Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells
Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells
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DOI：
10.1016/j.heliyon.2024.e25761
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发表时间：
2024-02-15
期刊：
影响因子：
4
通讯作者：
Imoukhuede，Princess I.
中科院分区：
文献类型：
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作者：
Fang，Yingye;Reinl，Erin L.;Imoukhuede，Princess I.
催产素通过催产素受体（OXTR）调节子宫收缩力。我们先前鉴定了OXTR遗传变体，并表明在HEK 293 T细胞中，两种OXTR蛋白变体定位于细胞表面的程度低于野生型OXTR。在这里，我们试图在细胞表面和整个细胞上测量更天然的人子宫肌层平滑肌细胞（HMSMC）系中的OXTR，并使用CRISPR编辑将HA标签添加到内源性OXTR基因中用于抗HA测量。定量流式细胞术显示这些细胞具有55，000 ± 3200个总OXTR和4900 ± 390个细胞表面OXTR/细胞。为了鉴定野生型与变体定位的任何差异，我们瞬时转染HMSMC以外源表达具有HA和绿色荧光蛋白标签的野生型或变体OXTR。野生型OXTR和所有测试的变体的总蛋白表达是相似的。然而，在HEK 293 T细胞中具有较低表面定位的两种变体在HMSMCs中也呈现较低的表面定位。总体而言，我们证实了差异的表面定位的变体OXTR在一个更天然的细胞类型，并进一步证明，定量流式细胞术技术是适用于全细胞测量。
Oxytocin acts through the oxytocin receptor (OXTR) to modulate uterine contractility. We previously identified OXTR genetic variants and showed that, in HEK293T cells, two of the OXTR protein variants localized to the cell surface less than wild-type OXTR. Here, we sought to measure OXTR in the more native human myometrial smooth muscle cell (HMSMC) line on both the cell-surface and across the whole cell, and used CRISPR editing to add an HA tag to the endogenous OXTR gene for anti-HA measurement. Quantitative flow cytometry revealed that these cells possessed 55,000 ± 3200 total OXTRs and 4900 ± 390 cell-surface OXTRs per cell. To identify any differential wild-type versus variant localization, we transiently transfected HMSMCs to exogenously express wild-type or variant OXTR with HA and green fluorescent protein tags. Total protein expression of wild-type OXTR and all tested variants were similar. However, the two variants with lower surface localization in HEK293T cells also presented lower surface localization in HMSMCs. Overall, we confirm the differential surface localization of variant OXTR in a more native cell type, and further demonstrate that the quantitative flow cytometry technique is adaptable to whole-cell measurements.