Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation

Filipe Ferreira, Matthew Foley, Alex Cooke, Margaret Cunningham, Gemma Smith, Robert Woolley, Graeme Henderson, Eamonn Kelly, Stuart Mundell, Elizabeth Smythe

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Signaling by transmembrane receptors such as G protein-coupled receptors (GPCRs) occurs at the cell surface and throughout the endocytic pathway, and signaling from the cell surface may differ in magnitude and downstream output from intracellular signaling. As a result, the rate at which signaling molecules traverse the endocytic pathway makes a significant contribution to downstream output. Modulation of the core endocytic machinery facilitates differential uptake of individual cargoes. Clathrin-coated pits are a major entry portal where assembled clathrin forms a lattice around invaginating buds that have captured endocytic cargo. Clathrin assembles into triskelia composed of three clathrin heavy chains and associated clathrin light chains (CLCs). Despite the identification of clathrin-coated pits at the cell surface over 30 years ago, the functions of CLCs in endocytosis have been elusive.  In this work, we identify a novel role for CLCs in the regulated endocytosis of specific cargoes. Small interfering RNA-mediated knockdown of either CLCa or CLCb inhibits the uptake of GPCRs. Moreover, we demonstrate that phosphorylation of Ser204 in CLCb is required for efficient endocytosis of a subset of GPCRs and identify G protein-coupled receptor kinase 2 (GRK2) as a kinase that can phosphorylate CLCb on Ser204. Overexpression of CLCb(S204A) specifically inhibits the endocytosis of those GPCRs whose endocytosis is GRK2-dependent.  Together, these results indicate that CLCb phosphorylation acts as a discriminator for the endocytosis of specific GPCRs.

LanguageEnglish
Pages1361-1370
Number of pages10
JournalCurrent Biology
Volume22
Issue number15
DOIs
Publication statusPublished - 7 Aug 2012

Fingerprint

Clathrin Light Chains
clathrin
Phosphorylation
endocytosis
G-Protein-Coupled Receptors
Endocytosis
Clathrin
phosphorylation
G-Protein-Coupled Receptor Kinase 2
Clathrin Heavy Chains
phosphotransferases (kinases)
Discriminators
Small Interfering RNA
Machinery
Phosphotransferases
Modulation
G-protein coupled receptors
cells
small interfering RNA
Molecules

Keywords

  • animals
  • clathrin light chains
  • clathrin-coated vesicles
  • endocytosis
  • G-protein-coupled receptor kinase 2
  • phosphorylation
  • receptors, G-protein-coupled

Cite this

Ferreira, Filipe ; Foley, Matthew ; Cooke, Alex ; Cunningham, Margaret ; Smith, Gemma ; Woolley, Robert ; Henderson, Graeme ; Kelly, Eamonn ; Mundell, Stuart ; Smythe, Elizabeth. / Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation. In: Current Biology. 2012 ; Vol. 22, No. 15. pp. 1361-1370.
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Ferreira, F, Foley, M, Cooke, A, Cunningham, M, Smith, G, Woolley, R, Henderson, G, Kelly, E, Mundell, S & Smythe, E 2012, 'Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation' Current Biology, vol. 22, no. 15, pp. 1361-1370. https://doi.org/10.1016/j.cub.2012.05.034

Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation. / Ferreira, Filipe; Foley, Matthew; Cooke, Alex; Cunningham, Margaret; Smith, Gemma; Woolley, Robert; Henderson, Graeme; Kelly, Eamonn; Mundell, Stuart; Smythe, Elizabeth.

In: Current Biology, Vol. 22, No. 15, 07.08.2012, p. 1361-1370.

Research output: Contribution to journalArticle

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T1 - Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation

AU - Ferreira, Filipe

AU - Foley, Matthew

AU - Cooke, Alex

AU - Cunningham, Margaret

AU - Smith, Gemma

AU - Woolley, Robert

AU - Henderson, Graeme

AU - Kelly, Eamonn

AU - Mundell, Stuart

AU - Smythe, Elizabeth

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N2 - Signaling by transmembrane receptors such as G protein-coupled receptors (GPCRs) occurs at the cell surface and throughout the endocytic pathway, and signaling from the cell surface may differ in magnitude and downstream output from intracellular signaling. As a result, the rate at which signaling molecules traverse the endocytic pathway makes a significant contribution to downstream output. Modulation of the core endocytic machinery facilitates differential uptake of individual cargoes. Clathrin-coated pits are a major entry portal where assembled clathrin forms a lattice around invaginating buds that have captured endocytic cargo. Clathrin assembles into triskelia composed of three clathrin heavy chains and associated clathrin light chains (CLCs). Despite the identification of clathrin-coated pits at the cell surface over 30 years ago, the functions of CLCs in endocytosis have been elusive.  In this work, we identify a novel role for CLCs in the regulated endocytosis of specific cargoes. Small interfering RNA-mediated knockdown of either CLCa or CLCb inhibits the uptake of GPCRs. Moreover, we demonstrate that phosphorylation of Ser204 in CLCb is required for efficient endocytosis of a subset of GPCRs and identify G protein-coupled receptor kinase 2 (GRK2) as a kinase that can phosphorylate CLCb on Ser204. Overexpression of CLCb(S204A) specifically inhibits the endocytosis of those GPCRs whose endocytosis is GRK2-dependent.  Together, these results indicate that CLCb phosphorylation acts as a discriminator for the endocytosis of specific GPCRs.

AB - Signaling by transmembrane receptors such as G protein-coupled receptors (GPCRs) occurs at the cell surface and throughout the endocytic pathway, and signaling from the cell surface may differ in magnitude and downstream output from intracellular signaling. As a result, the rate at which signaling molecules traverse the endocytic pathway makes a significant contribution to downstream output. Modulation of the core endocytic machinery facilitates differential uptake of individual cargoes. Clathrin-coated pits are a major entry portal where assembled clathrin forms a lattice around invaginating buds that have captured endocytic cargo. Clathrin assembles into triskelia composed of three clathrin heavy chains and associated clathrin light chains (CLCs). Despite the identification of clathrin-coated pits at the cell surface over 30 years ago, the functions of CLCs in endocytosis have been elusive.  In this work, we identify a novel role for CLCs in the regulated endocytosis of specific cargoes. Small interfering RNA-mediated knockdown of either CLCa or CLCb inhibits the uptake of GPCRs. Moreover, we demonstrate that phosphorylation of Ser204 in CLCb is required for efficient endocytosis of a subset of GPCRs and identify G protein-coupled receptor kinase 2 (GRK2) as a kinase that can phosphorylate CLCb on Ser204. Overexpression of CLCb(S204A) specifically inhibits the endocytosis of those GPCRs whose endocytosis is GRK2-dependent.  Together, these results indicate that CLCb phosphorylation acts as a discriminator for the endocytosis of specific GPCRs.

KW - animals

KW - clathrin light chains

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KW - endocytosis

KW - G-protein-coupled receptor kinase 2

KW - phosphorylation

KW - receptors, G-protein-coupled

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