Rapid agonist mediated phosphorylation of the metabotropic glutamate receptor 1α by protein kinase C in permanently transfected BHK cells

Alaluf, Simon; Mulvihill, Eileen R.; McIlhinney, R. A. Jeffrey

doi:10.1016/0014-5793(95)00575-t

Cited by 51 publications

(41 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, the antimGluR1␣ antiserum identified similar levels of a protein of ϳ150 kDa in both control and PTX-treated BHK cells. In agreement with a previous report (24), additional immunoreactive material ran toward the top of the gel (Fig. 6).…”

Section: Agonistsupporting

confidence: 93%

Enhanced Type 1α Metabotropic Glutamate Receptor-Stimulated Phosphoinositide Signaling after Pertussis Toxin Treatment

et al. 1997

View full text Add to dashboard Cite

SUMMARYThe regulation of phosphoinositide hydrolysis by the type 1␣ metabotropic glutamate receptor (mGluR1␣) was investigated in stably transfected baby hamster kidney (BHK) cells. Incubation of the cells with L-glutamate, quisqualate, and 1-aminocyclopentane-1S,3R-dicarboxylic acid resulted in a marked accumulation of 3 H]InsP 1 responses were similar in control and PTXtreated BHK-mGluR1␣ cells. These changes in the concentration-effect curves for mGluR agonists are consistent with a model in which the receptor associates with PTX-sensitive inhibitory (G i/o ) and PTX-insensitive stimulatory (G q/11 ) G proteins that can each influence PIC activity. The present observations are consistent with a dual regulation of mGluR1␣-mediated PIC activity that could be fundamental in controlling the output of phosphoinositide-derived messengers.The recent cloning of eight subtypes of mGluR has not only opened up new avenues for exploration of the central actions of this excitatory neurotransmitter but also expanded the potential to target drugs against specific receptor-mediated actions (1-3). Recently, novel synthetic glutamate analogues have been developed as ligands at different mGluRs, and of particular significance has been the development of competitive antagonists (4 -7) and their use in identifying the involvement and roles of different mGluR subtypes in fundamental mechanisms such as long term potentiation and long term depression (8 -12).The mGluRs form a distinct branch of the G protein-coupled receptor superfamily, sharing topological organization but little sequence homology with other G protein-coupled receptors. Sequence homology and pharmacological profiling have allowed three subgroups of mGluRs, termed I, II, and III, to be described (2, 3). Group II mGluRs (types 2 and 3) and group III MGluRs (types 4, 6, 7 and 8) both couple to G proteins of the G i/o family to inhibit adenylyl cyclase or modulate ion channel activities (2, 3); in contrast, group I mGluRs (types 1 and 5) activate PIC with the subsequent generation of the second messengers Ins(1,4,5)P 3 and diacylglycerol (2, 3, 13-17). The G protein or proteins responsible for coupling group I mGluRs to PIC have been the subject of some debate. Thus, although the phosphoinositide responses elicited by agonist stimulation of mGluR5 and the mGluR1␤ splice variant seem to be little affected by PTX treatment (14,15), the response to mGluR1␣ activation is substantially

show abstract

Section: Agonistsupporting

confidence: 93%

Enhanced Type 1α Metabotropic Glutamate Receptor-Stimulated Phosphoinositide Signaling after Pertussis Toxin Treatment

et al. 1997

View full text Add to dashboard Cite

show abstract

“…These data not only ®rmly establish that the AT 1A receptor is a substrate for PKC, but also indicate that PKC participates in agonist-induced phosphorylation. Similar ®ndings have been described for other receptors coupled to phospholipase C (Ali et al, 1993;Alaluf et al, 1995). However, such ®ndings are not universal.…”

Section: Receptor Phosphorylation By Angiotensin IIsupporting

confidence: 79%

Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT_1A receptor

Balmforth

Shepherd

Warburton

et al. 1997

British J Pharmacology

View full text Add to dashboard Cite

1 The role of protein kinase C (PKC) in the mechanism underlying rapid agonist-induced desensitization of angiotensin AT 1 receptors remains unresolved. A major problem has been to isolate these receptors in a su ciently puri®ed form to allow study of their phosphorylation state. 2 A cleavable (His) 6 a nity tag was introduced into the N-terminus of the recombinant AT 1A receptor and stably expressed in human embryonic kidney cells. This a nity tag allowed rapid isolation, puri®cation and determination of the phosphorylation state of the AT 1A receptor. Using these cells, we determined the role of PKC in both agonist-induced receptor phosphorylation and desensitization under identical conditions. 3 Agonist-induced phosphorylation of the AT 1A receptor was observed at both low and high concentrations of angiotensin II (AII). Preincubation of cells with Ro-31-8220 (a PKC speci®c inhibitor) revealed that at low concentrations of AII (1 nM), PKC appeared to be the main kinase involved in receptor phosphorylation. In contrast, at high concentrations of AII (100 nM), although PKC-mediated phosphorylation of the receptor was observed, this was overshadowed by a second kinase. 4 In preliminary desensitization studies we observed that at a low concentration of AII, preincubation with Ro-31-8220 attenuated desensitization, whilst at high concentrations of AII (100 nM) it had little or no e ect on the level of desensitization observed.5 These data directly demonstrate an association between PKC-induced receptor phosphorylation and desensitization at low concentrations of AII. Since circulating concentrations of AII are in the picomolar range, we propose that PKC is the physiologically relevant mediator of AT 1 receptor desensitization.

show abstract

“…Further studies using a range of recombinant G-proteinlinked receptors are presently underway to investigate the substrate specificity of MRK. Particular attention is being directed toward the metabotropic glutamate 1␣ receptor, which is abundantly expressed in Purkinje cells of the cerebellum and has recently been reported to undergo agonist-driven phosphorylation in transfected BHK cells, although in this system a role for protein kinase C has been implicated (39).…”

Section: Discussionmentioning

confidence: 99%

Identification of a Novel Receptor Kinase That Phosphorylates a Phospholipase C-linked Muscarinic Receptor

Tobin

Keys

Nahorski

1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

Phosphorylation of G-protein-linked receptors is thought to play a central role in receptor regulation and desensitization. Unlike the case of the extensively studied ␤-adrenergic receptor/adenylate cyclase pathway, in which receptor-specific phosphorylation is known to be mediated by ␤-adrenergic receptor kinase (␤-ARK), the kinases responsible for phosphorylation of phospholipase C-linked receptors have yet to be identified, although a role for ␤-ARK has been implicated. This study describes the purification of a novel 40-kDa receptor kinase from porcine cerebellum that is able to phosphorylate the phospholipase C-linked m3-muscarinic receptor in an agonist-dependent manner. The assay for kinase activity was based on the ability of the kinase to phosphorylate a bacterial fusion protein, Ex-m3, containing amino acids Ser 345 -Leu 463 of the third intracellular loop of the m3-muscarinic receptor. Purification of the muscarinic receptor kinase from a high speed supernatant fraction of porcine cerebellum was achieved using the following steps: (i) 30 -60% ammonium sulfate cut and successive chromatography on (ii) butyl-Sepharose (iii) Resource Q, (iv) Resource S, and (v) heparinSepharose. The purified protein kinase represented an ϳ18,600-fold purification and was a single polypeptide with a molecular weight of ϳ40 kDa. Based on the chromatographic mobility, molecular weight, and kinase inhibitor studies, the kinase, designated MRK, was shown to be distinct from previously characterized second messenger regulated protein kinases, ␤-ARK, and other members of the G-protein-linked receptor kinase family. It therefore represents a new class of receptor kinase.Many cell surface neurotransmitter and hormone receptors respond to agonist occupation by activation of phospholipase C. The subsequent hydrolysis of the lipid substrate phosphatidylinositol 4,5-bisphosphate releases the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) 1 and diacylglycerol (reviewed in Ref. 1). The second messenger response to a number of phospholipase C-linked receptors, including the m3-muscarinic receptor, is often complex, consisting of a burst of Ins(1,4,5)P 3 production reaching a peak within the first few seconds of receptor stimulation followed by a lower sustained phase of Ins(1,4,5)P 3 generation that is maintained for tens of minutes to hours (2-4). Similar patterns of Ins(1,4,5)P 3 generation are seen in response to agonist occupation of receptors for GRH (5), angiotensin (6), bombesin, and CCK (7).Little is known of the molecular mechanisms underlying these complex second messenger responses, although recent evidence suggests that some phospholipase C-linked receptors undergo a rapid partial desensitization that results in decreased phospholipase C activity within seconds of agonist occupation (3,4,7,8). Consistent with this notion are studies from our laboratory demonstrating that the early peak phase of Ins(1,4,5)P 3 production in response to m3-muscarinic receptor stimulation can be desensitized by a short pre-expos...

show abstract

Rapid agonist mediated phosphorylation of the metabotropic glutamate receptor 1α by protein kinase C in permanently transfected BHK cells

Cited by 51 publications

References 33 publications

Enhanced Type 1α Metabotropic Glutamate Receptor-Stimulated Phosphoinositide Signaling after Pertussis Toxin Treatment

Enhanced Type 1α Metabotropic Glutamate Receptor-Stimulated Phosphoinositide Signaling after Pertussis Toxin Treatment

Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT_1A receptor

Identification of a Novel Receptor Kinase That Phosphorylates a Phospholipase C-linked Muscarinic Receptor

Contact Info

Product

Resources

About

Rapid agonist mediated phosphorylation of the metabotropic glutamate receptor 1α by protein kinase C in permanently transfected BHK cells

Cited by 51 publications

References 33 publications

Enhanced Type 1α Metabotropic Glutamate Receptor-Stimulated Phosphoinositide Signaling after Pertussis Toxin Treatment

Enhanced Type 1α Metabotropic Glutamate Receptor-Stimulated Phosphoinositide Signaling after Pertussis Toxin Treatment

Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT1A receptor

Identification of a Novel Receptor Kinase That Phosphorylates a Phospholipase C-linked Muscarinic Receptor

Contact Info

Product

Resources

About

Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT_1A receptor