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N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake

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@article{3648ea2d820347df82da8167acb2c8f3,
title = "N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake",
abstract = "The P2X7 receptor is a frequently studied member of the purinergic receptor family signalling via channel opening and membrane pore formation. Fluorescent imaging is an important molecular method for studying cellular receptor expression and localization. Fusion of receptors to fluorescent proteins might cause major functional changes and requires careful functional evaluation such as has been done for the rat P2X7 receptor. This study examines fusion constructs of the human P2X7 receptor. We assessed surface expression, channel opening with calcium influx, and pore formation using YO-PRO-1 dye uptake in response to BzATP stimulation in transfected cells. We found that tagging at the N-terminal of the human P2X7 receptor with the enhanced green fluorescent protein (eGFP) disturbed channel opening and pore formation despite intact surface expression. A triple hemagglutinin (3HA) fused to the N-terminal also disrupted pore formation but not channel opening showing that even a small tag alters the normal function of the receptor. Together, this suggests that in contrast to what has been observed for the rat P2X7 receptor, the human P2X7 receptor contains N-terminal motifs important for signalling that prevent the construction of a functionally active fusion protein.",
keywords = "ATP, Flow cytometry, Fluo4, Purinergic",
author = "Karin Dreisig and Kristensen, {Nikolaj Pagh} and Dommer, {Maja Wallentin} and J{\o}rgensen, {Niklas Rye} and Kornum, {Birgitte Rahbek}",
year = "2018",
month = "3",
day = "1",
doi = "10.1007/s11302-017-9598-8",
language = "English",
volume = "14",
pages = "83--90",
journal = "Purinergic Signalling",
issn = "1573-9538",
publisher = "Springer Netherlands",
number = "1",

}

RIS

TY - JOUR

T1 - N-terminal tagging of human P2X7 receptor disturbs calcium influx and dye uptake

AU - Dreisig, Karin

AU - Kristensen, Nikolaj Pagh

AU - Dommer, Maja Wallentin

AU - Jørgensen, Niklas Rye

AU - Kornum, Birgitte Rahbek

PY - 2018/3/1

Y1 - 2018/3/1

N2 - The P2X7 receptor is a frequently studied member of the purinergic receptor family signalling via channel opening and membrane pore formation. Fluorescent imaging is an important molecular method for studying cellular receptor expression and localization. Fusion of receptors to fluorescent proteins might cause major functional changes and requires careful functional evaluation such as has been done for the rat P2X7 receptor. This study examines fusion constructs of the human P2X7 receptor. We assessed surface expression, channel opening with calcium influx, and pore formation using YO-PRO-1 dye uptake in response to BzATP stimulation in transfected cells. We found that tagging at the N-terminal of the human P2X7 receptor with the enhanced green fluorescent protein (eGFP) disturbed channel opening and pore formation despite intact surface expression. A triple hemagglutinin (3HA) fused to the N-terminal also disrupted pore formation but not channel opening showing that even a small tag alters the normal function of the receptor. Together, this suggests that in contrast to what has been observed for the rat P2X7 receptor, the human P2X7 receptor contains N-terminal motifs important for signalling that prevent the construction of a functionally active fusion protein.

AB - The P2X7 receptor is a frequently studied member of the purinergic receptor family signalling via channel opening and membrane pore formation. Fluorescent imaging is an important molecular method for studying cellular receptor expression and localization. Fusion of receptors to fluorescent proteins might cause major functional changes and requires careful functional evaluation such as has been done for the rat P2X7 receptor. This study examines fusion constructs of the human P2X7 receptor. We assessed surface expression, channel opening with calcium influx, and pore formation using YO-PRO-1 dye uptake in response to BzATP stimulation in transfected cells. We found that tagging at the N-terminal of the human P2X7 receptor with the enhanced green fluorescent protein (eGFP) disturbed channel opening and pore formation despite intact surface expression. A triple hemagglutinin (3HA) fused to the N-terminal also disrupted pore formation but not channel opening showing that even a small tag alters the normal function of the receptor. Together, this suggests that in contrast to what has been observed for the rat P2X7 receptor, the human P2X7 receptor contains N-terminal motifs important for signalling that prevent the construction of a functionally active fusion protein.

KW - ATP

KW - Flow cytometry

KW - Fluo4

KW - Purinergic

UR - http://www.scopus.com/inward/record.url?scp=85039727953&partnerID=8YFLogxK

U2 - 10.1007/s11302-017-9598-8

DO - 10.1007/s11302-017-9598-8

M3 - Journal article

VL - 14

SP - 83

EP - 90

JO - Purinergic Signalling

JF - Purinergic Signalling

SN - 1573-9538

IS - 1

ER -

ID: 55697530