Role of G protein-coupled receptor kinase 4 and β-arrestin 1 in agonist-stimulated metabotropic glutamate receptor 1 internalization and activation of mitogen-activated protein kinases

Luisa Iacovelli, Lorena Salvatore, Loredana Capobianco, Antonietta Picascia, Eliana Barletta, Marianna Storto, Stefania Mariggiò, Michele Sallese, Antonio Porcellini, Ferdinando Nicoletti, Antonio De Blasi

Research output: Contribution to journalArticle

Abstract

The metabotropic glutamate 1 (mGlu1) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu1 receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu1 receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Gαq. In cells transfected with GRK4 and exposed to agonist, β-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu1 receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of β-arrestin V53D dominant negative mutant, which did not affect the mGlu1 receptor internalization, reduced by 70-80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu1 receptor. The agonist-stimulated differential sorting of the mGlu1 receptor and β-arrestin as well as the activation of MAP kinases by mGlu1 agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of β-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing β-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu1 receptor by different mechanisms and that β-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule.

Original languageEnglish
Pages (from-to)12433-12442
Number of pages10
JournalJournal of Biological Chemistry
Volume278
Issue number14
DOIs
Publication statusPublished - Apr 4 2003

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G-Protein-Coupled Receptor Kinase 4
Arrestin
Mitogen-Activated Protein Kinases
Chemical activation
Purkinje Cells
Glutamic Acid
Phosphotransferases
G-Protein-Coupled Receptor Kinase 2
G-Protein-Coupled Receptor Kinases
Excitatory Amino Acid Agonists
metabotropic glutamate receptor type 1
HEK293 Cells
Cell membranes
Sorting
Adenoviridae

ASJC Scopus subject areas

  • Biochemistry

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Role of G protein-coupled receptor kinase 4 and β-arrestin 1 in agonist-stimulated metabotropic glutamate receptor 1 internalization and activation of mitogen-activated protein kinases. / Iacovelli, Luisa; Salvatore, Lorena; Capobianco, Loredana; Picascia, Antonietta; Barletta, Eliana; Storto, Marianna; Mariggiò, Stefania; Sallese, Michele; Porcellini, Antonio; Nicoletti, Ferdinando; De Blasi, Antonio.

In: Journal of Biological Chemistry, Vol. 278, No. 14, 04.04.2003, p. 12433-12442.

Research output: Contribution to journalArticle

Iacovelli, Luisa ; Salvatore, Lorena ; Capobianco, Loredana ; Picascia, Antonietta ; Barletta, Eliana ; Storto, Marianna ; Mariggiò, Stefania ; Sallese, Michele ; Porcellini, Antonio ; Nicoletti, Ferdinando ; De Blasi, Antonio. / Role of G protein-coupled receptor kinase 4 and β-arrestin 1 in agonist-stimulated metabotropic glutamate receptor 1 internalization and activation of mitogen-activated protein kinases. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 14. pp. 12433-12442.
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AU - Capobianco, Loredana

AU - Picascia, Antonietta

AU - Barletta, Eliana

AU - Storto, Marianna

AU - Mariggiò, Stefania

AU - Sallese, Michele

AU - Porcellini, Antonio

AU - Nicoletti, Ferdinando

AU - De Blasi, Antonio

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N2 - The metabotropic glutamate 1 (mGlu1) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu1 receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu1 receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Gαq. In cells transfected with GRK4 and exposed to agonist, β-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu1 receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of β-arrestin V53D dominant negative mutant, which did not affect the mGlu1 receptor internalization, reduced by 70-80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu1 receptor. The agonist-stimulated differential sorting of the mGlu1 receptor and β-arrestin as well as the activation of MAP kinases by mGlu1 agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of β-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing β-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu1 receptor by different mechanisms and that β-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule.

AB - The metabotropic glutamate 1 (mGlu1) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu1 receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu1 receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Gαq. In cells transfected with GRK4 and exposed to agonist, β-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu1 receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of β-arrestin V53D dominant negative mutant, which did not affect the mGlu1 receptor internalization, reduced by 70-80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu1 receptor. The agonist-stimulated differential sorting of the mGlu1 receptor and β-arrestin as well as the activation of MAP kinases by mGlu1 agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of β-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing β-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu1 receptor by different mechanisms and that β-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule.

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