The effect of N-linked glycosylation on activity of the Na(+)- and Cl(-)-dependent serotonin transporter expressed using recombinant baculovirus in insect cells.

摘要: The rat Na(+)- and Cl(-)-dependent serotonin transporter was expressed in Sf9 insect cells using the baculovirus system. Expression of caused to accumulate [3H]serotonin (Km 78 nM) bind specific transport inhibitor [125I]RT155 (2 beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane) (Kd 0.22 nM). Ligand binding assays on isolated membranes showed 500,000 copies transporter/cell (9 pmol/mg membrane protein). Immunoreactive bands apparent M(r) 54,000 (unglycosylated) 60,000 (glycosylated) were observed Western blots proteins from infected cells. 54-kDa band significantly smaller than expected 72,500 predicted cDNA sequence. shown represent intact by expressing a recombinant that contained c-Myc FLAG epitope tags engineered at N C termini, respectively. Both present protein migrated slightly slower previously band, consistent with extra mass added tags. did not affect Kd for binding. effect N-linked glycosylation ligand level expression studied. tunicamycin-treated resulted low levels activity (0.2 pmol/mg) but unchanged Kd. Similarly, mutated transporters reduced numbers sites had whether there 2, 1, or 0 transporter. In contrast, Bmax dramatically reduced; unglycosylated (0.4 20-fold lower compared fully glycosylated Km uptake also unchanged. These data indicate is required optimal stability per se.