title: Superactive mutants of thromboxane prostanoid receptor: functional and computational analysis of an active form alternative to constitutively active mutants.
abstract: In class A GPCRs the E/DRY motif is critical for receptor activation and function. According to experimental and computational data, R3.50 forms a double salt bridge with the adjacent E/D3.49 and E/D6.30 in helix 6, constraining the receptor in an inactive state. The disruption of this network of interactions facilitates conformational transitions that generate a signal or constitutive activity. Here we demonstrate that non-conservative substitution of either E129((3.49)) or E240((6.30)) of thromboxane prostanoid receptor (TP) resulted in mutants characterized by agonist-induced more efficient signaling properties, regardless of the G protein coupling. Results of computational modeling suggested a more effective interaction between G(q) and the agonist-bound forms of the TP mutants, compared to the wild type. Yet, none of the mutants examined revealed any increase in basal activity, precluding their classification as constitutively active mutants. Here, we propose that these alternative active conformations might be identified as superactive mutants or SAM.
authors.affiliation: Laboratory of Molecular Pharmacology, Department of Pharmacological Sciences, Università degli Studi di Milano, Via Balzaretti 9, 20133, Milan, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Ambrosio
authors.forename: Francesca
authors.initials: F
authors.lastname: Fanelli
authors.forename: Silvia
authors.initials: S
authors.lastname: Brocchetti
authors.forename: Francesco
authors.initials: F
authors.lastname: Raimondi
authors.forename: Mario
authors.initials: M
authors.lastname: Mauri
authors.forename: G Enrico
authors.initials: GE
authors.lastname: Rovati
authors.forename: Valérie
authors.initials: V
authors.lastname: Capra
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019295:Computational Biology / Q000379:methods; D019204:GTP-Binding Proteins / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D011487:Protein Conformation; D017482:Receptors, Thromboxane / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D009841:Oligonucleotides; D017482:Receptors, Thromboxane; D019204:GTP-Binding Proteins
references.citation: J Am Chem Soc. 2008 Apr 2;130(13):4310-25
references.pmid: 18335928
references.citation: Nature. 2008 Jul 10;454(7201):183-7
references.pmid: 18563085
references.citation: Trends Pharmacol Sci. 1998 Sep;19(9):365-9
references.pmid: 9786024
references.citation: Biochim Biophys Acta. 2000 Apr 17;1496(2-3):164-82
references.pmid: 10771086
references.citation: Proteins. 2003 Jul 1;52(1):80-7
references.pmid: 12784371
references.citation: Science. 2007 Nov 23;318(5854):1258-65
references.pmid: 17962520
references.citation: Mol Pharmacol. 2007 Apr;71(4):959-64
references.pmid: 17192495
references.citation: Biochemistry. 2003 Mar 18;42(10):2759-67
references.pmid: 12627940
references.citation: Nature. 2008 Jul 24;454(7203):486-91
references.pmid: 18594507
references.citation: Trends Pharmacol Sci. 1997 Nov;18(11):416-7
references.pmid: 9426468
references.citation: Trends Pharmacol Sci. 2007 Aug;28(8):397-406
references.pmid: 17629961
references.citation: Mol Pharmacol. 1999 Feb;55(2):339-47
references.pmid: 9927627
references.citation: Cell Signal. 2005 Nov;17(11):1373-83
references.pmid: 15893915
references.citation: EMBO J. 1996 Jul 15;15(14):3566-78
references.pmid: 8670860
references.citation: Mol Pharmacol. 1992 Mar;41(3):549-60
references.pmid: 1312216
references.citation: Cell. 1995 Dec 15;83(6):1047-58
references.pmid: 8521505
references.citation: Biochem Biophys Res Commun. 2002 May 17;293(4):1233-41
references.pmid: 12054508
references.citation: Proc Natl Acad Sci U S A. 1989 Oct;86(19):7321-5
references.pmid: 2552439
references.citation: Trends Pharmacol Sci. 2004 Aug;25(8):413-22
references.pmid: 15276710
references.citation: J Mol Biol. 1993 Dec 5;234(3):779-815
references.pmid: 8254673
references.citation: Biochemistry. 2001 Jul 3;40(26):7761-72
references.pmid: 11425302
references.citation: J Biol Chem. 1994 Sep 30;269(39):23879-81
references.pmid: 7929034
references.citation: Chem Rev. 2005 Sep;105(9):3297-351
references.pmid: 16159154
references.citation: Biophys J. 2003 Nov;85(5):2900-18
references.pmid: 14581194
references.citation: Nature. 2007 Nov 15;450(7168):383-7
references.pmid: 17952055
references.citation: Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):808-13
references.pmid: 9023338
references.citation: Science. 2008 Nov 21;322(5905):1211-7
references.pmid: 18832607
references.citation: J Recept Signal Transduct Res. 2002 Feb-Nov;22(1-4):1-16
references.pmid: 12503605
references.citation: Med Chem. 2005 Jul;1(4):405-21
references.pmid: 16789897
references.citation: Am J Physiol. 1978 Aug;235(2):E97-102
references.pmid: 686171
references.citation: Science. 2000 Aug 4;289(5480):739-45
references.pmid: 10926528
references.citation: J Biol Chem. 2009 Oct 16;284(42):28801-9
references.pmid: 19706606
references.citation: Trends Pharmacol Sci. 1994 May;15(5):140-4
references.pmid: 7754531
references.citation: Proc Natl Acad Sci U S A. 1990 Apr;87(8):2896-900
references.pmid: 2158097
references.citation: Mol Pharmacol. 2003 Dec;64(6):1271-6
references.pmid: 14645655
references.citation: J Biol Chem. 1993 Mar 5;268(7):4625-36
references.pmid: 8095262
references.citation: Mol Pharmacol. 2004 Oct;66(4):880-9
references.pmid: 15229298
references.citation: Trends Pharmacol Sci. 1993 Aug;14(8):303-7
references.pmid: 8249148
references.citation: Nat Rev Drug Discov. 2002 Feb;1(2):103-10
references.pmid: 12120091
references.citation: Mol Pharmacol. 2009 Jan;75(1):1-12
references.pmid: 18945819
references.citation: J Mol Biol. 2008 Jul 18;380(4):648-55
references.pmid: 18554610
references.citation: Nature. 2009 May 21;459(7245):356-63
references.pmid: 19458711
references.citation: Annu Rev Pharmacol Toxicol. 2008;48:107-41
references.pmid: 17848137
references.citation: Anal Biochem. 1980 Sep 1;107(1):220-39
references.pmid: 6254391
references.citation: Trends Pharmacol Sci. 2005 Dec;26(12):618-24
references.pmid: 16260046
references.citation: Pharmacol Ther. 2008 Apr;118(1):18-35
references.pmid: 18374420
references.citation: Biochem Soc Trans. 2001 Nov;29(Pt 6):641-54
references.pmid: 11709048
references.citation: Nature. 2008 Sep 25;455(7212):497-502
references.pmid: 18818650
references.citation: Trends Pharmacol Sci. 2004 Apr;25(4):186-92
references.pmid: 15063082