Selective recruitment of arrestin-3 to clathrin coated pits upon stimulation of G protein-coupled receptors.
Non-visual arrestins (arrestin-2 and arrestin-3) play critical roles in the desensitization and internalization of many G protein-coupled receptors. In vitro experiments have shown that both non-visual arrestins bind with high and approximately comparable affinities to activated, phosphorylated forms of receptors. They also exhibit high affinity binding, again of comparable magnitude, to clathrin. Further, agonist-promoted internalization of many receptors has been found to be stimulated by exogenous over-expression of either arrestin2 or arrestin3. The existence of multiple arrestins raises the question whether stimulated receptors are selective for a specific endogenous arrestin under more physiological conditions. Here we address this question in RBL-2H3 cells, a cell line that expresses comparable levels of endogenous arrestin-2 and arrestin-3. When (beta)(2)-adrenergic receptors are stably expressed in these cells the receptors internalize efficiently following agonist stimulation. However, by immunofluorescence microscopy we determine that only arrestin-3, but not arrestin-2, is rapidly recruited to clathrin coated pits upon receptor stimulation. Similarly, in RBL-2H3 cells that stably express physiological levels of m1AChR, the addition of carbachol selectively induces the localization of arrestin-3, but not arrestin-2, to coated pits. Thus, this work demonstrates coupling of G protein-coupled receptors to a specific non-visual arrestin in an in vivo setting.
Santini
F
F
Department of Microbiology and Immunology and the Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA. jim.keen@mail.tju.edu
Penn
R B
RB
Gagnon
A W
AW
Benovic
J L
JL
Keen
J H
JH
D019390:Arrestins / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D043684:Receptor, Adenosine A3; D043643:Receptor, Muscarinic M1; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism; D018047:Receptors, Purinergic P1 / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014407:Tumor Cells, Cultured
D019390:Arrestins; D002966:Clathrin; D010750:Phosphoproteins; D043684:Receptor, Adenosine A3; D043643:Receptor, Muscarinic M1; D018343:Receptors, Adrenergic, beta-2; D011956:Receptors, Cell Surface; D011976:Receptors, Muscarinic; D018047:Receptors, Purinergic P1; C082333:arrestin3; D019204:GTP-Binding Proteins