Yao Jia, Erickson Jeffrey D, Hersh Louis B
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536-0298, USA.
Traffic. 2004 Dec;5(12):1006-16. doi: 10.1111/j.1600-0854.2004.00240.x.
Previous studies have shown that the vesicular monoamine transporter 2 (VMAT2) is localized to both large dense core vesicles and synaptic vesicles in vivo. However, when exogenously expressed in PC12 cells, VMAT2 localizes only to large dense core vesicles. This distribution is similar to that of the endogenous vesicular monoamine transporter 1 (VMAT1) in PC12 cells. When VMAT2 was expressed in a protein kinase A (PKA)-deficient PC12 cell line it localized to synaptic-like microvesicles. Expression of recombinant VMAT1 in the same cell line showed a heterogeneous distribution to both large dense core vesicles and synaptic-like microvesicles. Coexpression of the PKA catalytic subunit partially restored trafficking of both VMAT2 and VMAT1 to large dense core vesicles; treatment of wild-type PC12 cells with the PKA inhibitor H89 increased VMAT2 on synaptic-like microvesicles. The VMAT1 and VMAT2 in large dense core vesicles exhibit a larger molecular size than those located on synaptic-like microvesicles. This difference is due to differential N-linked glycosylation. In vitro phosphorylation experiments show that PKA does not phosphorylate VMAT2. A chimera containing the VMAT2 cytoplasmic C-terminus fused to vesicular acetylcholine transporter (VAChT) shows mislocalization to synaptic-like microvesicles and VAChT-like glycosylation in the PKA-deficient cell line. However, coexpression with PKA changes the chimera's trafficking to large dense core vesicles and increases the molecular size. These results suggest that protein kinase A affects the formation and/or composition of VMAT trafficking complexes.
先前的研究表明，囊泡单胺转运体2（VMAT2）在体内定位于大的致密核心囊泡和突触囊泡。然而，当在PC12细胞中外源表达时，VMAT2仅定位于大的致密核心囊泡。这种分布类似于PC12细胞中内源性囊泡单胺转运体1（VMAT1）的分布。当VMAT2在蛋白激酶A（PKA）缺陷的PC12细胞系中表达时，它定位于突触样微囊泡。在同一细胞系中重组VMAT1的表达显示其在大的致密核心囊泡和突触样微囊泡中呈异质分布。PKA催化亚基的共表达部分恢复了VMAT2和VMAT1向大的致密核心囊泡的运输；用PKA抑制剂H89处理野生型PC12细胞增加了突触样微囊泡上的VMAT2。大的致密核心囊泡中的VMAT1和VMAT2表现出比突触样微囊泡上的更大的分子大小。这种差异是由于不同的N-糖基化。体外磷酸化实验表明PKA不使VMAT2磷酸化。一种包含与囊泡乙酰胆碱转运体（VAChT）融合的VMAT2细胞质C末端的嵌合体在PKA缺陷细胞系中表现出定位于突触样微囊泡的错误定位和VAChT样糖基化。然而，与PKA共表达改变了嵌合体向大的致密核心囊泡的运输并增加了分子大小。这些结果表明蛋白激酶A影响VMAT运输复合物的形成和/或组成。
