Understanding the functional activation of G protein-coupled receptors (GPCRs) in the context of their lipid bilayer environment

Human beings are made up of millions of cells. To sustain life these cells need to be able to work together. Each cell has its own machinery and is surrounded by a water-impermeable membrane forming a physical boundary. This membrane consists of a lipid bilayer into which a large number of water-insoluble proteins are embedded, so-called membrane proteins. These are essential for transmitting required nutrients, energy and information across the membrane. To work in a coordinated fashion, cells need to be able to adjust themselves to changes in their surroundings. This requires the ability to communicate environmental variations across the membrane bilayer. A large family of ca. 800 membrane embedded proteins is tasked to do this. These so-called G protein-coupled receptors (GPCRs) have the ability to sense the presence of a wide range of extracellular stimuli in the form of chemicals, peptides and proteins, for example odorants, pheromones, neurotransmitters, hormones, light (amongst others) and to communicate their presence to the cell interior. As cellular sensors, GPCRs are key players in the regulation of a wide range of normal, physiological and disease-related processes. Located on the cell surface they are already targeted by many of the currently available drugs. However, there is vast potential to develop this further in order to tackle many more diseases or improve existing treatments, with the promise to lead to dramatic improvements in health in the future.

To achieve this, there is an urgent need to obtain a better understanding of how GPCRs function. Typically this involves obtaining information on these proteins at a molecular level, and chemists and biologists are using a range of sophisticated methodologies that generate such insight. For an increasing number of these GPCRs it has recently become possible to visualize the structural aspects of these highly unstable and difficult to handle proteins in the form of static snapshot pictures. Based on these, one would assume that GPCRs function as simple on/off switches. However, GPCRs are highly mobile, shape-shifting proteins and this trademark characteristic lies at the heart of their function. Accordingly, many questions remain to be answered as the simple on/off picture is gradually replaced by one portraying GPCRs as rheostats i.e. continuous regulators.

In our proposal we will investigate the dynamic nature of these receptors using a particular GPCR called b1 adrenergic receptor (b1AR). This receptor plays an important role in the regulation of heart function, is involved in many diseases and is targeted by the famous beta blocker drugs. To understand the role of the dynamic nature for GPCR function we will mimic the natural cellular membrane environment and embed the receptor in small particles that resemble lipid bilayer rafts. We will then use a technique called nuclear magnetic resonance (NMR) spectroscopy to investigate how the shape-shifting properties of these proteins contribute to their function. Using such small membrane bilayer particles will allow us to study b1AR under realistic conditions and to focus on the role of the lipid environment for GPCR function. NMR spectroscopy will give us insight on how this receptor interacts with a range of proteins that couple to the receptor and how the initial signal sensed by the GPCR is transmitted from the cell exterior across the membrane to the inside of the cell. We will be able to study regions of the receptor that for technical reasons are inaccessible to other investigation methods, which is particularly valuable. Our study will improve our understanding of how this receptor works and will create a basis for the development of new drugs. While some of our findings will be specific to the b1AR receptor we are anticipating that many of the discoveries will also advance our general understanding of how GPCRs work.

G protein-coupled receptors (GPCRs) are membrane-embedded signalling sensors that communicate the presence of extracellular stimuli to the cell interior, regulating many signalling cascades. Conformational plasticity is key to their function, but our current understanding of GPCRs is strongly biased towards a static view that primarily focuses on the transmembrane regions. Hence, there is an urgent need for complementary studies that will reveal the dynamic nature of these receptors and the role their mobile intra- and extracellular loop regions play.

NMR spectroscopy is highly suited to study conformational dynamics. However, most studies so far have used GPCRs solubilized in detergents and relied on engineered, highly stabilized receptors borrowed from crystallography that had mobile regions such as intracellular loop 3 removed. To capture the genuine dynamic nature of GPCRs, and how their function is affected by their native lipid environment, requires NMR studies conducted with receptors embedded in a medium that faithfully mimics a phospholipid bilayer.

To advance our understanding of GPCR function and activation, we will use NMR to study the dynamic nature of a b1 adrenergic receptor (b1AR) in a bilayer mimic, employing a range of lipids. Little is known about how lipids interact with individual regions of GPCRs and how they influence their function. Interactions with intracellular binding partners (e.g. G proteins), and the conformations of loop regions and helix 8, are all modulated by the lipid bilayer environment, and are considered to be an essential part of GPCR function. Exploring these areas in the context of a native-like bilayer environment is essential to advancing understanding of these important proteins. We will conduct our NMR investigations with a minimally thermostabilised b1AR with intact native loop regions, with the receptor embedded in phospholipid bilayer-forming Salipro nanoparticles, recently developed in our lab.

GPCRs are the largest family of proteins in humans. They regulate most aspects of physiology and are implicated in a wide range of diseases. Understanding how GPCRs work is key for improving health and tackling illnesses, but obtaining a molecular perspective has been challenging. Our research investigates the mechanism of action of GPCRs. Our research outcomes will increase understanding of GPCR function through mechanistic insight and novel methodology that will contribute towards the development of new therapeutics and compounds that improve health.

Our work will impact on several areas:

1. Academia and the support of UK research excellence
Researchers in the fields of GPCR study, structural biology, biochemistry, pharmacology, drug development and computational chemistry will benefit from our investigations that generate new insight and methodology for studying GPCRs. GPCR function is strongly linked to conformational plasticity which is modulated by the receptor environment. Hence, investigating receptors embedded in lipid membrane-like environments makes our approach particularly precious, creating strong synergy between the fields of membrane protein structural biology and lipid/membrane research.

2. Pharmaceutical and biotechnology industry, drug development
Accessible at the cell surface and with a critical role in so many physiological functions GPCRs are the largest drug target. With over 34% of all available drugs directed at GPCRs this is a multi-billion market. However, as only a small fraction of receptors are targeted there is vast potential to tackle untreated diseases and to improve existing drugs by reducing their side effects. Mechanistic insight is the key to drug development and our study will add to the knowledge base of how these receptors work. Our research will establish an NMR platform that will allow the validation of new drugs via spectroscopic tools. It will help in guiding drug development. Our research into how GPCRs interact with non-G proteins that are involved in signaling processes will extend the range of possible targets further. Together this harbours untapped potential for biotech and pharmaceutical industry to expand drug therapeutic interventions. This will increase revenue in the UK, secure employment in the future, generate wealth and prosperity and maintain a leading role for the UK knowledge and technology driven industry.

3. Health and society
Our research will assist in developing novel drugs and the improvement of existing ones. These will target new diseases for which there is currently no therapy. Existing treatments will be refined e.g. in the context of b1 adrenergic receptor our research will contribute towards the development of beta blockers with improved selectivity and reduced side effects. Ultimately, the development of new drug therapies will improve general health in our society. Our methods will be applicable to other membrane proteins causing disease e.g. ion channels.

4. Training of research staff
Development of our society relies on the presence of a technically skilled workforce. This project provides direct technical training of a postdoc researcher in structural biology, molecular biology and biophysics techniques, preparing him for a future in the pharmaceutical and biotech industry or in academia. Adding to the trained pool of skilled researchers in the UK we will increase the economic competitiveness of the country by strengthening its position in the global academic and pharmaceutical market.

5. Educating the general public
Many of the effects related to GPCR mediated signaling in health and disease can be experienced in a way that can easily be appreciated by the general public e.g. vision, taste, cardiac output etc. Issues related to addictive drugs targeting GPCRs are another topic to engage the general public. This will raise awareness on the relevance of our research and the need for modern approaches to biomedical research.