Glycosyl Modification Facilitates Homo- and Hetero-oligomerization of the Serotonin Transporter: A SPECIFIC ROLE FOR SIALIC ACID RESIDUES

Deniz Ozaslan,Sophie Wang,Billow A Ahmed,Arif M Kocabas,John C Mccastlain,Anca Bene,Fusun Kilic

doi:10.1074/jbc.m306360200

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.

Highlights

The serotonin transporter (SERT) is an oligomeric rine (NET) and dopamine (DAT) [1,2,3,4,5,6,7,8,9]
The transport system for serotonin is the target of many to the structure and function of SERT in two model clinically important drugs used in the treatment of a variety of systems: wild-type SERT expressed in sialic acid-defec- disorders such as cocaine, amphetamines, and antidepressants tive Lec Chinese hamster ovary (CHO) cells and a mu- [1,2,3,4,5,6,7,8,9,10,11]
We demonstrated that defects in sialylated N-glycans do not alter whole cell or plasma membrane expression of SERT proteins, but do decrease serotonin uptake function

Summary

A SPECIFIC ROLE FOR SIALIC ACID RESIDUES*

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 alterations in SERT and/or its antidepressant-binding activity are reported in patients with major neuropsychiatric disorders, including affective disorders, anxiety disorders, obsessive-comhomo-oligomeric forms. Sialylated N-Glycans and the Serotonin Transporter required for normal cell-surface expression; and (iii) sialylated glycans are required for normal cytoskeletal associations of SERT with myosin IIA. Based on these findings, we propose a structural role for sialylated N-glycans in conferring an optimum conformation to SERT that facilitates homo-oligomerization upon biosynthesis and, in turn, exposes the domain(s) for myosin cytoskeletal associations. Utilizing the Lec mutant cells in this study was a novel approach to elucidate the significance of sialic acid residues in SERT function

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION