Data di Pubblicazione:
2008
Citazione:
Oligomerization of thromboxane A2 receptor : from computational modelling to biochemistry / M. Parenti, M. Mauri, F. Guzzi, G.E. Rovati, M. Ambrosio, V. Capra, F. Raimondi, F. Fanelli. - In: THE FEBS JOURNAL. - ISSN 1742-464X. - 275:Suppl. 1(2008), pp. 356-356. ((Intervento presentato al 33. convegno FEBS Congress & IUBMB Conference tenutosi a Athens nel 2008.
Abstract:
Thromboxane A2 induces platelet aggregation and constriction
of vascular and bronchial smooth muscle through the interaction
with a G protein-coupled receptor that exists as two alternatively
splice variants, termed TPa and TPb with different C-terminal cytoplasmic
tails. TPa and TPb are usually co-expressed in native cells and
form homo- and hetero-oligomers in transfected cells.
Methods: Computational modelling techniques [1, 2] were first used
to generate models of TPa monomer in free- and agonist-bound conformations,
then subjected to docking simulations for modelling of
TPa homo-dimers. Theoretical predictions were challenged by sitedirected mutagenesis of TPa and TPb. Wild type and mutant receptors
fused to fluorescent proteins (GFP, CFP, YFP) were transiently
expressed in HEK293 cells and their subcellular localization and dimerization
studied by a combination of microscopy and fluorescence resonance
energy transfer (FRET) techniques.
Results: The most supported model of TPa homo-dimer was characterized
by contacts between helices 1 of monomers, whereby cysteine35
residues lie at a distance predictive of an inter-helical disulphide bridge.
Upon substitution of cysteine35 with alanine (C35A) homo- and hetero-
dimerization of TPa and TPb as well as TPa trafficking was greatly
impaired. However, C35A mutant TP receptors did not significantly
differ from wild type in terms of [3H]-SQ29, 548 antagonist binding
and accumulation of inositol phosphates induced by U46619 agonist
stimulation.
Conclusions: Mutation of cysteine35 greatly impairs dimerization of
TP receptors as anticipated by computational modelling but leaves
unaffected the binding and signalling properties of monomeric receptors.
References:
1. Casciari et al., BMC Bioinformatics 2006; 7: 340, 2006.
2. Fanelli Mol Cell Endocrinol 2007, 260–262 59–64.
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
Thromboxane receptor; dimerization; GPCR
Elenco autori:
M. Parenti, M. Mauri, F. Guzzi, G.E. Rovati, M. Ambrosio, V. Capra, F. Raimondi, F. Fanelli
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