Robert F. Margolskee
- ADJUNCT PROFESSOR Neuroscience
- ADJUNCT PROFESSOR Pharmacology and Systems Therapeutics
- ADJUNCT PROFESSOR Structural and Chemical Biology
Ph.D., Johns Hopkins School of Medicine
M.D., Johns Hopkins School of Medicine
ResearchTaste Cells of the Gut. The sensation of taste is initiated by the interaction of tastants with receptors and ion channels in the apical microvilli of taste receptor cells found within taste buds in the oral cavity. This fundamental sense enables organisms to avoid toxins and find nutrients. Vertebrate taste is comprised of five distinct qualities (sweet, sour, bitter, salty and umami (the taste of glutamate)). Recently, we have begun to examine chemosensory responses of taste cell-like enteroendocrine cells of the gastrointestinal (GI) tract to determine their role in nutrient-specific satiety and hormonal/physiological responses to feeding. Our work on GI chemosensation may have important implications for appetite control, obesity and diabetes.
Making Sense of Taste. Many taste transduction pathways convert chemical information into cellular second messenger codes utilizing cyclic nucleotides or inositol trisphosphate. These messengers are typically part of a signaling cascade that leads to taste cell depolarization and Ca++ release. Our studies and those of other investigators have shown that responses to bitter, sweet and umami compounds are transduced by specific receptors linked to guanine nucleotide binding regulatory proteins (G proteins).
We use bioinformatics, biochemistry, molecular cloning, structural biology, electrophysiology and behavioral analysis of transgenic mice to identify and characterize functionally taste cell components involved in taste transduction and coding. Using these methods, we have identified taste transduction elements of the following types: G protein subunits, G protein-coupled receptors, effector enzymes, and ion channels.
Sweet receptors. The mouse sac gene is the primary genetic determinant of sensitivity of mice to sweeteners. Sac encodes the type 1 taste receptor T1R3 (mT1R3 in mouse and hT1R3 in humans). The T1R receptors are family 3 GPCRs with ligand-binding sites within large extracellular amino-terminal domains. Heterologous expression of T1R2 plus T1R3 yields a sweet-responsive receptor, while expression of T1R1 plus T1R3 yields an umami-responsive receptor.
A wide variety of chemically diverse compounds taste sweet, including natural sugars such as glucose and sucrose, sugar alcohols, small molecule artificial sweeteners such as saccharin, and proteins such as brazzein. Brazzein is a naturally occurring plant protein that only humans, apes and old world monkeys perceive as tasting sweet. Differential sensitivity of mice and humans to brazzein's sweetness provided us with a means to identify the portions of the T1R2 + T1R3 sweet receptor that interact with brazzein. Using inter-species pairs of human and mouse T1Rs we determined that hT1R2 + hT1R3 responds to brazzein, but hT1R2 + mT1R3 does not, indicating that residues in hT1R3 are required for receptor activity toward brazzein. Using mouse/human chimeric receptors we determined that human-specific residues within a limited segment of the C-rich region of T1R3 are required for sensitivity to brazzein. Replacement of the C-rich region of mouse T1R3 with the corresponding human segment enabled hT1R2 + humanized mT1R3 to respond to brazzein. Pairing humanized mT1R3 with hT1R2 better supports brazzein activity than pairing with mT1R2. Our studies indicate that brazzein physically interacts with both hT1R2 and hT1R3.
GI chemosensation: gut-expressed taste proteins mediate GI responses. We have found that nearly all of the receptors and downstream signalling elements involved in taste detection and transduction are expressed also in enteroendocrine cells where they underlie chemosensory functions of the gut. It is known that glucose givenorally, but not systemically, induces secretion of GIP (glucose-dependent insulinotropic peptide) and GLP-1 (glucagon like peptide 1), which in turn regulate appetite, insulin secretion and gut motility. We have found that duodenal L cells express sweet taste receptors, the taste G-protein gustducin, and several other taste transduction elements. Knockout mice lacking gustducin or the sweet taste receptor subunit T1R3 have deficiencies in secretion of GLP-1 and GIP, and in the regulation of plasma levels of insulin and glucose. Isolated small intestine and intestinal villi from gustducin null mice displayed markedly defective GLP-1 secretion in response to glucose. The human L cell line NCI-H716 expresses gustducin, taste receptors and several other taste signaling elements. GLP-1 release from NCI-H716 cells was promoted by sugars and the non-caloric sweetener sucralose, and blocked by the sweet receptor antagonist lactisole or siRNA for alpha-gustducin.
Dietary sugars are transported from the intestinal lumen into absorptive enterocytes by the sodium-dependent glucose transporter isoform 1 (SGLT1). Expression of SGLT1 is well known to be regulated by dietary monosaccharides, however, the luminal glucose sensor mediating this process previously was unknown. We found that enteroendocrine cell-expressed gustducin and T1R3 underlie intestinal sugar sensing and regulation of SGLT1 mRNA and protein. Dietary sugar and artificial sweeteners increased SGLT1 mRNA and protein expression, and glucose absorptive capacity in wild-type mice, but not in knockout mice lacking T1R3 or gustducin. Furthermore, artificial sweeteners, acting upon sweet taste receptors expressed in enteroendocrine GLUTag cells, stimulated secretion of gut hormones implicated in SGLT1 upregulation. Modulating hormone secretion from gut's taste cells may provide novel treatments for obesity, diabetes and malabsorption syndromes.
Egan JM, Margolskee RF. Taste cells of the gut and gastrointestinal chemosensation. Mol Interv 2008 Apr; 8(2): 78-81.
Margolskee RF, Dyer J, Kokrashvili Z, Salmon KS, Ilegems E, Daly K, Maillet EL, Ninomiya Y, Mosinger B, Shirazi-Beechey SP. T1R3 and gustducin in gut sense sugars to regulate expression of Na+-glucose cotransporter 1. Proc Natl Acad Sci U S A 2007 Sep 18; 104(38): 15075-15080.
Jang HJ, Kokrashvili Z, Theodorakis MJ, Carlson OD, Kim BJ, Zhou J, Kim HH, Xu X, Chan SL, Juhaszova M, Bernier M, Mosinger B, Margolskee RF, Egan JM. Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1. Proc Natl Acad Sci U S A 2007 Sep 18; 104(38): 15069-15074.
Damak S, Rong M, Yasumatsu K, Kokrashvili Z, Varadarajan V, Zou S, Jiang P, Ninomiya Y, Margolskee RF. Detection of sweet and umami taste in the absence of taste receptor T1r3. Science 2003 Aug 8; 301(5634): 850-853.
Margolskee RF. Molecular mechanisms of bitter and sweet taste transduction. J Biol Chem 2002 Jan 4 277(1):1-4.
Perez CA, Huang L, Kozak JA, Preuss AK, Zhang H, Max M, Margolskee RF. A transient receptor potential channel expressed in taste receptor cells. Nat Neurosci 2002 Nov; 5(11): 1169-1176.We used differential screening of cDNAs from individual taste receptor cells to identify candidate taste transduction elements in mice. Among the differentially expressed clones, one encoded Trpm5, a member of the mammalian family of transient receptor potential (TRP) channels. We found Trpm5 to be expressed in a restricted manner, with particularly high levels in taste tissue. In taste cells, Trpm5 was coexpressed with taste-signaling molecules such as alpha-gustducin, Ggamma13, phospholipase C-beta2 (PLC-beta2) and inositol 1,4,5-trisphosphate receptor type III (IP3R3). Our heterologous expression studies of Trpm5 indicate that it functions as a cationic channel that is gated when internal calcium stores are depleted. Trpm5 may be responsible for capacitative calcium entry in taste receptor cells that respond to bitter and/or sweet compounds.
Max M, Shanker YG, Rong M, Liu Z, Campagne F, Weinstein H, Damak S, Margolskee RF. Tas1r3, encoding a new candidate taste receptor, is allelic to the sweet responsiveness locus Sac. Nat Genet 2001 May; 28(1): 58-63.The ability to taste the sweetness of carbohydrate-rich foodstuffs has a critical role in the nutritional status of humans. Although several components of bitter transduction pathways have been identified, the receptors and other sweet transduction elements remain unknown. The Sac locus in mouse, mapped to the distal end of chromosome 4 (refs. 7-9), is the major determinant of differences between sweet-sensitive and -insensitive strains of mice in their responsiveness to saccharin, sucrose and other sweeteners. To identify the human Sac locus, we searched for candidate genes within a region of approximately one million base pairs of the sequenced human genome syntenous to the region of Sac in mouse. From this search, we identified a likely candidate: T1R3, a previously unknown G protein-coupled receptor (GPCR) and the only GPCR in this region. Mouse Tas1r3 (encoding T1r3) maps to within 20,000 bp of the marker closest to Sac (ref. 9) and, like human TAS1R3, is expressed selectively in taste receptor cells. By comparing the sequence of Tas1r3 from several independently derived strains of mice, we identified a specific polymorphism that assorts between taster and non-taster strains. According to models of its structure, T1r3 from non-tasters is predicted to have an extra amino-terminal glycosylation site that, if used, would interfere with dimerization.
Smith DV, Margolskee RF. Making sense of taste. Sci Am 2001 Mar 284(3):32-39.
Huang L, Shanker YG, Dubauskaite J, Zheng JZ, Yan W, Rosenzweig S, Spielman AI, Max M, Margolskee RF. Ggamma13 colocalizes with gustducin in taste receptor cells and mediates IP3 responses to bitter denatonium. Nat Neurosci 1999 Dec; 12: 1055-1062.
Wong GT, Gannon KS, Margolskee RF. Transduction of bitter and sweet taste by gustducin. Nature 1996 Jun; 381: 796-800.
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