Role of arrestins in endocytosis and signaling of alpha2-adrenergic receptor subtypes.
We investigated the role of arrestins in the trafficking of human alpha2-adrenergic receptors (alpha2-ARs) and the effect of receptor trafficking on p42/p44 MAP kinase activation. alpha2-ARs expressed in COS-1 cells demonstrated a modest level of agonist-mediated internalization, with alpha2c > alpha2b > alpha2a. However, upon coexpression of arrestin-2 (beta-arrestin-1) or arrestin-3 (beta-arrestin-2), internalization of the alpha2b AR was dramatically enhanced and redistribution of receptors to clathrin coated vesicles and endosomes was observed. Internalization of the alpha2c AR was selectively promoted by coexpression of arrestin-3, while alpha2a AR internalization was only slightly stimulated by coexpression of either arrestin. Coexpression of GRK2 had no effect on the internalization of any alpha2-AR subtype, either in the presence or absence of arrestins. Internalization of the alpha2b and alpha2c ARs was inhibited by coexpression of dominant negative dynamin-K44A. However, alpha2-AR-mediated activation of either endogenous or cotransfected p42/p44 mitogen-activated protein (MAP) kinase was not affected by either dynamin-K44A or arrestin-3. Moreover, activation of p42/p44 MAP kinase by endogenous epidermal growth factor, lysophosphatidic acid, and beta2-adrenergic receptors was also unaltered by dynamin-K44A. In summary, our data suggest that internalization of the alpha2b, alpha2c, and to a lesser extent alpha2a ARs, is both arrestin- and dynamin-dependent. However, endocytosis does not appear to be required for alpha2-adrenergic, epidermal growth factor, lysophosphatidic acid, or beta2-adrenergic receptor-mediated p42/p44 MAP kinase activation in COS-1 cells.
DeGraff
J L
JL
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Gagnon
A W
AW
Benovic
J L
JL
Orsini
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000502:physiology*; D019556:COS Cells; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008246:Lysophospholipids / Q000502:physiology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D018341:Receptors, Adrenergic, alpha-2 / Q000502:physiology*; D018343:Receptors, Adrenergic, beta-2 / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*
D019390:Arrestins; D008246:Lysophospholipids; D018341:Receptors, Adrenergic, alpha-2; D018343:Receptors, Adrenergic, beta-2; D004815:Epidermal Growth Factor; D020794:Receptor Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins