Chemical Senses Advance Access originally published online on April 18, 2006
Chemical Senses 2006 31(6):505-513; doi:10.1093/chemse/bjj053
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Expression and Purification of Functional Ligand–Binding Domains of T1R3 Taste Receptors
1 Department of Anatomy and Neurobiology and Graduate Programs in Life Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, USA 3 Faculty of Life Sciences, University of Manchester, Manchester, M60 1QD, UK 2 Present address: Department of Physiology, University of California, Los Angeles, Rm 6720 MacDonald Research Laboratories, 675 Charles E. Young Drive South, Los Angeles, CA 90095, USA
Correspondence to be sent to: Steven D. Munger, Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA. e-mail: smung001{at}umaryland.edu
Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein–coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.
Key words: G protein–coupled receptor (GPCR), protein expression, sugar, sweet taste, T1R3, umami taste
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  X. Li, D. Glaser, W. Li, W. E. Johnson, S. J. O'Brien, G. K. Beauchamp, and J. G. Brand Analyses of Sweet Receptor Gene (Tas1r2) and Preference for Sweet Stimuli in Species of Carnivora J. Hered., July 1, 2009; 100(suppl_1): S90 - S100. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-i. Nakajima, Y. Morita, A. Koizumi, T. Asakura, T. Terada, K. Ito, A. Shimizu-Ibuka, J.-i. Maruyama, K. Kitamoto, T. Misaka, et al. Acid-induced sweetness of neoculin is ascribed to its pH-dependent agonistic-antagonistic interaction with human sweet taste receptor FASEB J, July 1, 2008; 22(7): 2323 - 2330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Palmer The Pharmacology and Signaling of Bitter, Sweet, and Umami Taste Sensing Mol. Interv., April 1, 2007; 7(2): 87 - 98. [Abstract] [Full Text] [PDF]
|
|