Stannard, Corinne; (2003) An investigation of modification to endothelin receptors A and B, and downstream proteins, using proteomic techniques. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes the investigation of post-translational modifications to human endothelin (ET) receptors A and B occurring on binding of endothelin-1, and the changes in protein phosphorylation downstream of the receptors that result from ligand binding. Preliminary experiments confirmed the expression of endothelin receptors A and B by our cell system and gathered some basic kinetic data. A synthetic ligand ((dA)30-5'-S-EMC-ET) was successfully produced, and we were able to use it simultaneously as a stimulus and as an affinity-purification 'fish-hook'. A calcium imaging study showed the validity of the fish-hook as a stimulus. Differences in phosphorylation and palmitoylation were observed between ETA and ETB receptors, and between unstimulated controls and cells that had been stimulated with (dA)30-5'-S-EMC-ET. These observations are of particular interest in the context of differences in the intracellular fates of the receptors, and in their G-protein coupling. The number of post-translational modifications identified on both receptors was much larger than expected, and was shown to exist in several different combinations. The identification of several concurrent patterns of modification to the receptors is potentially representative of a great diversity of receptor species. This may be relevant to the observation that receptors of the same type do not always couple to the same G-protein, and is indicative of the complexity of receptor post-translational modification in living cells. Phosphorylation and dephosphorylation of a number of proteins downstream of the receptors was demonstrated on stimulation with ET-1. Protein identity was determined by mass fingerprinting and confirmed where necessary by partial nanospray sequencing. In particular, changes in the phosphorylation of several proteins involved in cell growth and division were identified. A new model for the action of ET-1 to promote cell cycle progression is proposed, and may offer an insight into the mechanism by which ET-1 exerts its mitogenic effect.

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