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Stuart R. Maudsley, Ph.D., Investigator Head, Receptor Pharmacology Unit Laboratory of Neurosciences E-mail: maudsleyst@mail.nih.gov |
Receptor Pharmacology Unit Overview |
| Biography: Dr. Maudsley received his Ph.D. in 1997 in Pharmacology from the Department of Pharmacology at the University of Leeds where he studied the molecular mechanisms of tachykinin receptor activation and desensitization. With a Howard Hughes Medical Institute Fellowship, he moved to Duke University to work with Professor Robert J. Lefkowitz on the connectivity of G protein-coupled receptor (GPCR) signaling to tyrosine kinase pathways. During this period Dr. Maudsley developed new theories of GPCR signaling based upon the creation of higher order superstructures. Dr. Maudsley then accepted an Investigator position at the Medical Research Council at the University of Edinburgh in the United Kingdom. There he furthered the development of his concepts of the organization of GPCRs into discrete signaling structures for specific physiological functions. This work forms the basis of his research into the alteration of the GPCR signaling structures during healthy and pathological aging. |
| Research Overview: For the majority of its experimental lifetime, information flow through G protein-coupled receptors (GPCRs) has been envisioned as unidirectional, i.e., changes in receptor conformation produced by extracellular agonist binding promotes the transfer of information from outside the cell inwards. Recent experimentation however, has demonstrated that receptor conformation is also controlled by protein-protein interactions occurring inside the cell. Receptor dimerization and interactions with intracellular scaffolding and signaling proteins can modify receptor structure and ligand selectivity and predetermine, from a menu of available options, which intracellular responses will predominate. In essence, the influences on receptor conformation are bi-directional; internal factors change the conformation of the receptor to reflect the status of the intracellular milieu, while extracellular factors, i.e., agonists, convey information to the cell about the external environment. This concept has critical implications for receptor theory and the design of therapeutics. Thus in complex physiological processes, e.g., aging or neurodegenerative disease, in which multiple proteins expression patterns are changed it is more likely than previously thought that GPCR signal conditioning could be affected. Therefore if indeed there is an alteration of GPCR pharmacology in these states then perhaps drug design should be targeted toward this new pharmacology rather than the standard models previously used. |
| G Protein-coupled Receptors and Their Therapeutic Importance: The heptahelical G protein-coupled receptors constitute the most diverse form of transmembrane signaling protein. Approximately 1% of the mammalian genome encodes GPCRs, and about 450 of the approximately 950 predicted human GPCRs are expected to be receptors for endogenous ligands. GPCRs allow organisms to detect an extraordinarily diverse set of stimuli in the external environment, from photons of light and ions to small molecule neurotransmitters, peptides, glycoproteins, and phospholipids. Emphasizing their importance as therapeutic targets, nearly 40% of all current drugs target GPCRs for their actions. Thus the manipulation of transmembrane signaling by GPCRs constitutes perhaps the single most important therapeutic target in medicine. Therapeutics acting on GPCRs have traditionally been classified as agonists, partial agonists, or antagonists based on a two state model of receptor function embodied in the ternary complex model. However many lines of investigation have shown that GPCR signaling exhibits greater diversity and 'texture' than previously appreciated. |
| Additional Protein Factors Add 'Texture' to Receptor Signaling: Signaling diversity from GPCRs arises from numerous factors, among them the ability of receptors themselves to adopt multiple 'active' states with different effector coupling profiles, the formation of receptor dimers that exhibit unique pharmacology, signaling, and trafficking, the dissociation of receptor 'activation' from desensitization and internalization, and perhaps most importantly the discovery that non-G protein effectors mediate some aspects of GPCR signaling. At the same time, clustering of GPCRs with their downstream effectors in membrane microdomains, and interactions between receptors and a plethora of multidomain scaffolding proteins and accessory/chaperone molecules confers signal pre-organization, efficiency, and specificity. |
| It is these interactions with proteins that organize GPCRs into greater signaling entities that are of prime interest for our laboratory as their effects upon GPCR signaling provide a gateway into new realms of therapeutic pharmacology. More importantly it is likely that alteration in the interactions of these proteins with GPCRs may occur in aging or neurodegenerative disorders, thus defining a distinct 'pharmacology' from that seen in younger organisms or normal physiology. In this context, the concept of agonist selective trafficking of receptor signaling, which recognizes that a bound ligand may select between a menu of 'active' receptor conformations and induce only a subset of the possible response profile, presents the opportunity to develop drugs that change the quality as well as the quantity of therapeutic efficacy. As a more comprehensive understanding of the complexity of GPCR signaling is developed, the rational design of ligands possessing increased specific efficacy and attenuated side effects may become the standard mode of drug development. Therefore one of our primary goals is to specifically enhance these drug qualities for age-related disorders such as Alzheimer's, Huntington's and Parkinson's disease. |
| We are studying the ability of multi-protein complexes to condition receptor signaling in three major programs, these include the identification and classification of GPCR signaling complexes (also known as receptorsomes), the role of intracellular scaffolding proteins in the integration of multiple receptor inputs and the ability of lipid raft microdomains to control receptor signal transduction and neurotransmission. |
| GPCR Receptorsome Structure in Aging and Neurodegeneration: The functional unit of the GPCR has been hypothesized for many years as a ternary complex of stimulating hormone (agonist), receptor and the G protein effector. However both our research and that of many others has demonstrated that many other protein factors are required for the generation of the full spectrum of agonist-mediated intracellular signaling events. GPCRs have now been shown to physically interact with other GPCRs, receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) and scaffolding proteins such as PSD-95 (post-synaptic density protein of 95kDa). The multiprotein complexes the GPCRs are involved in to create their full activity status are called receptorsomes and are now thought to be the true functional receptor signaling unit. Many of the factors that GPCRs interact with in these receptorsomes have been shown to fluctuate in expression during aging and neurodegeneration, e.g., b-arrestin and RGS (regulator of G protein signaling) proteins. As the pharmacology and signaling of the GPCR is dictated by the composition of the receptorsome we are therefore studying, using proteomic screening technologies such as differential in-gel electrophoresis (DIGE) and tandem mass spectrometry, how the structure of receptorsomes of GPCRs implicated in neurodegenerative diseases (muscarinic acetylcholine, dopamine and serotonin) changes with age and pathophysiology. With this knowledge we are then attempting to alter currently existing therapeutics to enhance their activity at these different receptorsome states. |
| Mechanisms and Patterns of Complex Signal Integration in the Central Nervous System: In the central nervous system there are up to 60 different identified neurotransmitters that are involved in modifying neuronal activity through direct synaptic transmission or neuromodulation. For each neurotransmitter there is a wide variety of specific receptors that it can interact with to affect neuronal cellular signaling. The presence of these receptors often typifies the specific neuronal type. At many synapses there is a co-release of several signaling hormones and over larger areas there is a diffusion of neuromodulating hormones and neurotrophic factors. Hence the activity of neurons is likely to be a function of the summated actions of multiple cellular inputs. However much of cellular neurophysiology has been studied employing in vitro scenarios in which the signal transduction activity of receptor signaling pathways is studied in isolation of other inputs. This approach has yielded a great understanding of the linear pathways of cell signaling yet it does not provide sufficiently reliable information with respect to how multiple inputs integrate to generate the eventual physiology of the neuron when exposed to multiple neurotransmitters or neurotrophic factors. We are attempting to identify how neuronal signaling is controlled by the application of multiple hormones to the cells in a progressive manner. An emerging principle underlying cellular physiology is that signal transduction cascades do not operate as self-contained linear units of information transmission, but rather function as integrative networks, interfacing at multiple levels both with themselves and with other signaling modules to effect context-appropriate functional outputs. |
| The molecular integration of these distinct multiple inputs (mediated by specific plasma membrane embedded receptors) occurs at the level of their associated signal transduction cascades. There has been over the past few years a realization that signal transduction cascades, including kinases, phosphatases and their substrates are actually pre-assembled into higher order structures by molecular scaffolds, e.g., AKAP (A-kinase anchoring protein), POSH (plenty of SH3 domains), JIP (c-Jun N-terminal kinase interacting protein), b-arrestins or 14-3-3 proteins. These proteins compartmentalize signaling pathways in the cell, enhance specificity of target-substrate interaction and improve the speed and efficiency of signal transduction. Conceptually we have therefore a funneling of the complex and diverse signaling inputs from hormones and their specific receptors at the plasma membrane into the higher order multi-protein signaling scaffolds attached either to cytoskeletal proteins or the plasma membrane itself. Thus the complex neuronal signaling traffic is likely to converge at cytoplasmic nexi, represented by these scaffolding proteins. Clustering of signaling molecules in multiprotein complexes eliminates delays that would otherwise occur as a result of random diffusion in the cytoplasm. An understanding of how multiple inputs works also may give us a more true appreciation of how neurotransmitters/neurotrophic factors actually mediate intracellular signaling events in the physiological setting. Recent evidence has implicated many of these scaffolding proteins in mediating neurological disorders such as Parkinson's and Alzheimer's and therefore we are undertaking a detailed approach to understand how protein-protein interaction at these scaffolds both controls signal integration from cell surface receptors and also signal transfer within the cell. It is likely that both the qualitative and quantitative nature of hormonal effects on cells are dictated by the specific composition of these signaling nexi. |
| Control of Synaptic Transmission by Lipid Raft Microdomain Structure: Lipid rafts and caveolae are cholesterol and sphingomyelin-rich membrane microdomains found in many tissues and are thought to be involved in lipid and protein trafficking, signal transduction, cell surface proteolysis and the organization of higher order multi-protein complexes. Caveolae are small flask-shaped membrane invaginations that contain a high density of a cholesterol-binding protein called caveolin. Lipid rafts tend to have a flat membrane structure, predominate in the central nervous system (CNS) and are mostly caveolin free. In the CNS lipid rafts are characterized by their high concentrations of a protein called flotillin. Flotillin appears to act as a scaffolding protein within the raft. Due to their high densities of cholesterol and sphingomyelin compared to the rest of the cells plasma membrane lipid rafts can induce and maintain the clustering of membrane components upon certain stimuli. This compartmentalization capacity is important for the generation of intracellular signals and the recruitment of downstream effector molecules. Thus as accessory proteins can interact with GPCR structures to create receptorsomes then multiple receptorsomes can be then organized by lipid raft microdomains. |
| Recent evidence has shown that alterations in lipid rafts may be associated with various diseases, e.g., diabetes, certain forms of cancer, atherosclerosis and degenerative muscular dystrophies. Flotillin and caveolin levels are both observed to be altered in relation to disease processes and with aging, suggesting a concomitant alteration in the lipid raft/caveolae structure. Caveolin knock out mice also can display a considerably reduced lifespan compared to wild type animals as well as severe cardiovascular disorders and pulmonary fibrosis. |
| More importantly lipid rafts have also been proposed to play a role in neurodegenerative disorders such as Alzheimer's disease (AD). In AD disruptions of lipid rafts are thought to contribute to the production and aggregation of the neurotoxic amyloid Ab protein. Primitive senile plaques in non-demented persons also show strong flotillin expression and in AD patients there is an elevated flotillin presence in the cortex. Studies have also shown an accumulation of flotillin in lysosomes of neurons having neurofibrillary tangles, a second hallmark of AD. Not only are flotillins involved in amyloid processing but through their capacity to cluster in lipid rafts they are also thought to possess a structural scaffolding action. Flotillins can therefore control the membrane organization of c-src tyrosine kinases, GPCRs, structural proteins and even GLUT-4 glucose transporters. Therefore not only are the physico-chemical properties of the rafts themselves important, i.e., high lipid density to enforce juxtaposition of related signaling proteins, for cell signaling but also the functional state of the intrinsic scaffolding proteins such as flotillin is important. We are investigating whether flotillin-mediated alterations in lipid rafts can change the correct protein constituency and stoichiometry of neuroprotective signaling mechanisms. |
| It is highly likely that lipid rafts themselves crucially control individual neurotransmission events, in aging and disease models. Lipid raft structure appears to be coupled to the dynamics of the actin-cytoskeleton, this is of specific importance for synaptic plasticity as dendritic spines are strongly influenced by the capacity of the cell to regulate its actin cytoskeleton. Lipid rafts control the location of AMPA-glutamate receptors at the post-synaptic area as well as many other crucial neuronal signaling factors, e.g., phospholipase D1, adenylyl cyclase and the EGFR. This relationship between signaling factors and the raft enables efficient signal processing by promoting cross-talk between the different signaling cascades. Therefore any pathological disruption of the protein stoichiometry in the raft will result in a reduction of synaptic efficiency that may be a precursor for the loss of neuronal functions in aging and degeneration. It is therefore clear that a detailed quantitative analysis, using isotopic mass label mass spectrometry e.g., iTRAQ, of the actual levels and ratios of protein components of the rafts is required to understand the role of these organizing structures in synaptic transmission in aging and disease. As with our interest in receptorsomes, it is likely that therapeutics can even be directed towards idiosyncratic receptor systems created by their presence in distinct forms of lipid raft microdomains. |
| Summary: The laboratories interest lies in the appreciation that receptor signaling systems do not have a static profile and their response to ligands and the downstream signals they create are plastic. Natural events such as aging as well as neuropathophysiology are likely to affect this plasticity to generate new pharmacological profiles for receptor systems. It is our primary thesis that it may be possible to use this knowledge to create therapeutic agents specific to these new pharmacological states. |
Dr. Maudsley at the Intel Science Fair |
NIH Training Initiatives |
Graduate Research Programs at NIH |
Dr. Maudsley Research Profiles |
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