis in which the effect of each mutation (20 in all) was separately investigated by changes in agonist potency in the intracellular calcium release assay. Five PAR2 mutants showed more than a 5-fold reduction in agonist potency, while three others showed up to a 7-fold reduction. Mutations found to be important for agonist activity were mapped back to the model. Because there was extensive clustering of these key mutated amino acids, it is likely that this study has pinpointed the precise binding site of the agonist peptide in PAR2. Interestingly, this site is within the transmembrane region of the receptor.
Chapter 5 reports the design, discovery and development of novel PAR2 agonists and antagonists and their regulatory effects in a diverse array of cell types. Structure-activity relationships were used to examine influences on the first, sixth and seventh positions of a PAR2 agonist peptide. At least five compounds were found herein to be equiopotent with the most potent PAR2 agonist reported. Knowledge obtained from this study was then used to create the first non-peptidic agonists for PAR2. The most potent nonpeptidic agonist (retaining one natural amino acid) was at least equipotent with the best peptide agonists. Conversion to nonpeptidic antagonists proved to be successful and this chapter reports the most potent known nonpeptide antagonist, which was selective for PAR2 and active at low micromolar concentrations. It inhibited intracellular Ca2+ release induced by different PAR2 agonists (trypsin, 2f-LIGRLO-NH2, nonpeptide agonists) in multiple cell lines (HT29, Panc-1, A549, MKN1, MKN45, MDA-MB-231, HUVEC) that have been physiologically associated with PAR2. It also inhibited release of inflammatory cytokines IL-8 and IL-6 and shows antiproliferative activity against primary human cells. The antagonist is competitive, reversible and surmountable (pA2 6.11).
This thesis summarizes a large body of work that provides valuable molecular insights to PAR2 regulation, and lays the groundwork for rational design and development of novel nonpeptidic agonists and antagonists of PAR2 as potentially valuable pharmacological probes in vivo and as useful leads to development of therapeutics for inflammatory diseases and cancers.