An investigation into the conformational changes and lipid dependence of NTS1 activation by its agonist

Many of our neurological functions are controlled through receptor proteins (called GPCRs) residing in the brain, and for this reason it has been estimated that about 30% of all drugs we use act on these receptors, of which there are more than 800. The functions controlled by GPCRs are numerous, and one receptor may be involved in many responses. We need to find out how these receptors work, and it has been said that this is the major challenge of current structural biology (Lagerstrom & Schioth, 2008, Nature Reviews Drug Discovery, 7, 339-57).

To help in that process of understanding, and as a result of recent breakthroughs, it is now possible to make a very limited number of GPCRs so that we can start to discover how they function. The one we have elected to work on is involved in pain management, neuromuscular diseases such as Parkinson s and the control of appetite, and hence obesity control. We have been able to express this mammalian brain receptor in simple E. coli bacterial cells, and it is functionally active to make any of the work we do relevant to its function in the brain. As with many of these receptors, they are activated by the binding of small molecules, and although we can monitor this binding, the important aspect is to understand and investigate how the protein is then activated and how it sends its signal to other proteins and then ultimately the cell.

We will, therefore, express and purify the receptor, activate it with hormone, and then use state-of-the-art methods to probe the receptor changes during the activation process. These methods can be used to measure distances at the nanoscale (0.5-8nm +/- 0.01nm) in these nanodetectors , as well as the time scale (in microseconds - nanoseconds) for the activation process. Since this protein normally sits in a membrane of the brain cell, it needs some of the lipid components of the membrane to function properly, and we will also investigate which of these components are required. Finally, if time permits, we will determine how the activated receptor passes the signal to the next component in the chain of signalling molecules, to unravel a little more about this vital signalling mechanism. All the information from this cutting edge project will add to our general understanding of brain signal transmission, and help in future drug design and disease control.

The G-protein coupled receptor (GPCR), rat brain neurotensin receptor type I (NTS1) is one of a small number of GPCRs that have been successfully expressed in E.coli as a functional, ligand-binding receptor. The NTS1 receptor has been expressed and purified for lipid-protein reconstitutions by detergent removal at a range of molar ratios and characterized with density gradient centrifugation and ligand binding capacity.

Nitroxide spin-labels will be placed at either single sites or double cys sites on the receptor and ligand binding monitored. ESR spectra from labelled proteins are sensitive to backbone motions. Helical and beta-sheet sites having characteristic and more restricted motions than loop sites as a result of the differential anisotropy averaging of the nitroxide hyperfine interactions on a timescale (1/tau ~ (Azz - Axx,yy) ~ microsec - ns) which is relevant to backbone motions for isotropic proteins in solution such as a detergent solubilized receptor (r ~ 4nm). For membrane-embedded receptor, the spectra are additionally sensitive to probe motion due to being buried or accessible. Using this data, homology modeling of NTS1 will be based on the recently reported adrenergic receptors.

Doubly labeled NTS1 will then be studied to resolve the conformational changes in NTS1 on activation, by placing labels at the ends of helices to report on helical movements, as shown for rhodopsin on light activation. Distances of 1-8nm can be measured, and some indication of which helices are involved in activation of the receptor will be determined. About 8-10 such samples will be required, and a triangulation approach used to show how NTS1 is activated.

Finally, we will study the lipid dependence of NTS1 ligand binding and activation since we know that it is active in brain lipids, but not in any single component lipid studied. The possibility that cholesterol has a binding site will also be explored.