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    Evolution, Structure and fonction of GPCRs

    Evolution, Structure and fonction of GPCRs

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    Marie Chabbert

     

     

     

     

     

     

     

    Team members : 

    Marie Chabbert, Ph.D.

    Patrice Rodien, M.D., Ph.D.

    Mathilde Munier,  Ph.D, Postdoctoral Fellow

    Louis Gourdin, Technician

    Valentine Suteau-Courant, Undergraduate student

    Marine Sarfati-Lebreton, Undergraduate student

    Antoine Garnier, Undergraduate student

    Asma Tiss, Undergraduate student

     

    With about 800 members, the super-family of G-protein-coupled receptors (GPCRs) is involved in most physio-pathological functions and is the target of about a quarter of available drugs. GPCRs play a key role in vascular regulation:

    • directly through receptors of vasoactive peptides (AT1, AT2, BK1, BK2, UR2R, ETA, ETB…) and purinergic receptors (P2YR2, P2YR6)
    • indirectly through the immune response (e.g chemokine receptors), adaptive response to stress (catecholamine receptors) and endocrine regulation of the metabolic control.
    • directly through receptors of vasoactive peptides (AT1, AT2, BK1, BK2, UR2R, ETA, ETB…) and purinergic receptors (P2YR2, P2YR6)
    • indirectly through the immune response (e.g chemokine receptors), adaptative response to stress (catecholamine receptors) and endocrine regulation of the metabolic control.

     

    We are interested in the sequence-structure-function relationships of GPCRs. We aim at deciphering molecular mechanisms of receptor activation and function, with application to drug design. Our approach is based on the analysis of evolutionary information. In particular, we study the evolutionary mechanisms that led to the expansion and diversification of such a large family, in order to understand the specificity of each sub-family.

     

    Previously, using computational approaches (Fig. 1), we have shown that two major pathways of GPCR evolution are related to mutations of or near proline residues (Pelé et al., 2011). A first pathway is related to the deletion of one residue in helix 2, leading to receptors with the P2.58 pattern in transmembrane helix 2 (TM2). By divergent evolution, this pathway led to vasoactive peptide, chemokine and purinergic receptors (Devillé et al., 2009). A second pathway is related to the correlated mutations of proline residues in TM2 and TM5, in several independent sub-families (Pelé et al., 2011).

     


    The human non-olfactory class A receptors are projected onto the first two dimensions of the sequence space. The color code indicates the proline pattern of TM2 (left) and TM5 (right). The spanning ellipses indicate the receptor clusters corresponding to the P2.58 pattern and to the correlated absence of proline in TM2 and TM5. Adapted from Pelé et al., 2011, Plos One, 6 : 1110.

     

    How these mutations affect receptor activation remains to be determined. To answer this question, we use both computational and molecular biology approaches. Computational approaches are based on multidimensional scaling and correlation analysis of GPCR sequences from cnidarians to humans to determine key residues whose mutation led to emergence of new sub-families and the role of these residues in the activation mechanism. Molecular modelling of receptors of interest relies on our classification of helical distortions into a limited number of conformations with precise structural requirement (Devillé et al., 2008; Rey et al., 2010). Experimentally, we study the relationship between helical reorientation and activation, in particular for the receptors of glycoprotein hormones.

     

    In a process of understanding the activation mechanisms of GPCR and more particularly to identify which molecules could activate or inhibit them, we are interested in the effect of small molecules present in our environment on the functionality of GPCR. We speak of endocrine disruptors chemicals (EDC). These compounds mimic the hormone and disturb the endocrine system. Research on EDC often focuses on their estrogenic or anti-androgenic effects. However, they have others potential action mechanisms, including interactions with G protein-coupled receptors. In this context, we have evaluated the action of DDT on receptor for the FSH (follicle stimulating hormone). Indeed, DDT displays certain structural similarities with small molecules used by the pharmaceutical industry, known to have an action on FSH receptor (FSHR). DDT, for dichlorodiphenyltrichloroethane, is probably the best known pesticide. Abundantly used in the middle of the 20th century, this organochloride is now banned in most developed countries: it is indeed an EDC. However, it is still used in developing countries, and its high persistence allows it to be detected in areas where it has been banned for years. Therefore, we have revealed a new interaction of DDT with the hormonal system: the pesticide interacts with the FSHR. The FSH is a hormone essential for spermatogenesis and ovulation. Our work indicated that DDT binds to the transmembrane part of the FSH receptor and thus potentiates the action of the receptor. By using an experimental approach coupled with an in silico approach, we identified more precisely the transmembrane domains in contact with DDT. From this in vitro data it is tempting to speculate that this could have a role in the phenomena of ovarian hyperstimulation that can be seen in early pregnancy or in stimulation protocols within the framework of medical assistance to procreation. Indeed, we showed that when DDT binds to the FSH receptor, the latter becomes sensitive to hCG (hormone secreted during pregnancy). In addition, this illegitimate stimulation of the FSH receptor by hCG in utero is likely to disrupt the development of the gonads.


    3D representation of the mode of DDT binding in the transmembrane domain of the FSH receptor.

    We also demonstrated that bisphenol A (BPA), which has a structure similar to DDT, decreases the activity of the receptor. BPA is one of the most widely produced chemical compounds in the world. Thanks to its polymerization properties, it is used in polycarbonate plastics and epoxy resins, as well as in cash tickets or dental fillings. Thus, BPA appears to be a negative allosteric modulator of the FSH receptor. This effect could be one of the mechanisms of action of the BPA envisaged in its deleterious effects demonstrated in vivo on the postnatal ovary: inhibition of follicular growth, increase of the atresia phenomenon, which can then lead to primary amenorrhea. Finally, the hCG and LH receptor is structurally similar to that of FSH, and also plays a key role in controlling development and gonadal function (testosterone production, ovulation). Very interestingly, DDT blocks the action of the receptor suggesting a new mechanism of reprotoxic action of the EDC in the alteration of the establishment of the male reproductive system (cryptorchidism, hypospadias, micropenis).

    Our research thus provides new fundamental data in the understanding of the role of GPCR in the mechanisms of action of environmental compounds likely to affect endocrine system. Now, we extend our studies to the analysis of the effect of EDC on the function of metabolic GPCRs.

    Collaborations:
    • H. Abdi (University of Texas at Dallas),
    • H. Castel (INSERM U982, Rouen),
    • I. Milazzo (CNRS 6014COBRA, Rouen).
    • Grouleff J, Schiøtt B. Interdisciplinary Nanoscience Center, Center for Insoluble Protein Structures, and Department of Chemistry, Aarhus University, Aarhus, Denmark
    References:
    • Munier M, Grouleff J, Gourdin L, Fauchard M, Chantreau V, Henrion D, Coutant R, Schiøtt B, Chabbert M, Rodien P. In Vitro Effects of the Endocrine Disruptor p,p'-DDT on Human Follitropin Receptor. Environ Health Perspect. 2016 Jul;124(7):991-9. doi: 10.1289/ehp.1510006.
    • Munier M, Rodien P. Perturbateurs Endocriniens. Les voies inexplorées du DDT. Sciences et Santé. 2016. Numéro 31.
    • Pelé J., Bécu J.M., Abdi H. and Chabbert M. (2012) Bios2mds: an R package for comparing orthologous protein families by metric multidimensional scaling. BMC Bioinformatics, 13:133.
    • Chabbert M., Castel H., Pelé J., Devillé J., Legendre R. and Rodien P. (2012) Evolution of class A G-protein-coupled receptors: implications for molecular modeling. Curr. Med. Chem., 19:1110-8.
    • Pelé J., Abdi H., Moreau M., Thybert D. and Chabbert M. (2011) Multidimensional scalling reveals the main evolutionary pathways of class A G-protein coupled receptors. PLoS One 6(4):e19094.
    • Rey J., Devillé J., and Chabbert M. (2010) Structural determinants stabilizing helical distortions related to proline. J. Struct. Biol., 171: 266-76.
    • Rodien P, Beau I, Vasseur C. (2010) Ovarian hyperstimulation syndrome (OHSS) due to mutations in the follicle-stimulating hormone receptor. Ann Endocrinol 71:206-9.
    • Devillé J., Rey J. and Chabbert M. (2009) An indel in transmembrane helix 2 helps to trace molecular evolution of class A G-protein coupled receptors. J. Mol. Evol., 68: 475-89.
    • Devillé J., Rey J. and Chabbert M. (2008) Comprehensive analysis of the helix-X-helix motif in soluble proteins. PROTEINS, 72: 115-135.
    • Royer J, Lefevre-Minisini A, Caltabiano G, Lacombe T, Malthiery Y, Savagner F, Pardo L, Rodien P. (2008) The cloned equine thyrotropin receptor is hypersensitive to human chorionic gonadotropin; identification of three residues in the extracellular domain involved in ligand specificity. Endocrinology. 149:5088-96.