title: The predicted N-terminal signal sequence of the human α₂C-adrenoceptor does not act as a functional cleavable signal peptide.
abstract: The N-terminal region of the human α(2C)-adrenoceptor has a 22 amino acid sequence MASPALAAALAVAAAAGPNASG. This stretch is predicted to be a cleavable signal peptide. Signal peptides facilitate the translocation of membrane proteins from ribosomes into the endoplasmatic reticulum (ER) for further transport to the plasma membrane. However, recently it has been suggested that the hydrophobic stretch ALAAALAAAAA in the N-tail of the rat α(2C)-adrenoceptor, rather than being part of a signal peptide, is an ER retention signal (Angelotti, 2010). Here, we have investigated the functionality of the N-terminal region of the human α(2C)-adrenoceptor further. The predicted signal peptide was found to be non-cleavable, as shown for a modified α(2C)-adrenoceptor construct equipped with a FLAG epitope. The influence of the N-terminal region on receptor translocation to the plasma membrane was investigated by rebuilding the N-tail and then by analyzing the expression level of binding-competent receptors in transfected COS-7 cell membranes. Truncated α(2C)-adrenoceptor constructs showed decreased expression levels as compared to the wild type α(2C)-adrenoceptor. Addition of, or exchange for, the influenza virus hemagglutinin signal peptide to the α(2C)-adrenoceptor had no effect, respectively decreased, the expression level of binding-competent receptor in the membranes. Our analysis supports the conclusions that the predicted signal peptide in the N-terminal tail of the α(2C)-adrenoceptor does not act as a cleavable signal peptide. In addition, the results indicate that the presence of an intact N-tail is augmenting the amount of binding-competent α(2C)-adrenoceptors at the cell surface.
authors.affiliation: Section of Pharmacology, Institute of Medicine, Faculty of Medicine and Dentistry, University of Bergen, Norway. jan.jahnsen@med.uib.no
authors.forename: Jan Anker
authors.initials: JA
authors.lastname: Jahnsen
authors.forename: Staffan
authors.initials: S
authors.lastname: Uhlén
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D019329:Idazoxan / Q000031:analogs & derivatives / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D059748:Proteolysis; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D008024:Ligands; D021382:Protein Sorting Signals; D018341:Receptors, Adrenergic, alpha-2; C058525:2-methoxyidazoxan; D019329:Idazoxan