Expression and Intracellular Localization of Catechol O‐methyltransferase in Transfected Mammalian Cells

Abstract

The intracellular localization of soluble and membrane-bound isoforms of rat and human catechol 0-methyltransferase (COMT) was studied by expressing the recombinant COMT proteins either separately or together in mammalian cell lines (HeLa and COS-7 cells) and in rat primary neurons. The distribution of soluble and membrane-bound COMT enzyme was visualized by immunocytochemistry. For comparison, the localization of native COMT was studied in rat C6 glioma cells by immunoelectron microscopy. Staining of cells expressing membrane-bound COMT with a COMT-specific antiserum revealed an immunofluorescence signal in intracellular reticular structures and in the nuclear membrane. Double-staining of the cells with antisera against proteins specific for the rough endoplasmic reticulum indicated that they colocalized with membrane-bound COMT, suggesting that it resided in the endoplasmic reticulum. Notably, no COMT-specific fluorescence of plasma membranes was detected. The signal in the endoplasmic reticulum was also evident in the cells expressing both recombinant COMT forms. Intracellular native COMT reaction was detected by immunoelectron microscopy in rat C6 glioma cells and an intense cytoplasmic signal was seen in the primary neurons infected with the recombinant Semliki Forest virus. The cells expressing recombinant soluble COMT revealed intense nuclear staining together with diffuse cytoplasmic immunoreactivity, suggesting that a part of soluble COMT is transported to nuclei. Western blotting from rat liver and brain revealed soluble COMT in the nuclei. Enzyme activity measurements from liver cytoplasmic and nuclear fractions suggested that about 5 % of the soluble COMT resided in nuclei. The intracellular localization of both COMT forms implies that COMT acts in the cytoplasm and possibly also in the nuclear compartment, and that the physiological substrates of COMT enzymes may have to be internalized before their methylation by COMT.

Schizophrenia genes, gene expression, and neuropathology: on the matter of their convergence

“…However, the precise distribution of COMT remains unclear; for example, whether COMT can 'see' synaptic dopamine; whether and where S and MB forms are differentially expressed, and the role, if any, of COMT within dopaminergic neurons. 178,188 Similarly, there is still uncertainty about the role of COMT in metabolism of norepinephrine and other catechols in brain. These issues may limit the conclusions that can be drawn regarding the cellular and molecular basis for the effect of COMT variation on cortical function and schizophrenia, discussed below.…”

Site-Specific Role of Catechol-O-Methyltransferase in Dopamine Overflow within Prefrontal Cortex and Dorsal Striatum

“…Both MAO and COMT are intracellular enzymes; soluble COMT can be found in cytoplasm and nucleus whereas membrane-bound COMT is located in rough endoplasmic reticulum (Ulmanen et al, 1997). Cellular localization of COMT in the brain is still poorly characterized.…”

Haplotypic association spanning the 22q11.21 genes COMT and ARVCF with schizophrenia

“…5 COMT resides within both neurons and glial cells in the brain, 6,7 but in the prefrontal cortex and striatum most COMT mRNAs are found in neurons in humans and mice. 7,8 There are two major forms of COMT, a soluble shorter form (S-COMT) found in the cytoplasm and a longer membrane-bound form (MB-COMT) found on the cytoplasmic side of the rough endoplasmic reticulum, [9][10][11][12] with the latter being the predominant form, B70% of total COMT protein, expressed in the brain. 13 In addition, catecholamines have a higher affinity, but a lower reaction velocity, with MB-COMT than with S-COMT, 14 making MB-COMT more effective at the lower physiological dopamine concentrations found in the brain, especially in the prefrontal cortex, 14,15 all of which points the greater importance of MB-COMT in the brain.…”

“…16 COMT, whether MB-COMT or S-COMT, is an intracellular enzyme; 15 no MB-COMT has been found in the cell membrane. 12 So, at least in the prefrontal cortex where SLC6A3 is scarce, another method of getting dopamine into the cell is necessary to invoke, 7 perhaps the organic cation transporters, solute carrier family 22 (organic cation transporter), member 2 (SLC22A2, also known as OCT2) or solute carrier family 22 (extraneuronal monoamine transporter), member 3 (SLC22A3, also known as OCT3), which are expressed in cortical neurons and might represent background transporters for monoamine neurotransmitters. [17][18][19] COMT has six exons, the first two of which are noncoding; exon 3 begins with the start codon for the 1.5 kb (kilobase) MB-COMT mRNA and 150 bp later in exon 3 is the start codon for the 1.3 kb S-COMT mRNA (see review Lundstrom et al 20 ).…”