BrisSynBio: a BBSRC/EPSRC-funded Synthetic Biology Research Centre.

BrisSynBio is the Bristol-based Biotechnology and Biological Sciences Research Council (BBSRC)/Engineering and Physical Sciences Research Council (EPSRC)-funded Synthetic Biology Research Centre. It is one of six such Centres in the U.K. BrisSynBio's emphasis is on rational and predictive bimolecular modelling, design and engineering in the context of synthetic biology. It trains the next generation of synthetic biologists in these approaches, to facilitate translation of fundamental synthetic biology research to industry and the clinic, and to do this within an innovative and responsible research framework.

Plasma membrane expression of GnRH receptors: regulation by antagonists in breast, prostate, and gonadotrope cell lines.

In heterologous expression systems, human GnRH receptors (hGnRHRs) are poorly expressed at the cell surface and this may reflect inefficient exit from the endoplasmic reticulum. Here, we have defined the proportion of GnRHRs at the cell surface using a novel assay based on adenoviral transduction with epitope-tagged GnRHRs followed by staining and semi-automated imaging. We find that in MCF7 (breast cancer) cells, the proportional cell surface expression (PCSE) of hGnRHRs is remarkably low (<1%), when compared with Xenopus laevis (X) GnRHRs ( approximately 40%). This distinction is retained at comparable whole cell expression levels, and the hGnRHR PCSE is increased by addition of the XGnRHR C-tail (h.XGnRHR) or by a membrane-permeant pharmacological chaperone (IN3). The IN3 effect is concentration- and time-dependent and IN3 also enhances the hGnRHR-mediated (but not h.XGnRHR- or mouse GnRHR-mediated) stimulation of [(3)H]inositol phosphate accumulation and the hGnRHR-mediated reduction in cell number. We also find that the PCSE for hGnRHRs and h.XGnRHRs is low and is greatly increased by IN3 in two hormone-dependent cancer lines, but is higher and less sensitive to IN3 in a gonadotrope line. Finally, we show that the effect of IN3 on hGnRHR PCSE is not mimicked or blocked by two peptide antagonists although they do increase the PCSE for h.XGnRHRs, revealing that an antagonist-occupied cell surface GnRHR conformation can differ from that of the unoccupied receptor. The low PCSE of hGnRHRs and this novel peptide antagonist effect may be important for understanding GnRHR function in extrapituitary sites.

Intracellular gonadotropin-releasing hormone receptors in breast cancer and gonadotrope lineage cells.

Gonadotropin-releasing hormone receptors (GnRHRs) are expressed in gonadotropes and several extra-pituitary sites. They are assumed to be cell surface proteins but the human (h) GnRHR lacks features favoring plasma membrane localization and receptor location varies with cell type. When expressed in mammary (MCF7) cells, cell surface hGnRHR binding was much lower than that of mouse and sheep GnRHRs (type I GnRHRs without C-terminal tails), Xenopus (X) and marmoset type II GnRHRs (type II GnRHRs with C-tails) or chimeric receptors (type I GnRHRs with added XGnRHR C-tails). hGnRHR binding was higher in alphaT4 (gonadotrope-derived) cells and was increased less by C-tail addition. Whole cell levels of tagged human, Xenopus and chimeric GnRHRs were comparable (Western blotting) and confocal microscopy revealed that the hGnRHR is primarily intracellular (distribution similar to the endoplasmic reticulum marker, calreticulin), whereas most XGnRHR is at the plasma membrane, and adding the C-tail increased cell surface hGnRHR levels. A membrane-permeant antagonist increased cell surface hGnRHR number (>4-fold, t1/2 = 4 h) and also increased hGnRHR signaling and hGnRHR-mediated inhibition of proliferation. A more rapid increase in hGnRHR binding occurred when the temperature was raised from 4 to 37 degrees C (>5-fold, t1/2 = 15 min) and this effect was prevented by mutation to prevent signaling. Thus, cell surface GnRHR expression depends on receptor and cell type and the hGnRHR is primarily an intracellular protein that traffics to the cell surface for signaling in MCF7 cells. Manipulations favoring such trafficking may facilitate selective targeting of extra-pituitary GnRHRs.

Seven-transmembrane receptor signalling and ERK compartmentalization.

Vast numbers of extracellular signalling molecules exert effects on their target cells by activation of a relatively limited number of mitogen-activated protein kinase (MAPK) cascades, raising the question of how specificity is achieved. To a large extent, this appears to be attributable to differences in kinetics and compartmentalization of MAPK protein activation that are dictated by MAPK-associated proteins serving as scaffolds, anchors, activators or effectors. Here, we review spatiotemporal aspects of signalling via the Ras-Raf-extracellular signal-regulated kinase pathway, emphasizing recent work on roles of arrestins as scaffolds and transducers for seven transmembrane receptor signalling.

GnRH receptor signalling to ERK: kinetics and compartmentalization.

Many hormones, neurotransmitters and growth factors influence their target cells by activation of mitogen-activated protein kinase cascades. The consequences of such activation reflect not only the magnitude, but also the kinetics and cellular compartmentalization of kinase activity. Gonadotropin-releasing hormone (GnRH) receptors are seven-transmembrane receptors that have undergone a period of rapidly accelerated molecular evolution in which the advent of type I mammalian GnRH receptors has been associated with the loss of the carboxyl-terminal tail, a structure present in all other seven-transmembrane receptors. Here, we review spatiotemporal aspects of extracellular-signal-regulated kinase activation by gonadotropin-releasing hormone receptors, emphasizing how the absence or presence of the carboxyl-terminal tail dictates the receptors' ability to engage and signal via arrestins.

Arrestin-mediated ERK activation by gonadotropin-releasing hormone receptors: receptor-specific activation mechanisms and compartmentalization.

Activation of seven-transmembrane region receptors typically causes their phosphorylation with consequent arrestin binding and desensitization. Arrestins also act as scaffolds, mediating signaling to Raf and ERK and, for some receptors, inhibiting nuclear translocation of ERK. GnRH receptors (GnRHRs) act via Gq/11 to stimulate the phospholipase C/Ca2+/protein kinase C (PKC) cascade and the Raf/MEK/ERK cassette. Uniquely, type I mammalian GnRHRs lack the C-tails that are found in other seven-transmembrane region receptors (including nonmammalian GnRHRs) and are implicated in arrestin binding. Here we have compared ERK signaling by human GnRHRs (hGnRHRs) and Xenopus GnRHRs (XGnRHRs). In HeLa cells, XGnRHRs underwent rapid and arrestin-dependent internalization and caused arrestin/green fluorescent protein (GFP) translocation to the membrane and endosomes, whereas hGnRHRs did not. Internalized XGnRHRs were co-localized with arrestin-GFP, whereas hGnRHRs were not. Both receptors mediated transient ERK phosphorylation and nuclear translocation (revealed by immunohistochemistry or by imaging of co-transfected ERK2-GFP), and for both, ERK phosphorylation was reduced by PKC inhibition but not by inhibiting epidermal growth factor receptor autophosphorylation. In the presence of PKC inhibitor, Deltaarrestin-(319-418) blocked XGnRHR-mediated, but not hGnRHR-mediated, ERK phosphorylation. When receptor number was varied, hGnRHRs activated phospholipase C and ERK more efficiently than XGnRHRs but were less efficient at causing ERK2-GFP translocation. At high receptor number, XGnRHRs and hGnRHRs both caused ERK2-GFP translocation to the nucleus, but at low receptor number, XGnRHRs caused ERK2-GFP translocation, whereas hGnRHRs did not. Thus, experiments with XGnRHRs have revealed the first direct evidence of arrestin-mediated (probably G protein-independent) GnRHR signaling, whereas those with hGnRHRs imply that scaffolds other than arrestins can determine GnRHR effects on ERK compartmentalization.

Internalization of gonadotropin-releasing hormone receptors (GnRHRs): does arrestin binding to the C-terminal tail target GnRHRs for dynamin-dependent internalization?

Activation of seven-transmembrane receptors is typically followed by desensitization and arrestin-dependent internalization via vesicles that are pinched off by a dynamin collar. Arrestins also scaffold Src, which mediates dynamin-dependent internalization of beta2-adrenergic receptors. Type I mammalian gonadotropin-releasing hormone receptors (GnRHRs) do not rapidly desensitize or internalize (characteristics attributed to their unique lack of C-terminal tails) whereas non-mammalian GnRHRs (that have C-terminal tails) are rapidly internalized and desensitized. Moreover, internalization of Xenopus (X) GnRHRs is dynamin-dependent whereas that of human (h) GnRHRs is not, raising the possibility that binding of arrestin to the C-terminal tails of GnRHRs targets them to the dynamin-dependent internalization pathway. To test this we have compared wild-type GnRHRs with chimeric receptors (XGnRHR C-terminal tail added to the hGnRHR alone (h.XtGnRHR) or with exchange of the third intracellular loops (h.Xl.XtGnRHR)). We show that adding the XGnRHR C-terminal tail facilitates arrestin- and dynamin-dependent internalization as well as arrestin/green fluorescent protein translocation, but Src (or mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase) inhibition does not slow internalization, and h.XtGnRHR internalization is slower than that of the hGnRHR. Moreover, arrestin expression increased XGnRHR internalization even when dynamin was inhibited and h.Xl.XtGnRHR underwent rapid arrestin-dependent internalization without signaling to G(q/11). Thus, although the C-terminal tail can direct GnRHRs for arrestin- and dynamin-dependent internalization, this effect is not dependent on Src activation and arrestin can also facilitate dynamin-independent internalization.

Regulation of gonadotropin-releasing hormone receptors by protein kinase C: inside out signalling and evidence for multiple active conformations.

Desensitization and internalization of G protein-coupled receptors can be mediated by phosphorylation within the C-terminal tail, facilitating beta-arrestin binding and targeting the receptor for internalization. Type II GnRH receptors (GnRH-Rs) show such regulation, but type I GnRH-Rs lack C-tails and are not rapidly desensitized or internalized. Here we show contrasting susceptibility of type I (human and sheep) and II (Xenopus) GnRH-Rs to regulation by protein kinase C (PKC). When human (h) or Xenopus (X) GnRH-Rs were expressed using recombinant adenovirus, PKC activation increased radioligand binding to XGnRH-Rs but not to hGnRH-Rs. A dominant-negative dynamin mutant (K44A) inhibited internalization of XGnRH-Rs (but not hGnRH-Rs) without influencing PKC regulation of XGnRH-R binding. PKC activation increased the affinity of XGnRH-Rs for the type II GnRH ligand and increased effects of low concentrations of GnRH-II on the [Ca(2+)](i) but had no effect on type I ligand binding to hGnRH-Rs, sGnRH-Rs or XGnRH-Rs, or to chimeric receptors with the XGnRH-R C-tail added to a type I receptor. Binding of type II ligand to human or sheep receptors was also unaffected but was increased in the chimeras. Mutation of both PKC-phosphorylation consensus sites in the XGnRH-R tail did not prevent the PKC-mediated increases in binding or alter agonist-induced translocation of beta-arrestin2/green fluorescent protein or inhibition of inositol phosphate accumulation by beta-arrestin2/green fluorescent protein. Thus, it appears that there are two distinct active conformations of XGnRH-Rs (differing in affinity for type I and II ligands) and that these cells exhibit a novel form of inside-out signaling in which PKC feeds back to influence receptor affinity.