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


RESULTS
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.


CONCLUSIONS
Together, these results indicate that CLCb phosphorylation acts as a discriminator for the endocytosis of specific GPCRs.
Department of Biomedical Science, Centre for Membrane Interactions and Dynamics, University of Sheffield, Sheffield S10 2TN, UK.
Filipe
F
Ferreira
Matthew
M
Foley
Alex
A
Cooke
Margaret
M
Cunningham
Gemma
G
Smith
Robert
R
Woolley
Graeme
G
Henderson
Eamonn
E
Kelly
Stuart
S
Mundell
Elizabeth
E
Smythe
D000818:Animals; D033941:Clathrin Light Chains / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000502:physiology*; D004705:Endocytosis; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000378:metabolism*; D010766:Phosphorylation; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*
D033941:Clathrin Light Chains; D043562:Receptors, G-Protein-Coupled; D054769:G-Protein-Coupled Receptor Kinase 2