GTP‐Binding–Protein‐Coupled Receptor Kinases Two Mechanistic Models

Palczewski, Krzysztof

doi:10.1111/j.1432-1033.1997.00261.x

Cited by 108 publications

(98 citation statements)

References 103 publications

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“…Phosphorylation of rhodopsin is involved in lowering the catalytic efficiency of photoactivated rhodopsin in the activation of Gt (60,61). These authors suggest that most of the PKC in photoreceptor outer segments is of the conventional type and that most, if not all, of this conventional PKC activity comes from a novel isozyme(s) (34).…”

Section: Discussionmentioning

confidence: 96%

Identification of Protein Kinase C Isozymes Responsible for the Phosphorylation of Photoreceptor-specific RGS9-1 at Ser475

Sokal

Liang

et al. 2003

Journal of Biological Chemistry

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Inactivation of the visual G-protein transducin by GTP hydrolysis is regulated by the GTPase-accelerating protein (GAP) RGS9-1. Regulation of RGS9-1 itself is poorly understood, but we found previously that it is subject to a light-and Ca 2؉ -sensitive phosphorylation on Ser 475 . Because there are much higher RGS9-1 levels in cones than in rods, we investigated whether Ser 475 is phosphorylated in rods using Coneless mice and found that both the phosphorylation and its regulation by light occur in rods. Therefore, we used rod outer segments as the starting material for the purification of RGS9-1 kinase activity. Two major peaks of activity corresponded to protein kinase C (PKC) isozymes, PKC␣ and PKC. A synthetic peptide corresponding to the Ser 475 RGS9-1 sequence and RGS9-1 were substrates for recombinant PKC␣ and PKC. This phosphorylation was removed efficiently by protein phosphatase 2A, an endogenous phosphatase in rod outer segments, but not by PP1 or PP2B. Phosphorylation of RGS9-1 by PKC had little effect on its activity in solution but significantly decreased its affinity for its membrane anchor protein and GAP enhancer, RGS9-1 anchor protein (R9AP). PKC immunostaining was at higher levels in cone outer segments than in rod outer segments, as was found for the components of the RGS9-1 GAP complex. Thus, PKCmediated phosphorylation of RGS9-1 represents a potential mechanism for feedback control of the kinetics of photoresponse recovery in both rods and cones, with this mechanism probably especially important in cones.Transducin (Gt), 1 the visual G-protein, is inactivated when its bound GTP is hydrolyzed to GDP, a free phosphate dissociates from the catalytic site, and the ␣ subunit reassociates with its partner ␤␥ subunits. The rate of hydrolysis is regulated by the protein's association with a GTPase-accelerating protein (GAP). This GAP has been identified to be RGS9-1 (1), a member of a family of regulatory proteins for G␣ GAPs termed the regulators of G-protein signaling (RGS) family (reviewed in Refs. 2 and 3). The GAP activity of RGS9-1 is further enhanced by the ␥-subunit of the phototransduction effector cGMP phosphodiesterase (PDE␥) (1, 4). The acceleration of GTPase activity is essential for timely recovery of dark conditions of photoreceptor cells (5) (for recent reviews, see Refs. 6 and 7).RGS9-1 contains a G-protein-␥-subunit-like domain (8) that is employed for functional association with the G␤5L subunit (9 -12). This subunit regulates GAP activity by the effector subunit, PDE␥, and induces and stabilizes RGS9-1 (5, 10, 13). ROS membranes from knockout mice lacking RGS9 hydrolyze GTP more slowly than ROS membranes from control mice. Moreover, in electrophysiological measurements, the flash responses of RGS9Ϫ/Ϫ rods recovered much more slowly than normal after illumination (5). Alternative splicing of the RGS9 gene yields two molecular forms of RGS9, RGS9-1 and RGS9-2, that vary only in the amino acid sequence of the C terminus. RGS9-1 is a retina-specific transcript, whereas RGS9-2 is ex...

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Section: Discussionmentioning

confidence: 96%

Identification of Protein Kinase C Isozymes Responsible for the Phosphorylation of Photoreceptor-specific RGS9-1 at Ser475

Sokal

Liang

et al. 2003

Journal of Biological Chemistry

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“…Among the known GRKs that phosphorylate agonist occupied GPCRs, two classes have been identified. The first includes GRK2 and GRK3, which have domains for binding to ␤␥ subunits of G-protein, and their enzymatic activity is potentiated by the ␤␥ subunit upon activation and dissociation of the heterotrimeric G protein (32, 33); and the second includes GRK1, GRK4, GRK5, and GRK6, which do not have sites for binding to ␤␥ subunits (34). The PTX treatment did not affect CP-55,940-induced Ser 352 CB 2 phosphorylation, indicating that there is no involvement of the first class of GRK.…”

Section: Discussionmentioning

confidence: 99%

Regulation of Peripheral Cannabinoid Receptor CB2Phosphorylation by the Inverse Agonist SR 144528

Bouaboula

Dussossoy

Casellas

1999

Journal of Biological Chemistry

100

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We recently demonstrated that the selective cannabinoid receptor antagonist SR 144528 acts as an inverse agonist that blocks constitutive mitogen-activated protein kinase activity coupled to the spontaneous autoactivated peripheral cannabinoid receptor (CB 2 ) in the Chinese hamster ovary cell line stably transfected with human CB 2 . In the present report, we studied the effect of SR 144528 on CB 2 phosphorylation. The CB 2 phosphorylation status was monitored by immunodetection using an antibody specific to the COOH-terminal CB 2 which can discriminate between phosphorylated and non-phosphorylated CB 2 isoforms at serine 352. We first showed that CB 2 is constitutively active, phosphorylated, and internalized at the basal level. By blocking autoactivated receptors, inverse agonist SR 144528 treatment completely inhibited this phosphorylation state, leading to an up-regulated CB 2 receptor level at the cell surface, and enhanced cannabinoid agonist sensitivity for mitogen-activated protein kinase activation of Chinese hamster ovary-CB 2 cells. After acute agonist treatment, serine 352 was extensively phosphorylated and maintained in this phosphorylated state for more than 8 h after agonist treatment. The cellular responses to CP-55,940 were concomitantly abolished. Surprisingly, CP-55,940-induced CB 2 phosphorylation was reversed by SR 144528, paradoxically leading to a nonphosphorylated CB 2 which could then be fully activated by CP-55,940. The process of CP-55,940-induced receptor phosphorylation followed by SR 144528-induced receptor dephosphorylation kept recurring many times on the same cells, indicating that the agonist switches the system off but the inverse agonist switches the system back on. Finally, we showed that autophosphorylation and CP-55,940-induced serine 352 CB 2 phosphorylation involve an acidotropic GRK kinase, which does not use G i ␤␥. In contrast, SR 144528-induced CB 2 dephosphorylation was found to involve an okadaic acid and calyculin A-sensitive type 2A phosphatase. Several studies revealed that receptor activation can occur spontaneously in the absence of an agonist. This discovery led to a reclassification of antagonists as neutral antagonists or inverse agonists. Neutral antagonists block agonist action without any effect on constitutive activity (16 -19), whereas agonists block agonist action but also suppress constitutive activity.We recently demonstrated the agonist-independent activity of CB 1 and CB 2 receptors expressed in mammalian cells following transfection (20,21). We also showed that the CB 1 antagonist SR141716 and the CB 2 antagonist SR 144528 not only block the actions of cannabinoid agonists but they also suppress the constitutive activity of these receptors, indicating that these molecules act as inverse agonists. Furthermore, we revealed for the first time a novel property of these inverse agonists. We demonstrated that they also switch off the activation induced by other unrelated G i -dependent receptors such as insulin or insulin-like growth factor-1 receptors...

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“…Because GRK2 and GRK5 also preferentially phosphorylate serine or threonine residues surrounded by acidic amino acids (Palczewski, 1997), we also performed an in vitro phosphorylation reactions with purified active GRK2 and GRK5. GRK2 and GRK5 phosphorylated NR2B Ser1480 very weakly compared with the same amount of CK2 (Fig.…”

Section: Synaptic Nmdar Activation Increases Ck2 Phosphorylation Of Nmentioning

confidence: 99%

Regulation of the NMDA Receptor Complex and Trafficking by Activity-Dependent Phosphorylation of the NR2B Subunit PDZ Ligand

Chung¹,

Huang²,

Lau³

et al. 2004

J. Neurosci.

264

243

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GTP‐Binding–Protein‐Coupled Receptor Kinases Two Mechanistic Models

Cited by 108 publications

References 103 publications

Identification of Protein Kinase C Isozymes Responsible for the Phosphorylation of Photoreceptor-specific RGS9-1 at Ser475

Identification of Protein Kinase C Isozymes Responsible for the Phosphorylation of Photoreceptor-specific RGS9-1 at Ser475

Regulation of Peripheral Cannabinoid Receptor CB2Phosphorylation by the Inverse Agonist SR 144528

Regulation of the NMDA Receptor Complex and Trafficking by Activity-Dependent Phosphorylation of the NR2B Subunit PDZ Ligand

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