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 α2C-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 α2C-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 α2C-adrenoceptor further. The predicted signal peptide was found to be non-cleavable, as shown for a modified α2C-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 α2C-adrenoceptor constructs showed decreased expression levels as compared to the wild type α2C-adrenoceptor. Addition of, or exchange for, the influenza virus hemagglutinin signal peptide to the α2C-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 α2C-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 α2C-adrenoceptors at the cell surface.