Glycosyl modification facilitates homo- and hetero-oligomerization of the serotonin transporter. A specific role for sialic acid residues.
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.
Ozaslan
Deniz
D
Department of Biochemistry and Molecular Biology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Wang
Sophie
S
Ahmed
Billow A
BA
Kocabas
Arif M
AM
McCastlain
John C
JC
Bene
Anca
A
Kilic
Fusun
F
D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002822:Choriocarcinoma; D006224:Cricetinae; D006152:Cyclic GMP / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006031:Glycosylation; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000235:genetics; D009419:Nerve Tissue Proteins; D024507:Nonmuscle Myosin Type IIA / Q000235:genetics / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010766:Phosphorylation; D011247:Pregnancy; D011487:Protein Conformation; D051381:Rats; D012701:Serotonin / Q000378:metabolism; D050486:Serotonin Plasma Membrane Transport Proteins; D013329:Structure-Activity Relationship; D014162:Transfection; D014407:Tumor Cells, Cultured; D014594:Uterine Neoplasms
D002352:Carrier Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat; D012701:Serotonin; D017869:Cyclic GMP-Dependent Protein Kinases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D024507:Nonmuscle Myosin Type IIA; D019158:N-Acetylneuraminic Acid; D006152:Cyclic GMP