Do biased agonists at the mu-opioid receptor induce different patterns of receptor phosphorylation?

The mu-opioid receptor (MOPr) is a protein receptor found in the membrane of certain nerve cells in the brain and spinal cord. It interacts with (i.e. is receptive to) chemicals such as endorphins and enkephalins in the body causing a reduction in the electrical excitability of the nerve cells. In the whole organism, this leads to a number of outcomes, most notably the relief of pain, although MOPr also controls a number of other processes including mood and emotion, secretions in the stomach and intestine, the rate of breathing, and body temperature. The MOPr is therefore a very important receptor and consequently over the years many chemical compounds (ligands) have been made that can interact with this receptor. Although many of these ligands seem to produce similar effects when they interact with MOPr, we have recently found that some ligands interact with the receptor to produce significantly different effects from others. This can be seen as one ligand strongly activating one particular intracellular signaling pathway rather than another, whereas another ligand might activate the latter pathway more strongly. This phenomenon has been called 'biased agonism', but as yet the mechanisms involved in producing biased agonism are not known. We think that what happens is that the different ligands are able to bind to the MOPr and make the receptor take up different shapes. This not only makes the MOPr susceptible to a chemical process called phosphorylation, but the phosphorylation can take place on different parts of the MOPr, depending upon which ligand it is interacting with. Phosphorylation is where chemical groups called phosphate groups are added to a protein, and phosphorylation normally leads to the protein changing its function in some way, or the rate at which it does something. Phosphorylation of MOPr leads to it becoming less active in some regards, but more active in others. We think that different MOPr ligands make it become phosphorylated in different ways and this leads to distinct cell signaling outputs. This is what we want to measure in this proposal - we want to investigate whether different ligands phosphorylate MOPr in different ways, and whether this leads to different signaling and hence different effects in the organism. Knowledge gained from this work will allow us regard receptors such as MOPr in a completely new way, meaning that we could make ligands that bind to the same receptor (MOPr), but can lead to different effects in the whole organism. In addition, this phemomenon is likely to occur for many of the other types of related receptor (>800 in the mammalian genome) and so the MOPr can be used as an excellent model system to test out our hypothesis. The major aims of this work are therefore to: (1) identify exactly which parts of MOPr are phosphorylated when it interacts with different ligands (2) use this knowledge to make antibodies that will recognize the phosphorylated MOPr and use them to see how different ligands might produce different phosphorylation events on the receptor (3) investigate using cell imaging technology whether there is a correlation between the amount and type of phosphorylation of MOPr and where the MOPr is located in a nerve cell (4) investigate whether and how MOPr in brain is phosphorylated (5) identify the different cellular signals that biased ligands at MOPr might produce

The mu-opioid receptor (MOPr) is a GPCR found throughout the CNS and plays a crucial role in the regulation of a diverse range of physiological processes. Importantly, we have recently obtained evidence for biased agonism at MOPr, in that different ligands for MOPr possess distinct abilities to activate G protein signaling relative to the recruitment of arrestins to the receptor. Since GPCR phosphorylation is now known to play a pivotal role in both the desensitization of G protein-dependent signaling, as well as in the activation of arrestin-dependent signaling, it is logical to hypothesize that the ability of ligands with variable bias at MOPr to differentially activate signaling or regulatory pathways is dependent upon ligand-specific phosphorylation of the receptor. In this proposal we will determine whether biased and unbiased ligands at MOPr induce different patterns of receptor phosphorylation, by quantifying the agonist-induced phosphorylation of all phosphoacceptor residues in the intracellular regions of the intact MOPr. The major objectives of this proposal will be to: (1) use state-of-the-art mass spectrometry to identify the amino acid residues in the intracellular regions of MOPr that are phosphorylated in response to receptor occupation by a range of chemically diverse MOPr agonists displaying varying degrees of bias between G protein signaling and arrestin recruitment (2) use both quantifiable mass spectrometry and antiphosphoreceptor antibodies to quantify the kinetics and agonist concentration-dependence of phosphorylation induced by ligands of varying bias at individual residues (3) determine the relationship between the subcellular localization of MOPr in a neurone and its phosphorylation status, and to investigate the phosphorylation status of MOPr in CNS tissue (4) determine the role of phosphorylation/non-visual arrestin recruitment in MOPr signaling and regulation induced by ligands displaying differential bias

Who will benefit and how? The Post Doctoral Research Assistant, who will be equipped with new skills and so will be able to provide essential experience for research or related jobs in academia, education, healthcare, or industry. The immediate beneficiaries will thus be the wider UK and international academic communities, public and private education, the healthcare sector and industry. Public health and wealth creation. The findings of this work will be of great interest to the Pharmaceutical industry, a very important component of the UK economy. On the one hand the industry will be interested in the basic ideas about receptor theory, how receptors work, and the nature of biased agonism. This can inform them in their search for new drug targets as well as new ways in which to analyse drug action in their search. Opioid drugs are extremely important in the treatment of pain in animals and humans, whilst the abuse of opioid drugs has major health, welfare and economic consequences for the UK. Greater knowledge about these drugs and the way in which they interact with their receptor will also help to inform future public policy. The public. In more general terms our work is of benefit to the UK in terms of our commitment to public engagement. This includes speaking in schools, and serving as scientific advisors on public bodies. Participation in these activities helps to ensure our knowledge and influence passes beyond the laboratories and walls of the University. Communications and Engagement: New findings will be disseminated both through peer-reviewed publications and by presentations at international scientific meetings. Particularly noteworthy discoveries will be highlighted and presented in a form accessible to a lay audience via the University of Bristol and School of Physiology and Pharmacology web pages. Collaboration: The academic collaboration outlined in the proposal will be coordinated mainly by the Principal Investigator, with the Post Doctoral Research Assistant also taking responsibility for maintaining and developing these interactions. Importantly, the Kelly and Henderson laboratories have a number of other collaborations with researchers interested in understanding the molecular details of GPCR function or MOPr function in particular. It is likely that the research that these groups undertake will be directly affected by the knowledge and understanding obtained from the present application. For example, we have an ongoing collaboration with Dr William Dewey at Virginia Commonwealth University, Richmond, USA, to look at the role of protein kinase C in tolerance to opioid agonists. He will clearly be very interested in determining the effects of biased opioid ligands in model systems to measure analgesic tolerance in rodents. Also, we have collaborated with Dr Steve Charlton, Director of Receptor Biology at Novartis, Horsham in our initial studies to characterise biased agonism at MOPr (McPherson et al Mol Pharm 2010), and hope to continue doing so in relation to G protein/arrestin bias, possibly in relation to other physiologically relevant GPCRs. Clearly with relation to receptor theory the work in our present proposal is of great interest to his group and will help to inform some of his future directions. Exploitation and Application: In the longer term we anticipate potential impacts in the areas of receptor theory and drug discovery, as detailed earlier. Capability: The Investigators will assume overall responsibility for undertaking and delivering the impact activities, although it is expected that the Post Doctoral Research Assistant will make a very significant contribution, as discussed above. Resources: No additional resources required.