Superactive mutants of thromboxane prostanoid receptor: functional and computational analysis of an active form alternative to constitutively active mutants.
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.
Ambrosio
Manuela
M
Laboratory of Molecular Pharmacology, Department of Pharmacological Sciences, Università degli Studi di Milano, Via Balzaretti 9, 20133, Milan, Italy.
Fanelli
Francesca
F
Brocchetti
Silvia
S
Raimondi
Francesco
F
Mauri
Mario
M
Rovati
G Enrico
GE
Capra
Valérie
V
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
D009841:Oligonucleotides; D017482:Receptors, Thromboxane; D019204:GTP-Binding Proteins
J Am Chem Soc. 2008 Apr 2;130(13):4310-25
18335928
Nature. 2008 Jul 10;454(7201):183-7
18563085
Trends Pharmacol Sci. 1998 Sep;19(9):365-9
9786024
Biochim Biophys Acta. 2000 Apr 17;1496(2-3):164-82
10771086
Proteins. 2003 Jul 1;52(1):80-7
12784371
Science. 2007 Nov 23;318(5854):1258-65
17962520
Mol Pharmacol. 2007 Apr;71(4):959-64
17192495
Biochemistry. 2003 Mar 18;42(10):2759-67
12627940
Nature. 2008 Jul 24;454(7203):486-91
18594507
Trends Pharmacol Sci. 1997 Nov;18(11):416-7
9426468
Trends Pharmacol Sci. 2007 Aug;28(8):397-406
17629961
Mol Pharmacol. 1999 Feb;55(2):339-47
9927627
Cell Signal. 2005 Nov;17(11):1373-83
15893915
EMBO J. 1996 Jul 15;15(14):3566-78
8670860
Mol Pharmacol. 1992 Mar;41(3):549-60
1312216
Cell. 1995 Dec 15;83(6):1047-58
8521505
Biochem Biophys Res Commun. 2002 May 17;293(4):1233-41
12054508
Proc Natl Acad Sci U S A. 1989 Oct;86(19):7321-5
2552439
Trends Pharmacol Sci. 2004 Aug;25(8):413-22
15276710
J Mol Biol. 1993 Dec 5;234(3):779-815
8254673
Biochemistry. 2001 Jul 3;40(26):7761-72
11425302
J Biol Chem. 1994 Sep 30;269(39):23879-81
7929034
Chem Rev. 2005 Sep;105(9):3297-351
16159154
Biophys J. 2003 Nov;85(5):2900-18
14581194
Nature. 2007 Nov 15;450(7168):383-7
17952055
Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):808-13
9023338
Science. 2008 Nov 21;322(5905):1211-7
18832607
J Recept Signal Transduct Res. 2002 Feb-Nov;22(1-4):1-16
12503605
Med Chem. 2005 Jul;1(4):405-21
16789897
Am J Physiol. 1978 Aug;235(2):E97-102
686171
Science. 2000 Aug 4;289(5480):739-45
10926528
J Biol Chem. 2009 Oct 16;284(42):28801-9
19706606
Trends Pharmacol Sci. 1994 May;15(5):140-4
7754531
Proc Natl Acad Sci U S A. 1990 Apr;87(8):2896-900
2158097
Mol Pharmacol. 2003 Dec;64(6):1271-6
14645655
J Biol Chem. 1993 Mar 5;268(7):4625-36
8095262
Mol Pharmacol. 2004 Oct;66(4):880-9
15229298
Trends Pharmacol Sci. 1993 Aug;14(8):303-7
8249148
Nat Rev Drug Discov. 2002 Feb;1(2):103-10
12120091
Mol Pharmacol. 2009 Jan;75(1):1-12
18945819
J Mol Biol. 2008 Jul 18;380(4):648-55
18554610
Nature. 2009 May 21;459(7245):356-63
19458711
Annu Rev Pharmacol Toxicol. 2008;48:107-41
17848137
Anal Biochem. 1980 Sep 1;107(1):220-39
6254391
Trends Pharmacol Sci. 2005 Dec;26(12):618-24
16260046
Pharmacol Ther. 2008 Apr;118(1):18-35
18374420
Biochem Soc Trans. 2001 Nov;29(Pt 6):641-54
11709048
Nature. 2008 Sep 25;455(7212):497-502
18818650
Trends Pharmacol Sci. 2004 Apr;25(4):186-92
15063082