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Superactive mutants of thromboxane prostanoid receptor: functional and computational analysis of an active form alternative to constitutively active mutants

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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 Gq 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.

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Acknowledgments

We acknowledge Dr. Tommaso Costa (Laboratory of Pharmacology, Istituto Superiore di Sanità, Roma, Italy) for exhaustive and useful discussions of the data and for critical assessment of the paper. We thank Dr. Susanna Cotecchia (Département de Pharmacologie et de Toxicologie, Lausanne, Switzerland) for providing CAM of the α1B-AR. This work was supported in part by grants from EC FP6 (LSHM-CT-2004-005033 to GER). This study was also supported by a Telethon-Italy (S00068TELU to F.F.) and by a MIUR-FIRB grant (no. RBIN04CKYN to M.M.).

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Authors and Affiliations

  1. Laboratory of Molecular Pharmacology, Department of Pharmacological Sciences, Università degli Studi di Milano, Via Balzaretti 9, 20133, Milan, Italy

    Manuela Ambrosio, Silvia Brocchetti, G. Enrico Rovati & Valérie Capra

  2. Department of Chemistry and Dulbecco Telethon Institute, Università di Modena e Reggio Emilia, Modena, Italy

    Francesca Fanelli & Francesco Raimondi

  3. Department of Experimental Medicine, Università di Milano-Bicocca, Monza, Italy

    Mario Mauri

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  1. Manuela Ambrosio
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  2. Francesca Fanelli
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  3. Silvia Brocchetti
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  4. Francesco Raimondi
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  5. Mario Mauri
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  6. G. Enrico Rovati
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  7. Valérie Capra
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Correspondence to G. Enrico Rovati.

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Ambrosio, M., Fanelli, F., Brocchetti, S. et al. Superactive mutants of thromboxane prostanoid receptor: functional and computational analysis of an active form alternative to constitutively active mutants. Cell. Mol. Life Sci. 67, 2979–2989 (2010). https://doi.org/10.1007/s00018-010-0368-9

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