Glycosyl modification facilitates homo- and hetero-oligomerization of the serotonin transporter. A specific role for sialic acid residues

Abstract

The serotonin transporter (SERT) is an oligomeric glycoprotein with two sialic acid residues on each of two complex oligosaccharide molecules. In this study, we investigated the contribution of N-glycosyl modification to the structure and function of SERT in two model systems: wild-type SERT expressed in sialic acid-defective Lec4 Chinese hamster ovary (CHO) cells and a mutant form (after site-directed mutagenesis of Asn-208 and Asn-217 to Gln) of SERT, QQ, expressed in parental CHO cells. In both systems, SERT monomers required modification with both complex oligosaccharide residues to associate with each other and to function in homo-oligomeric forms. However, defects in sialylated N-glycans did not alter surface expression of the SERT protein. Furthermore, in heterologous (CHO and Lec4 cells) and endogenous (placental choriocarcinoma JAR cells) expression systems, we tested whether glycosyl modification also manipulates the hetero-oligomeric interactions of SERT, specifically with myosin IIA. SERT is phosphorylated by cGMP-dependent protein kinase G through interactions with anchoring proteins, and myosin is a protein kinase G-anchoring protein. A physical interaction between myosin and SERT was apparent; however, defects in sialylated N-glycans impaired association of SERT with myosin as well as the stimulation of the serotonin uptake function in the cGMP-dependent pathway. We propose that sialylated N-glycans provide a favorable conformation to SERT that allows the transporter to function most efficiently via its protein-protein interactions.

Fig. 1. Immunoblot analysis of SERT and QQ mutant transporters expressed in whole cell and plasma membranes of CHO and Lec4 cells

SERT (lanes 1 and 5) and QQ (lanes 4 and 7) transporters were expressed in either CHO or Lec4 cells using the cytomegalovirus transfection system and labeled with NHS-SS-biotin. They were fractionated by adsorption to streptavidin-agarose beads, and the biotinylated fraction was resolved by 10% SDS-PAGE and immunoblotted with affinity-purified anti-SERT antibody as described under “Experimental Procedures.” To compare whole cell and plasma membrane expression differences, soluble cell lysate proteins from SERT-expressing CHO cells (lane 2) were resolved and immunoblotted with affinity-purified biotinylated anti-SERT antibody. N-Glycosyl groups were stripped from the SERT proteins in CHO cells (lane 3) and Lec4 cells (lane 6) with PNGase F treatment. Immunoblot analyses were done with horseradish peroxidase-conjugated streptavidin as described under “Experimental Procedures.” All lanes contain protein recovered from the same number of cells equivalent to 30% of one well from a confluent 24-well dish. Three wells for each condition were pooled, and an aliquot of this mixture was run on the gel. The positions of molecular mass standards run on the same gel are shown in kilodaltons.

Fig. 2. Serotonin uptake function of CHO-QQ and Lec4-SERT cells

The uptake of serotonin by CHO-QQ cells or Lec4-SERT cells was greatly reduced compared with wild-type SERT in CHO cells. [³H]Serotonin uptake was measured in intact cells transiently expressing the transporters as described under “Experimental Procedures.” Background accumulation of [³H]serotonin was measured in the same experiment using mock-transfected cells and subtracted from each experimental value. Maximum background accumulation was 0.01 pmol/mg of protein/min. White bars represent CHO cells; hatched bars represent Lec4 cells (Origin plotting program, MicroCal Software). Mutation of both potential glycosylation sites caused a 70% decrease in the serotonin uptake function in CHO cells, but only 37% decrease in Lec4 cells. No difference was observed with Q1 or Q2.

Fig. 3. Standard curves for quantitation of SERT mutant expression

The indicated amounts of detergent-solubilized cell lysate from Myc-SERT-FLAG-expressing CHO and Lec4 cells were separated by SDS-PAGE and visualized by Western blot analysis. The integrated density value for each band was converted to an equivalent amount of Myc-SERT-FLAG for antibodies (Ab) against peptide-purified and biotinylated SERT (A), Myc (B), and FLAG (C). In this way, the relative amount of SERT constructs was determined in both total cell lysate and the cell-surface pool isolated using streptavidin-agarose (Table I), and the expression levels for SERT forms could be compared even though they were detected with different antibodies. Immunoblots were quantitated using a the VersaDoc 1000 system.

Fig. 4. Self-association ability of SERT proteins under different glycosylation patterns

CHO cells (lanes 1–4 and 6) and Lec4 cells (lane 5) were cotransfected with SERT-FLAG and Myc-SERT constructs at a 1:1 ratio where indicated, solubilized, and treated with mouse anti-Myc antibody (Ab)-coated rabbit anti-mouse protein A-Sepharose beads. The immunoprecipitates were separated and blotted with biotinylated anti-FLAG antibody as described under “Experimental Procedures.” Control experiments in which only SERT-FLAG-transfected CHO cells were tested did not reveal a visible band (lane 6) on the immunoblot. All lanes contain protein recovered from the same number of cells equivalent to 30% of one well from a confluent 6-well dish. The positions of molecular mass standards run on the same gel are shown in kilodaltons. MSH, β-mercaptoethanol.

Fig. 5. Effect of glycosylation on functional association of SERT proteins

Either CHO cells (A and C) or Lec4 cells (B) were transfected with different amounts of Res-FLAG and Sens-Myc cDNAs and assayed for serotonin uptake activity. Squares represent serotonin uptake rates; circles represent rates after treatment with 0.25 mM MTSEA for 10 min. Three lines were plotted according to predictions of the amount of activity remaining after MTSEA treatment. The dotted lines are the activity expected if no interaction occurred between the resistant and sensitive forms and if the amount of inactivation was equal to the amount of activity contributed by Sens-Myc. The solid lines represent the predicted activity if SERT was a dimer and if modification of both subunits was required for inactivation of activity in that dimer. The dashed lines represent the expected activity if modification of one subunit in a dimer inactivated all the activity of that dimer.

Fig. 6. Hetero-oligomerization of SERT and myosin IIA in heterologous and endogenous expression systems

A, either CHO cells (lanes 1 and 2) or Lec4 cells (lane 3) were cotransfected with myosin IIA and Myc-SERT (lanes 1 and 3) or with myosin IIA and Myc-QQ (lane 2). Cell lysate was treated with mouse anti-Myc antibody-coated rabbit anti-mouse protein A-Sepharose beads. Anti-Myc antibody-bound proteins were eluted and analyzed by Western blotting with rabbit polyclonal anti-myosin IIA antibody (Ab). Although transiently transfected myosin IIA protein was detected in Lec4 cells (lane 4), it was not detected in co-IP with Myc-SERT (lane 3). B, endogenous myosin IIA (lane 1), SERT (lane 2), and transient Myc-QQ (lane 3) expression was shown in JAR cells in blotting experiments. To demonstrate myosin IIA-SERT association in JAR cells, co-IP was performed with rabbit polyclonal anti-myosin IIA antibody. The immunoprecipitates were eluted and separated on 10% SDS-polyacrylamide gel under reducing (lane 4) or nonreducing (lane 7) conditions. Blots were probed first with biotinylated anti-SERT antibody and then with horseradish peroxidase-conjugated streptavidin as described under “Experimental Procedures.” To understand the involvement of glycosylation in myosin IIA-SERT association in JAR cells, Myc-QQ-expressing cells were subjected to IP assay with rabbit polyclonal anti-myosin IIA antibody, and then blots were probed with mouse monoclonal anti-Myc antibody (lane 5). The same analysis was done without including anti-myosin IIA antibody in the IP, and no protein band was labeled (lane 6).

Fig. 7. Effect of glycosyl modification on stimulation of SERT via cGMP

CHO or Lec4 cells expressing rSERT or the QQ mutant were preincubated where indicated with 1 mM 8-bromo-cGMP. After a 1-h preincubation in the dark at room temperature, transport was initiated by addition of [³H]serotonin to a final concentration of 20 nM. Transport was measured in a 10-min incubation in the continued presence of the cGMP donor.