Ligand binding directly stimulates ubiquitination of the inositol 1,4,5-trisphosphate receptor

Zhu, Chenming; Wojcikiewicz, Richard J.H.

doi:10.1042/bj3480551

Cited by 27 publications

(10 citation statements)

References 40 publications

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“…260 kDa) did not co-immunoprecipitate with IP 3 R1-HA (approx. 270 kDa, long exposure; Figure 1A, lanes [11][12][13][14][15], that down-regulation of endogenous IP 3 R1 was beginning to occur (short exposure; Figure 1A, lanes 4 and 5, and, 9 and 10) and that expression of IP 3 R1-HA did not markedly increase total IP 3 R1 immunoreactivity (short exposure; Figure 1A, compare lanes 6-10 with 1-5), consistent with the relatively low transfection efficiency. Overall, these experiments indicate that exogenous IP 3 R1-HA is expressed at relatively high levels in transiently transfected αT3-1 cells and that it is ubiquitinated in response to GnRH similarly to endogenous IP 3 R1.…”

Section: Figure 2 the Properties Ubiquitination And Down-regulation mentioning

confidence: 99%

“…IP 3 R1-HA was point-mutated at sites known to modify receptor properties. Arg 265 was mutated to glutamine (R265Q), since this has been reported to block IP 3 binding [21] and should allow for a more precise analysis of the role of IP 3 binding in triggering ubiquitination than that obtained from analysing deletion mutants [15]. Glu 2100 was mutated to asparagine (E2100D), since this decreases the binding affinity of Ca 2+ to the Ca 2+ sensor [5] by approx.…”

Section: Effects Of Mutations On Ip 3 R1-ha Ubiquitination and Down-rmentioning

confidence: 99%

“…However, it does appear that IP 3 plays an important role since only those GPCRs that persistently activate phosphoinositidase C and elevate IP 3 concentration cause IP 3 R ubiquitination [12]. In addition microinjection of an IP 3 analogue into mouse oocytes causes proteasome-mediated IP 3 R down-regulation [13,14] and a mutant IP 3 R1 lacking residues 316-352, that does not bind IP 3 , is resistant to ubiquitination [15]. Whether Ca 2+ also contributes to the triggering of ubiquitination has yet to be established, but involvement is hinted at by studies showing that IP 3 R ubiquitination is blocked by thapsigargin, which inhibits the Ca 2+ -ATPase activity that fills ER Ca 2+ stores [10,12].…”

Section: Introductionmentioning

confidence: 99%

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The role of Ca2+ in triggering inositol 1,4,5-trisphosphate receptor ubiquitination

Alzayady

Wojcikiewicz

2005

Biochemical Journal

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The IP3R (inositol 1,4,5-trisphosphate receptor) forms tetrameric Ca2+ channels in ER (endoplasmic reticulum) membranes, where channel activity is largely under the control of the co-agonists IP3 and Ca2+. In cells stimulated using extracellular ligands that persistently elevate phosphoinositidase C activity, IP3Rs are rapidly ubiquitinated and then degraded by the proteasome through as yet undefined mechanisms. Whereas binding of IP3 has been suggested to be a key event in the triggering of IP3R ubiquitination the role of Ca2+ in this process remains unknown. In the present study we use alphaT3-1 mouse pituitary cells expressing exogenous wild-type or mutant-type-I IP3Rs (IP3R1) to provide several lines of evidence that Ca2+ is also a trigger. Firstly, depletion of ER Ca2+ stores with thapsigargin blocked wild-type IP3R1 ubiquitination. Secondly, ubiquitination was blocked by mutating Glu2100 to Asp, which is known to markedly suppress Ca2+-binding to IP3R1 and the potency of Ca2+ as a stimulus for channel opening. Thirdly, mutating Asp2550 to Ala, which inhibits Ca2+ flux through the channel pore, partially inhibited ubiquitination indicating that Ca2+ released via wild-type IP3R1 contributes to triggering ubiquitination. Fourthly, and consistent with this conclusion, although suppression of increases in cytoplasmic Ca2+ concentration did not inhibit the ubiquitination of wild-type IP3R1, it strongly inhibited the ubiquitination of the Asp2550 to Ala mutant. Overall, these results show that Ca2+ plays an important role in triggering IP3R ubiquitination. Additional experiments with IP3R1 containing an Arg265 to Gln mutation, which decreases IP3-binding affinity, confirmed that IP3-binding also plays a role. Finally, the mutations at Glu2100, Asp2550 and Arg265 inhibited IP3R1 degradation to an extent that paralleled their inhibitory effects on ubiquitination. We conclude that IP3R ubiquitination and degradation are triggered by the concerted action of IP3- and Ca2+-binding.

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Section: Figure 2 the Properties Ubiquitination And Down-regulation mentioning

confidence: 99%

Section: Effects Of Mutations On Ip 3 R1-ha Ubiquitination and Down-rmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The role of Ca2+ in triggering inositol 1,4,5-trisphosphate receptor ubiquitination

Alzayady

Wojcikiewicz

2005

Biochemical Journal

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“…In somatic cells, mutational studies have shown that IP 3 R-1 down-regulation is mediated by IP 3 binding, which induces ubiquitination and subsequent degradation by the proteasome [22]. A similar mechanism, therefore, is believed to operate in mammalian eggs [20,21].…”

Section: Introductionmentioning

confidence: 96%

Fertilization and Inositol 1,4,5-Trisphosphate (IP3)-Induced Calcium Release in Type-1 Inositol 1,4,5-Trisphosphate Receptor Down-Regulated Bovine Eggs1

Malcuit

Knott

et al. 2005

Biology of Reproduction

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It is widely believed that stimulation of the phosphoinositide pathway and production of 1,4,5-inositol trisphosphate (IP(3)) underlies the oscillatory changes in the concentration of intracellular free calcium ions ([Ca(2+)](i)) seen during mammalian fertilization. IP(3) promotes Ca(2+) release in eggs by binding to its receptor, the type-1 IP(3) receptor (IP(3)R-1, also known as ITPR1), a ligand-gated Ca(2+) channel located in the membrane of the endoplasmic reticulum, the main Ca(2+) store of the cell. While IP(3)R-1 has been shown to mediate all Ca(2+) release during mouse fertilization, whether or not it plays such an essential role in fertilization-induced Ca(2+) release in large domestic species such as bovine and porcine is presently not known. Accordingly, we have generated metaphase II bovine eggs with a approximately 70%-80% reduction in the number of intact IP(3)R-1 by inducing receptor down-regulation during oocyte maturation. We did so by injecting the nonhydrolyzable IP(3) analogue, adenophostin A. Functional Ca(2+) release analysis revealed that IP(3)R-1 is the predominant Ca(2+) release channel in bovine eggs, requiring as little as 20% of total intact receptor to mount persistent [Ca(2+)](i) oscillations in response to fertilization, expression of PLCzeta (also known as PLCZ1), and adenophostin A. However, lower concentrations of IP(3) and near-physiological concentrations of porcine sperm extract were unable to trigger [Ca(2+)](i) oscillations in this reduced IP(3)R-1 model. Furthermore, we present evidence that the sensitivity of bovine IP(3)R-1 is impaired at the first embryonic interphase. Together, these results demonstrate the essential role of IP(3)R-1-mediated Ca(2+) release during fertilization in bovine eggs, and identify cell cycle regulatory mechanisms of [Ca(2+)](i) oscillations at the level of IP(3)R-1.

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“…The effect was, however, associated with (and apparently dependent upon) a GnRH-stimulated reduction in the number of Ins(1, 4, 5)P 3 -Rs (Willars et al, 2001). Sustained activation of several PLCactivating GPCRs has been shown to cause Ins(1, 4, 5)P 3 -R down-regulation by a process thought to involve a Ca 2+ -dependent proteolysis of the active Ins(1, 4, 5)P 3 -R con- formation (Wojcikiewicz & Oberdorf, 1996;Zhu & Wojcikiewicz, 2000). Although this effect is typically sluggish and modest the effect was more pronounced and rapid with GnRH-R (e.g., 80% reduction within 20-30 min).…”

Section: What Desensitisation Mechanisms Operate Down-stream Of the Gmentioning

confidence: 97%

The Gonadotrophin-Releasing Hormone Receptor: Signalling, Cycling and Desensitisation

McArdle

Franklin

Green

et al. 2002

Archives of Physiology and Biochemistry

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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.

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Ligand binding directly stimulates ubiquitination of the inositol 1,4,5-trisphosphate receptor

Cited by 27 publications

References 40 publications

The role of Ca2+ in triggering inositol 1,4,5-trisphosphate receptor ubiquitination

The role of Ca2+ in triggering inositol 1,4,5-trisphosphate receptor ubiquitination

Fertilization and Inositol 1,4,5-Trisphosphate (IP3)-Induced Calcium Release in Type-1 Inositol 1,4,5-Trisphosphate Receptor Down-Regulated Bovine Eggs1

The Gonadotrophin-Releasing Hormone Receptor: Signalling, Cycling and Desensitisation

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