Marianna Max - US grants

DESCRIPTION (Adapted from applicant's abstract): The daily cycle of light and darkness is the most consistent and pervasive signal from the environment to the central nervous system. Melatonin is the universal dark signal that informs the body of this change in illumination. It also prepares sensory and nervous responses to be ready for the changes in illumination by predicting the onset of darkness or day. It does this my being dually regulated by light and a circadian clock. The clock is itself synchronized to the diurnal cycle by light. Melatonin synthesis is regulated by a multi-enzyme pathway that is under control of the circadian clock and of opsin-like photopigments. Light is the most important entraining agent for the melatonin cycle since it both entrains the circadian clock and acutely suppresses melatonin synthesis. Two cell types, photoreceptors of the retina and pinealocytes of the pineal eye of lower vertebrates are known to synthesize melatonin rhythmically under regulation of an endogenous circadian oscillator and to photoregulate melatonin synthesis using opsin-like photopigments. This proposal puts forth the pineal eye of the chick as a model system in which to identify key elements in this transduction pathway. It takes advantage of the recent identification of the chick pineal eye photopigment (P-opsin) and seeks to identify other elements in the transduction pathway. The pineal eye has several advantages as a model system. The pineal eye's primary function is to transduce diurnal lighting information into a melatonin signal, thus many of the molecules expressed in the pineal will be devoted to this task. It is made up of relatively few cell types, primarily pinealocytes and it is relatively easy to culture. In culture, pinealocytes retain the full range of photoreceptive and circadian functions. The level of melatonin expression is sufficiently robust so as to make its measurement in a real time, flow through culture extremely reliable. Many pharmacological and perturbation type investigations overs the past few years provide the basis for the molecular identification of key transduction elements described in the following pages.

DESCRIPTION (applicant's abstract): Endogenous circadian clocks allow organisms to appropriately time their behavioral and physiological responses to environmental cycles such as daily light cycles and seasonal changes in day length. A prominent output of the clock in vertebrates is the pineal hormone melatonin. The rhythm of melatonin synthesis controlled by the circadian clock is reset by the daily light cycle. Melatonin synthesis is regulated independently by the circadian clock and by light. Thus, light regulates melatonin synthesis via two pathways. Pinealocyte cultures provide a useful system to study both these pathways; pinealocytes rhythmically release melatonin for weeks in culture and their rhythms in vitro are entrained by light. Pharmacological methods have been extensively used to study in vitro pineal rhythms; however, no genetic manipulation of this system has been achieved because transgenic or "knock out" methods to alter expression of specific genes have not been possible in chickens to genetically manipulate pinealocyte gene expression. Replication-deficient, recombinant adenoviruses that express sense, mutant, ribosomal or antisense versions of pineal cDNAs will be used to infect pinealocytes. P-opsin, a pineal-specific opsin, will be expressed and "knocked out" in pinealocyte cultures. The pineal-expressed G-protein alpha rod transducin will be knocked out and a GTPase-deficient rod transducin alpha mutant with prolonged activation will be expressed in pinealocytes. The wild type gamma subunit of cGMP PDE and two mutant forms, one that is hyperactive and one that is hypoactive, will be expressed in pinealocytes. The effects of these genetic manipulations on melatonin biosynthesis and rhythmicity and the photosensitivity of the pinealocytes will be assessed by both static and flow through cultures and by radioimmunoassay for melatonin. Specific predictions are made for the effects of these molecular-genetic manipulations on the pineal's response to light based upon the hypothesis that a retina-like phototransduction pathway exists in the chick pinealocyte.

The pineal gland lies in the brain of vertebrate animals and is involved in detecting light levels in the environment to regulate the circadian rhythm, or daily 'clock', of cyclic activity of an animal. Cells in the pineal contain a protein molecule called pineal opsin (P-opsin), which is related to the photosensitive pigment in the eye, and is activated by light. The P-opsin is believed to be the pigment that initiates light response in the pineal, including suppressing production of the hormone melatonin. This project uses biochemistry on isolated chick pineal cells as a model system to identify other protein molecules called G-proteins, which are activated by light and P-opsin, and which, in turn, regulate melatonin production and entrainment of the pineal circadian clock.
Results will help understand how molecular mechanisms in a single cell containing a photosensor, a rhythmic output, and an endogenous clock, can combine to provide an circadian oscillator, and will have an impact on the biology of rhythms as well as on neuroscience and cell biology.

DESCRIPTION (provided by applicant): The long term goal of this proposal is to develop a molecular understanding of the mechanisms of sweet receptor activation by combining experimental and computational approaches. The taste preference for sweet compounds allows animals to seek out high carbohydrate energy sources to exploit for food. The sweet receptor is composed of two type 1 taste receptor monomers (T1R2 plus T1R3), apparently as a heterodimer. This proposal uses mutagenesis of the sweet receptor, expression in HEK 293 cells, calcium imaging, bioluminescent-resonance-energy-transfer and surface expression of receptors, to probe the sweet receptor's interaction with ligands. Aim 1 uses computational approaches to homology model the large extracellular domain of the heterodimer, using as template the crystal structure of the extracellular domain of mGluRl , another member of this family of receptors. The resulting homology models are tested and refined by mutagenesis of residues in T1R2+T1R3 predicted to form the dimerization interface, and then the expressed receptors are assayed for responses to sweet ligands and formation of heterodimers. The resulting optimized models will be useful to explain effects of mutations on ligand-induced activity in subsequent Aims. Aim 2 seeks to discover T1R2 residues that influence ligand-receptor interaction and receptor activation. Aim 2a uses the alignment of the TIR s with mGluRl to choose potential ligand-interacting residues in T1R2, then mutate them to discover their effects on receptor responses to sweet ligands. Aim 2b employs differences in species-specific taste perception, and chimeric human/mouse T1R2 receptors to track portions of the receptor responsible for human-like responses to sweeteners. Aim 2c uses mutagenesis to scan the surface-accessible arginines and lysines that might interact with the brazzein dipole. Mutated receptors are expressed, assayed for loss of responsiveness toward brazzein, then brazzein mutants are tested for the ability to compensate for receptor mutations. Aim 3 follows up on our recent observation that two residues in the cysteine-rich linker region of human T1R3 are essential for receptor responses to brazzein. We have proposed makin g additional mutations in this region to identify and characterize those residues that enable the human receptor to respond to brazzein. The knowledge gained from these studies will provide a working model for sweet receptor activity that may lead to the design of superior artificial sweeteners. Our molecular studies of the sweet receptor may shed light on transduction mechanisms common to other members of this family of receptors, such as the calcium-sensing receptor, which regulates calcium metabolism, and the metabotropic glutamate receptors, which are involved in multiple neurological responses.

[unreadable] DESCRIPTION (provided by applicant): The human sweet receptor composed of the monomers T1R2 + T1R3, appears to be the main (and perhaps the only) receptor required to explain sweet taste in humans. When co-expressed with a reporter G-protein in heterologous systems, this heterodimeric receptor responds to the full range of sweet-tasting compounds sensed by humans at concentrations that humans taste. The sweet receptor responds to a surprisingly diverse set of ligands, from small amino acids to moderately sized sweet-tasting plant proteins. No common structure accounts for the sweetness of all of these compounds. Studies from our lab and others indicate that the sweet receptor can be activated by means of a variety of domains and distinct binding sites on the receptor. We have used this diversity in sweet receptor activity as a tool for understanding ligand receptor interactions as well as for probing the molecular events that lead to activation of this complex receptor. By using heterologous expression, calcium imaging, BRET, mutagenesis and computational modeling my colleagues and I have mapped sweetener binding to at least four domains of the sweet receptor: the venus fly trap module (VFTM) of hT1R2 (various small molecule artificial sweeteners, natural sugars and dipeptide sweeteners), the VFTM of hT1R3 (natural sugars), the cysteine-rich domain (CRD) of hT1R3 (brazzein) and the transmembrane domain (TMD) of hT1R3 (cyclamate and NHDC). To date, no sweeteners have definitively been shown to bind within the TMD of T1R2, however, our recent finding suggests that this domain is able to allosterically regulate ligand- induced activity in the sweet receptor. In this proposal, we outline a plan to further determine the characteristics of the hT1R2 TMD that promotes allosteric interactions with other domains of the receptor. We will also determine whether any sweeteners map to its putative intra-helical TMD binding site. In addition to our established techniques (heterologous expression of receptors, mutagenesis, functional assay and computational modeling) for exploring sweetener interactions with the sweet receptor, our collaborator, Dr. Fariba Assadi-Porter will use saturation transfer difference (STD) NMR to track ligand-binding to cells expressing full length T1R2 + T1R3 together, each monomer by itself or mutants receptors (using parent cells not expressing receptors as control). This exciting new development will allow us to monitor ligand binding separate from receptor activity. It will also allow us to identify the critical ligand-receptor binding residues that determine sensitivity and selectivity for ligands, in addition to the effect of sweet receptor mutations on the ligand-binding pocket environment(s) by monitoring changes in the NMR spectra for each ligand. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The human sweet receptor, composed of the monomers T1R2 + T1R3, appears to be the main (and perhaps the only) receptor underlying sweet taste in humans. When co-expressed with a reporter G-protein in heterologous systems, this heterodimeric receptor responds to the full range of sweet-tasting compounds sensed by humans at appropriate concentrations. The sweet receptor responds to a surprisingly diverse set of ligands, from small amino acids to moderately sized sweet-tasting plant proteins. No common structure accounts for the sweetness of all of these compounds. Studies from our lab and others indicate that the sweet receptor can be activated by means of a variety of domains and distinct binding sites on the receptor. Using heterologous expression, calcium imaging, BRET, mutagenesis and computational modeling, my lab and those of my colleagues have described at least 4 binding regions of the sweet receptor: the venus fly trap module (VFTM) of hT1R2, the cysteine-rich domain (CRD) of hT1R3 and the transmembrane domain (TMD) of hT1R3. The current proposal focuses and builds on our recent discovery of overlapping binding pockets within the TMD of hT1R3 for agonists (cyclamate and analogs) and antagonists (lactisole and analogs). This domain is of critical importance for sweet receptor activation since all of the diverse ligands that humans perceive as sweet can be blocked by binding lactisole in the T1R3 TMD pocket. This suggests that this domain is the key element in the final conformational change leading to receptor activation. Since the sweet receptor can also be activated by ligands that bind in the TMD of T1R3 and these binding pockets share common residues, characterization of these binding pockets at a molecular level will provide insight into the differences between the ground and active state requirements oft the sweet receptor. We propose here to characterize the environment of the hT1R3 TMD responsible for both active and inactive conformations of the receptor using mutagenesis, heterologous expression and activity assays and computational modeling of ligand docking sites. In addition, through the collaborative effort outlined in the proposal, we will directly monitor the binding environment and binding of ligands to the TMD of T1R3 using STD-NMR, a powerful tool recently adapted to monitor the taste system. Our long-term goal is to elucidate the molecular events that underlie ligand binding and ligand induced activity (or stabilization of the ground state) and the conformational changes of the receptor required for G-protein activation. PUBLIC HEALTH RELEVANCE: There is today in the affluent countries of the world an epidemic of obesity, insulin-resistant diabetes and diet-related disorders. This is only made worse by our species evolutionary love affair with high- carbohydrate/energy-rich sweet foods. Taste perception and taste preference undoubtedly contribute to sweet-seeking behavior and food consumption. The identification of T1R2+T1R3 as the sweet receptor provides the target for intercession in modifying a behavior, which is maladaptive because of the plentiful food in affluent countries. Understanding the sweet receptor at a molecular level will enable the design of better low calorie sweeteners.

DESCRIPTION (provided by applicant): The long-range goal of our research program is to understand the molecular events underlying function of the heterodimeric sweet taste receptor (T1R2+T1R3). The sweet receptor is a remarkably broadly acting receptor, capable of responding to native and artificial sweeteners. In vivo and in vitro studies suggest that this single heterodimeric receptor is the primary or only sweet taste receptor. We are interested in how so many chemically diverse ligands can bind to the sweet receptor, how binding at different sites leads to receptor activation, and how the domains of each T1R monomer contribute to binding, activation and signal transduction. To address these goals we have developed ligand binding and activity assays, and used these techniques in concert with mutagenesis and molecular modeling to begin to understand this complex receptor. The present proposal applies these several techniques to examine how the small molecule-binding site of T1R2 interacts with aspartame, neotame and alitame (so-called dipeptide sweeteners). This "canonical" binding site is found within the "venus fly trap module" (VFTM) of T1R2. In Aim 1 we will use the differential sensitivity of the human and mouse sweet receptors to dipeptide sweeteners, along with heterologous expression assays, to identify key residues within the VFTM of T1R2 involved in the interactions with dipeptide sweeteners. In Aim 2 we will use directed mutagenesis of T1R2, heterologous assays and molecular modeling to physically and chemically characterize the interaction of dipeptide sweeteners with the VFTM of T1R2. In Aim 3 we will use spectroscopic and calorimetric techniques to monitor binding of dipeptide sweeteners to the expressed VFTM of T1R2 and T1R2 mutants. There is today in the affluent countries of the world an epidemic of obesity, insulin-resistant diabetes and diet-related disorders. In our evolutionary past a strong drive to consume high-carbohydrate/energy-rich foods was advantageous for survival. Today, our more sedentary lives and the ready availability of food makes this sweet-seeking behavior a liability that may contribute significantly to obesity. Sweet taste perception mediated by the heterodimeric sweet taste receptor undoubtedly contributes to sweet-seeking behavior and food consumption. The studies in this proposal will enhance our understanding at the molecular level of sweet receptor function with the hope of future means to control our sweet cravings and the attendant diseases of over-consumption.