Distinct subcellular distribution of delta-opioid receptor fused with various tags in PC12 cells.
In small dorsal root ganglion neurons, delta-opioid receptors (DORs) have been found to be mainly distributed in the cytoplasm and often associated with the membrane of large dense-core vesicles (LDCVs) that contain neuropeptides. To study the distribution of DORs under various physiological or pharmacological conditions, the receptors fused with different tags are constructed, transfected into cells or animals, and examined with microscopy. In this study, we show that DOR with different tags have distinct patterns of subcellular distribution in neuroendocrine cells, PC12 cells. Both immunostaining and vesicle fraction analysis showed that the native DORs expressed in PC12 cells were mainly associated with LDCVs. In transfected PC12 cells, DOR tagged with Myc or hemagglutinin exhibited LDCV localization. However, DOR fused with GFP at N- or C-terminus was found to be mainly localized on the cell surface, and mediated the function of DOR agonist. Therefore, the distribution of DOR fused with GFP differs from the native DORs. These results suggest that the subcellular distribution of the receptor could be better presented by the fused tag with smaller molecular size.
Wang
Hai-Bo
HB
Institute of Biochemistry and Cell Biology and Laboratory of Molecular Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
Guan
Ji-Song
JS
Bao
Lan
L
Zhang
Xu
X
D000818:Animals; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006388:Hemagglutinins / Q000378:metabolism; D016716:PC12 Cells; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D051381:Rats; D017465:Receptors, Opioid, delta / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D006388:Hemagglutinins; D016271:Proto-Oncogene Proteins c-myc; D017465:Receptors, Opioid, delta; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins