Interaction of the ubiquitin carboxyl terminal esterase L1 with alpha(2)-adrenergic receptors inhibits agonist-mediated p44/42 MAP kinase activation.
Neuroprotective effects of alpha(2)-adrenergic receptor (AR) agonists are mediated via the alpha(2A)AR subtype, but the molecular mechanisms underlying these actions are still not elucidated. A two-hybrid screen was performed to identify new proteins that may control alpha(2)AR receptor function and trafficking. This screen identified the ubiquitin carboxyl-terminal hydrolase-L1 (Uch-L1), a protein associated with Parkinson's disease, as alpha(2)AR interacting protein. This interaction was confirmed and evaluated by GST pull down assays demonstrating that Uch-L1 binds preferentially to the alpha(2A)AR subtype and only with less affinity to alpha(2B)AR and alpha(2C)AR. Co-immunoprecipitation of epitope-tagged proteins confirmed the specificity of this interaction in vivo. Moreover, co-transfection of a truncated G-protein coupled receptor kinase-DNA preventing alpha(2)AR phosphorylation led to an increased signal-strength of coimmunoprecipitated Uch-L1. Confocal laser microscopy showed that interaction of alpha(2A)AR and Uch-L1 occurred in the cytoplasm. alpha(2)AR agonist mediated activation of p44/42 MAP Kinase was drastically decreased in the presence of Uch-L1 indicating a functional relevance of this interaction. These findings may present a mechanism contributing to subtype-specific alpha(2)AR trafficking and a potential pathway for the neuroprotective effects of alpha(2)AR agonists.
Weber
Bernd
B
Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany. brendiee@web.de
Schaper
Christian
C
Wang
Yi
Y
Scholz
Jens
J
Bein
Berthold
B
D000595:Amino Acid Sequence; D002460:Cell Line; D006801:Humans; D047468:Immunoprecipitation; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques; D043222:Ubiquitin Thiolesterase / Q000378:metabolism* / Q000502:physiology
C547768:ADRA2A protein, human; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; C507533:Ubiquitin carboxyl-Terminal Hydrolase L-1, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D043222:Ubiquitin Thiolesterase