Abstract
The adrenocorticotropin (ACTH) receptor (melanocortin 2 receptor, or MC2R) is the smallest G-protein-coupled receptor that, when activated by the peptide hormone ACTH, stimulates cAMP production and adrenal steroidogenesis. Receptor expression is dependent on a specific membrane trafficking process involving an accessory protein (melanocortin 2 receptor accessory protein, or MRAP) and other unidentified components. In an attempt to discover novel receptor interacting proteins, the C-terminal tail of the MC2R was used to screen a mouse adrenal Y6 cell cDNA library using the bacterial two-hybrid system. This identified the nucleoporin Nup 50 (Npap60) as the major full-length interacting protein. Interaction was confirmed by a GST pulldown assay and by coimmunoprecipitation in human H295R cells (which express both proteins endogenously). Deletion analysis identified the region between residues 143 and 466 in Nup50 as being required for interaction with the MC2R. Stimulation of H295R cells with ACTH (10(-6) M) was followed by a gradual translocation of the Nup50-MC2R complex from the membrane to the nucleus after 30 min. This time course is most consistent with MC2R internalization dynamics and may suggest a novel role for Nup50.