Molecular Pharmacology and Structure Function Modelling of the Leukotriene B4 Receptor BLT1

Abstract: Leukotrienes are membrane derived bioactive lipids that play an important role in immune responses by initiating and maintaining the inflammatory responses. Leukotriene B4 (LTB4) released at the site of an inflammatory response attracts, activates and prolongs the life of leukocytes and lymphocytes. LTB4 can bind to two recently cloned cell surface G-protein coupled receptors named BLT1 and BLT2. This thesis focuses on BLT1 and presents the original discovery of the receptor, the development of monoclonal antibodies against the receptor, an investigation of the signal transduction pathways activated by the receptor, and finally, a three-dimensional computer model of the receptor in an active conformation. Using degenerate primers and a homologous screening strategy, a B-cell lymphoblast cDNA library was examined, and a novel receptor was discovered. This thesis presents subsequent experiments involving radioligand binding, assays for intracellular calcium release, and antibodies that identified this protein as a receptor for LTB4. Given the importance of this receptor for inflammation we sought to produce monoclonal antibodies that could be used to easily identify tissues and cells expressing this receptor. Two antibodies were selected based on their superior utility in a number of common immunohistochemical techniques and pharmacological characterisation of these antibodies is presented. Both antibodies could inhibit a variety of functional responses in both cells that endogenously expressed BLT1 and cells that had been transfected with BLT1. Only one of the antibodies (7B1) antagonised the binding of LTB4 however. The other antibody (14F11) was a non-competitive antagonist. Both antibodies were found to require the complex epitopes represented by the tertiary structure of the receptor. The signal transduction mechanisms of BLT1 were examined with a focus on intra-cellular calcium measured in transfected HeLa cells. HeLa cells were used because they are compatible with BLT1 and, unlike most leukocytes, do not express BLT2. BLT1 induces an increase in intracellular calcium concentrations ([Ca2+]i) through both PTX sensitive and insensitive G-proteins. Calcium is initially released from internal stores followed by an influx of extracellular calcium through store-operated channels. This function of the receptor was not sensitive to the redox state of the extracellular environment, but it was sensitive to treatments that modulated membrane cholesterol levels, or membrane physiology. A number of different kinases were also found to have an effect on BLT1 induced changes in [Ca2+]i including protein kinase C (PKC), protein kinase A (PKA) and protein-tyrosine kinases (PTKs). Inhibiting MAP kinases, Rho-associated kinases, or phosphoinositol-3-kinases (PI3K) had no effect on BLT1 induced calcium signalling. In order to explore how LTB4 binds to BLT1 a three-dimensional computer model was created based on a theoretical model of the active conformation of BLT1. LTB4 was then docked into the receptor’s ligand binding pocket and amino acids that could interact with LTB4 were identified. Several receptors with point mutations of key amino acids were then produced and examined using functional and ligand binding experiments to confirm the ligand-receptor interactions suggested by the theoretical studies.