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J. Biol. Chem., Vol. 269, Issue 42, 26303-26310, 10, 1994
CG Tate and RD Blakely
The rat Na(+)- and Cl(-)-dependent serotonin transporter was expressed in
Sf9 insect cells using the baculovirus system. Expression of the serotonin
transporter caused the Sf9 cells to accumulate [3H]serotonin (Km 78 nM) and
to bind the 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 of the serotonin transporter/cell
(9 pmol/mg of membrane protein). Immunoreactive bands of apparent M(r)
54,000 (unglycosylated) and 60,000 (glycosylated) were observed in Western
blots of membrane proteins from infected cells. The 54-kDa band was
significantly smaller than the expected M(r) of 72,500 predicted from the
cDNA sequence. The 54-kDa band was shown to represent the intact serotonin
transporter by expressing a recombinant serotonin transporter that
contained c-Myc and FLAG epitope tags engineered at the N and C termini,
respectively. Both tags were present on a membrane protein that migrated
slightly slower than the previously observed 54-kDa band, consistent with
the extra mass added by the tags. The tags did not affect the Kd for
[125I]RT155 binding. The effect of N-linked glycosylation on ligand binding
and the level of expression were studied. The expression of the serotonin
transporter in tunicamycin-treated Sf9 cells resulted in low levels of
ligand binding activity (0.2 pmol/mg) but unchanged Kd. Similarly, mutated
serotonin transporters that contained reduced numbers of N- linked
glycosylation sites had unchanged Kd for [125I]RT155 binding whether there
were 2, 1, or 0 N-linked glycosylation sites present on the serotonin
transporter. In contrast, Bmax was dramatically reduced; levels of
expression of the unglycosylated serotonin transporter (0.4 pmol/mg) were
20-fold lower compared with levels of the fully glycosylated serotonin
transporter. The Km for [3H]serotonin uptake was also unchanged. These data
indicate that glycosylation is required for optimal stability of the
serotonin transporter in the membrane but not for serotonin transport or
ligand binding per se.
The effect of N-linked glycosylation on activity of the Na(+)- and Cl(- )-dependent serotonin transporter expressed using recombinant baculovirus in insect cells
Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.
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