Glycine residues G338 and G342 are important determinants for serotonin transporter dimerisation and cell surface expression.
Compelling evidence has been provided that Na(+) and Cl(-)-dependent neurotransmitter transporter proteins form oligomeric complexes. Specific helix-helix interactions in lipid bilayers are thought to promote the assembly of integral membrane proteins to oligomeric structures. These interactions are determined by selective transmembrane helix packing motifs one of which is the Glycophorin A motif (GxxxG). This motif is present in the sixth transmembrane domain of most transporter proteins. In order to investigate, whether this motif is important for proper expression and function of the serotonin transporter (SERT), we have analysed the effect of mutating the respective glycine residues Gly338 and Gly342 to valine upon transient expression of the respective cDNAs in HEK293 cells. As revealed by western blotting, wildtype SERT is found in monomeric and dimeric forms while both mutants are expressed as monomers solely. Confocal microscopy revealed that the wildtype SERT is expressed at the cell surface, whereas both mutant proteins are localised in intracellular compartments. Failure of integration into the cell membrane is responsible for a total loss of [(3)H]5HT uptake capability by the mutants. These findings show that in the SERT protein the integrity of the GxxxG motif is essential for dimerisation and proper targeting of the transporter complex to the cell surface.
Horschitz
Sandra
S
Biochemical Laboratory, Central Institute of Mental Health, J5, 68159 Mannheim, Germany.
Lau
Thorsten
T
Schloss
Patrick
P
D019943:Amino Acid Substitution / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D015283:Citalopram / Q000378:metabolism; D003627:Data Interpretation, Statistical; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005998:Glycine / Q000378:metabolism*; D006021:Glycophorins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D018613:Microscopy, Confocal; D009154:Mutation; D011869:Radioligand Assay; D050486:Serotonin Plasma Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D017367:Selective Serotonin Reuptake Inhibitors / Q000378:metabolism; D014633:Valine / Q000378:metabolism
D015283:Citalopram; D005998:Glycine; D006021:Glycophorins; D049452:Green Fluorescent Proteins; D008562:Membrane Glycoproteins; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; D014633:Valine