Sustained stimulation of G-protein-coupled receptors (GPCRs) typically causes receptor desensitisation, which is mediated by phosphorylation, often within the C-terminal tail of the receptor. The consequent binding of -arrestin not only prevents the receptor from activating its G protein (causing desensitisation), but can also target it for internalisation via clathrin-coated vesicles and can mediate signalling to proteins regulating endocytosis and mitogenactivated protein kinase (MAPK) cascades. GnRH acts via phospholipase C (PLC)-coupled GPCRs on pituitary gonadotrophs to stimulate a Ca 2+ -mediated increase in gonadotrophin secretion. The type I GnRH receptors (GnRH-Rs), found only in mammals, are unique in that they lack C-terminal tails and apparently do not undergo agonist-induced phosphorylation or bind -arrestin; they are therefore resistant to receptor desensitisation and internalise slowly. In contrast, the type II GnRH-Rs, found in numerous vertebrates, possess such tails and show rapid desensitisation and internalisation, with concomitant receptor phosphorylation (within the C-terminal tails) or binding of -arrestin, or both. The association with -arrestin may also be important for regulation of dynamin, a GTPase that controls separation of endosomes from the plasma membrane. Using recombinant adenovirus to express GnRH-Rs in Hela cells conditionally expressing a dominant negative mutant of dynamin (K44A), we have found that blockade of dynamindependent endocytosis inhibits internalisation of type II (xenopus) GnRH-Rs but not type I (human) GnRH-Rs. In these cells, blockade of dynamin-dependent internalisation also inhibited GnRH-R-mediated MAPK activation, but this effect was not receptor specific and therefore not dependent upon dynamin-regulated GnRH-R internalisation. Although type I GnRH-Rs do not desensitise, sustained activation of GnRH-Rs causes desensitisation of gonadotrophin secretion, and we have found that GnRH can cause down-regulation of inositol (1,4,5) trisphosphate receptors and desensitisation of Ca 2+ mobilisation in pituitary cells. The atypical resistance of the GnRH-R to desensitisation may underlie its atypical efficiency at provoking this downstream adaptive response. GnRH-Rs are also expressed in several extrapituitary sites, and these may mediate direct inhibition of proliferation of hormone-dependent cancer cells. Infection with type I GnRH-R-expressing adenovirus facilitated expression of high-affinity, PLC-coupled GnRH-R in mammary and prostate cancer cells, and these mediated pronounced antiproliferative effects of receptor agonists. No such effect was seen in cells transfected with a type II GnRH-R, implying that it is mediated most efficiently by a nondesensitising receptor. Thus it appears that the mammalian GnRH-Rs have undergone a period of rapidly accelerated molecular evolution that is of functional relevance to GnRH-Rs in pituitary and extrapituitary sites.
Although rapid-onset, short-term regulation of neuronal Ca currents by neurotransmitters and second messengers is well documented, little is known about conditions that can cause longer-lasting changes in Ca channel function. We report here that persistent depolarization is accompanied by slowly developing long-term reduction of neuronal Ca currents. Rat myenteric neurons grown in cell culture for 1-7 d were studied with the tight-seal whole-cell recording technique. Macroscopic Ca-channel currents had decaying and sustained components at all days studied. When the neurons were grown in medium containing 25 mM KCl, which depolarized them to -40 mV and caused significant elevation of intracellular Ca, the densities of both components of Ca-channel current decreased by 40-80%. Several results suggest that different mechanisms underlie the downregulation of the two components. (1) The density of the decaying component decreased approximately four times faster than did that of the sustained component. (2) When neurons were returned to control medium, which contained 5 mM KCl, the density of the sustained component returned to control levels within 24 hr, while that of the decaying component did not recover significantly. (3) Inhibitors of RNA and protein synthesis reduced or prevented downregulation of the sustained but not of the decaying component. (4) The dihydropyridine antagonist nitrendipine, which prevented the sustained elevation of intracellular Ca in neurons grown in 25 mM KCl, prevented downregulation of the sustained component but had no effect on downregulation of the decaying component. We suggest that these forms of regulation of Ca current density could help neurons adapt to altered levels of electrical activity and may contribute to changes in synaptic strength that occur during periods of increased or decreased electrical activity.
Sustained stimulation of G-protein coupled receptors (GPCRs) typically causes receptor desensitisation that is mediated by phosphorylation, often within the C-terminal tail of the receptor. The consequent binding of beta-arrestin not only prevents the receptor from activating its G-protein (causing desensitisation) but can also target it for internalisation via clathrin-coated vesicles and can mediate signalling to proteins regulating endocytosis and mitogen-activated protein kinase (MAPK) cascades. GnRH acts via phospholipase C coupled GPCRs on pituitary gonadotrophs. The type I GnRH-receptors (GnRH-Rs) found only in mammals, are unique in that they lack C-terminal tails and apparently do not undergo agonist-induced phosphorylation or bind beta-arrestin. They are therefore resistant to receptor desensitisation and internalise slowly. In contrast, the type II GnRH-Rs, found in numerous vertebrates, possess such tails and show rapid desensitisation and internalisation with concomitant receptor phosphorylation (within the C-terminal tails) and/or binding of beta-arrestin. The binding to beta-arrestin may also be important for association with dynamin, a GTPase that controls cleavage of endosomes from the plasma membrane. Using recombinant adenovirus to express GnRH-R, we have found that blockade of dynamin-dependent endocytosis inhibits internalisation of type II (Xenopus) GnRH-Rs but not type I (human) GnRH-Rs, revealing the existence of functionally distinct routes through which these receptors are internalised. Although type I GnRH-R do not rapidly desensitise, sustained activation of GnRH receptors does cause desensitisation of gonadotrophin secretion, an effect which must therefore involve adaptive responses distal to the receptor. One such response is the GnRH-induced down regulation of inositol 1, 4, 5 trisphosphate receptors that apparently underlies desensitisation of Ca2+ mobilisation in a gonadotroph-derived cell line. Although activation of other GPCRs can down-regulate inositol 1, 4, 5 trisphosphate receptors, the effect of GnRH is atypically rapid and pronounced, presumably because of the receptor's atypical resistance to desensitisation. GnRH-Rs are also expressed in several extra-pituitary sites and these may mediate direct inhibition of proliferation of hormone-dependent cancer cells. Infection with type I GnRH-R expressing adenovirus facilitated expression of high affinity, PLC-coupled GnRH-R in mammary and prostate cancer cells and these mediated pronounced antiproliferative effects of receptor agonists. No such effect was seen in cells transfected with a type II GnRH-R, implying that it is mediated most efficiently by a non-desensitising receptor. Thus it appears that the GnRH-Rs have undergone a period of rapidly accelerated molecular evolution that is of functional relevance to GnRH-R signalling in pituitary and extra-pituitary sites.
Gonadotrophin-releasing hormone receptors (GnRH-Rs) are found in cancers of reproductive tissues, including those of the prostate, and gonadotrophin-releasing hormone (GnRH) can inhibit growth of cell lines derived from such cancers. Although pituitary and extra-pituitary
1. Inward currents of myenteric neurons that had been grown in cell cultures prepared from the small intestines of neonatal or young adult rats were studied with tight seal whole-cell recordings. The kinetic and pharmacological properties of these neurons were analyzed. 2. All neurons had rapidly inactivating, tetrodotoxin (TTX)-sensitive Na+ currents that could be evoked by steps to potentials more positive than -50 mV. Holding potentials more negative than -65 mV were necessary to remove steady-state inactivation. No TTX-insensitive Na+ currents were observed, thus the ability of subsets of myenteric neurons to fire action potentials in TTX must depend upon their density of Ca2+ channels. 3. Ca2+ and Ba2+ currents were studied in neurons perfused internally with CsCl to block K+ currents and bathed with solutions containing TTX and antagonists of K+ channels. Currents were significantly larger when Ba2+ replaced Ca2+ as the charge carrier. Cd2+ and Gd3+ blocked Ca2+ and Ba2+ currents rapidly and reversibly. High-voltage-activated (HVA) Ca2+ and Ba2+ currents were observed in all neurons. Too few neurons possessed detectable low-voltage-activated Ca2+ currents to permit detailed study. 4. HVA Ca2+ and Ba2+ currents evoked from holding potentials more negative than -50 mV could be divided into two kinetically distinguishable components with very different rates of inactivation. A "decaying" component inactivated relatively rapidly with a t1/2 of 25-75 ms. A "sustained" component inactivated quite slowly with a t1/2 of 1-5 s. At more positive holding potentials, only the sustained component was observed. Although the two kinetically distinguishable components had different current-voltage relationships, they had indistinguishable rates of deactivation: a single time constant was sufficient to fit the decay of tail currents. The relative amplitudes of the two components varied considerably among different neurons. 5. Ca2+ and Ba2+ currents could be divided into two pharmacologically distinct components on the basis of sensitivity to omega-conotoxin GVIA (I omega CgTX) and to dihydropyridine antagonists (IDHP). At holding potentials more positive than -70 mV, a combination of omega CgTX and DHPs completely blocked Ca2+ and Ba2+ currents in most neurons. At holding potentials more negative than -50 mV, I omega CgTX and IDHP each contained decaying and sustained components. I omega CgTX activated more slowly than did IDHP. The DHP agonist Bay K8644 increased the amplitude of IDHP and slowed its rate of deactivation. 6. The results suggest that myenteric neurons may have as few as two subtypes of HVA Ca2+ channels; omega CgTX-sensitive ones and DHP-sensitive ones.(ABSTRACT TRUNCATED AT 400 WORDS)
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