Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells
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...
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| Language: | English |
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Elsevier
2024-02-01
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| Series: | Heliyon |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405844024017924 |
| _version_ | 1797267845573246976 |
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| author | Yingye Fang Erin L. Reinl Audrey Liu Trinidi D. Prochaska Manasi Malik Antonina I. Frolova Sarah K. England Princess I. Imoukhuede |
| author_facet | Yingye Fang Erin L. Reinl Audrey Liu Trinidi D. Prochaska Manasi Malik Antonina I. Frolova Sarah K. England Princess I. Imoukhuede |
| author_sort | Yingye Fang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-08T00:23:20Z |
| format | Article |
| id | doaj.art-de72c2ef8acb44f8828eaa62a83993ef |
| institution | Directory Open Access Journal |
| issn | 2405-8440 |
| language | English |
| last_indexed | 2024-04-25T01:23:04Z |
| publishDate | 2024-02-01 |
| publisher | Elsevier |
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| series | Heliyon |
| spelling | doaj.art-de72c2ef8acb44f8828eaa62a83993ef2024-03-09T09:26:09ZengElsevierHeliyon2405-84402024-02-01104e25761Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cellsYingye Fang0Erin L. Reinl1Audrey Liu2Trinidi D. Prochaska3Manasi Malik4Antonina I. Frolova5Sarah K. England6Princess I. Imoukhuede7Department of Bioengineering, University of Washington, Seattle, WA, 98109, USADepartment of Obstetrics and Gynecology, Center for Reproductive Health Sciences, Washington University School of Medicine in St. Louis, St. Louis, MO, 63110, USADepartment of Obstetrics and Gynecology, Center for Reproductive Health Sciences, Washington University School of Medicine in St. Louis, St. Louis, MO, 63110, USADepartment of Obstetrics and Gynecology, Center for Reproductive Health Sciences, Washington University School of Medicine in St. Louis, St. Louis, MO, 63110, USADepartment of Obstetrics and Gynecology, Center for Reproductive Health Sciences, Washington University School of Medicine in St. Louis, St. Louis, MO, 63110, USADepartment of Obstetrics and Gynecology, Center for Reproductive Health Sciences, Washington University School of Medicine in St. Louis, St. Louis, MO, 63110, USADepartment of Obstetrics and Gynecology, Center for Reproductive Health Sciences, Washington University School of Medicine in St. Louis, St. Louis, MO, 63110, USADepartment of Bioengineering, University of Washington, Seattle, WA, 98109, USA; Corresponding author.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.http://www.sciencedirect.com/science/article/pii/S2405844024017924Oxytocin receptorQuantitative flow cytometryProtein surface localizationGenetic variants |
| spellingShingle | Yingye Fang Erin L. Reinl Audrey Liu Trinidi D. Prochaska Manasi Malik Antonina I. Frolova Sarah K. England Princess I. Imoukhuede Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells Heliyon Oxytocin receptor Quantitative flow cytometry Protein surface localization Genetic variants |
| title | Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells |
| title_full | Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells |
| title_fullStr | Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells |
| title_full_unstemmed | Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells |
| title_short | Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells |
| title_sort | quantification of surface localized and total oxytocin receptor in myometrial smooth muscle cells |
| topic | Oxytocin receptor Quantitative flow cytometry Protein surface localization Genetic variants |
| url | http://www.sciencedirect.com/science/article/pii/S2405844024017924 |
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