title: Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans.
abstract: New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.
authors.affiliation: Polgenix, Inc., Cleveland, Ohio, USA.
authors.forename: David
authors.initials: D
authors.lastname: Salom
authors.forename: Pengxiu
authors.initials: P
authors.lastname: Cao
authors.forename: Wenyu
authors.initials: W
authors.lastname: Sun
authors.forename: Kristopher
authors.initials: K
authors.lastname: Kramp
authors.forename: Beata
authors.initials: B
authors.lastname: Jastrzebska
authors.forename: Hui
authors.initials: H
authors.lastname: Jin
authors.forename: Zhaoyang
authors.initials: Z
authors.lastname: Feng
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D015870:Gene Expression; D006801:Humans; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D043705:Receptor, Adenosine A2A / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012243:Rhodopsin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D043705:Receptor, Adenosine A2A; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C042882:isorhodopsin; D012243:Rhodopsin
references.citation: Nature. 2010 Jun 10;465(7299):823-6
references.pmid: 20535212
references.citation: J Neurosci Methods. 2011 Apr 30;197(2):315-23
references.pmid: 21376755
references.citation: Trends Biotechnol. 2005 Feb;23(2):103-8
references.pmid: 15661348
references.citation: Br J Pharmacol. 1996 Apr;117(8):1693-701
references.pmid: 8732278
references.citation: Genetics. 1993 Oct;135(2):385-404
references.pmid: 8244003
references.citation: J Biol Chem. 2006 Apr 28;281(17):11917-22
references.pmid: 16495215
references.citation: Nat Methods. 2010 Jun;7(6):429-34
references.pmid: 20508636
references.citation: WormBook. 2006 Feb 11;:1-11
references.pmid: 18050451
references.citation: Development. 2005 Jul;132(14):3197-207
references.pmid: 15983401
references.citation: Mol Pharmacol. 2009 Jan;75(1):1-12
references.pmid: 18945819
references.citation: Curr Protein Pept Sci. 2006 Oct;7(5):465-70
references.pmid: 17073697
references.citation: J Biol Chem. 2008 Oct 24;283(43):29086-98
references.pmid: 18703511
references.citation: Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4689-94
references.pmid: 19258456
references.citation: J Biol Chem. 2001 Mar 30;276(13):10000-9
references.pmid: 11116153
references.citation: ChemMedChem. 2008 Jul;3(7):1021-3
references.pmid: 18409177
references.citation: Biochem Pharmacol. 1996 Feb 23;51(4):545-55
references.pmid: 8619901
references.citation: Neuropharmacology. 2002 Jan;42(1):60-73
references.pmid: 11750916
references.citation: Neuron. 2000 Jun;26(3):619-31
references.pmid: 10896158
references.citation: Nat Neurosci. 2008 Aug;11(8):916-22
references.pmid: 18604203
references.citation: EMBO J. 1989 Nov;8(11):3419-28
references.pmid: 2583105
references.citation: Nucleic Acids Res. 1994 Jul 11;22(13):2437-46
references.pmid: 8041603
references.citation: Vision Res. 1991;31(1):17-31
references.pmid: 2006550
references.citation: Biochem Pharmacol. 2009 Jul 1;78(1):11-20
references.pmid: 19447219
references.citation: J Struct Biol. 2007 Aug;159(2):166-78
references.pmid: 17591446
references.citation: J Cell Mol Med. 2007 Mar-Apr;11(2):224-38
references.pmid: 17488474
references.citation: Nature. 2011 Jan 13;469(7329):172-3
references.pmid: 21228868
references.citation: Science. 2000 Aug 4;289(5480):739-45
references.pmid: 10926528
references.citation: FASEB J. 2012 Feb;26(2):480-91
references.pmid: 22090313
references.citation: Science. 1998 Dec 11;282(5396):2028-33
references.pmid: 9851919
references.citation: Biochemistry. 2008 Dec 16;47(50):13296-307
references.pmid: 19053287
references.citation: J Mol Biol. 2004 Nov 5;343(5):1409-38
references.pmid: 15491621
references.citation: Photochem Photobiol. 2009 Mar-Apr;85(2):425-30
references.pmid: 19192200
references.citation: Proc Natl Acad Sci U S A. 1983 Apr;80(7):1887-91
references.pmid: 6572950
references.citation: Science. 1996 Nov 1;274(5288):768-70
references.pmid: 8864113
references.citation: Biochemistry. 2009 Jun 9;48(22):4789-97
references.pmid: 19378975
references.citation: J Mol Biol. 2004 Sep 10;342(2):571-83
references.pmid: 15327956
references.citation: Nat Rev Drug Discov. 2006 Dec;5(12):993-6
references.pmid: 17139284
references.citation: Biochemistry. 1984 Dec 18;23(26):6544-9
references.pmid: 6529569
references.citation: J Struct Funct Genomics. 2006 Jun;7(2):77-91
references.pmid: 17120110
references.citation: Biophys J. 2010 May 19;98(10):2347-55
references.pmid: 20483344
references.citation: Biochemistry. 1993 Jul 20;32(28):7229-36
references.pmid: 8343512
references.citation: PLoS One. 2011 Apr 08;6(4):e18478
references.pmid: 21494623
references.citation: J Gerontol. 1994 Jul;49(4):B145-56
references.pmid: 7516947
references.citation: Curr Opin Struct Biol. 2010 Aug;20(4):464-70
references.pmid: 20739007
references.citation: J Neurosci Methods. 2006 Jun 30;154(1-2):68-79
references.pmid: 16466809
references.citation: Methods Mol Biol. 2007;375:57-78
references.pmid: 17634596
references.citation: Cell. 2006 Nov 3;127(3):621-33
references.pmid: 17081982
references.citation: Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):4024-8
references.pmid: 8171029
references.citation: Ernst Schering Found Symp Proc. 2006;(2):145-61
references.pmid: 17703581