Direct demonstration of NCAM cis-dimerization and inhibitory effect of palmitoylation using the BRET2 technique

Nikolaj Kulahin; Lars Groth Grunnet; Morten Lundh; Dan Ploug Christensen; Rasmus Jorgensen; Anders Heding; Nils Billestrup; Vladimir Berezin; Elisabeth Bock; Thomas Mandrup-Poulsen

doi:10.1016/j.febslet.2010.11.043

Direct demonstration of NCAM cis-dimerization and inhibitory effect of palmitoylation using the BRET2 technique

Nikolaj Kulahin, Lars Groth Grunnet, Morten Lundh, Dan Ploug Christensen, Rasmus Jorgensen, Anders Heding, Nils Billestrup, Vladimir Berezin, Elisabeth Bock, Thomas Mandrup-Poulsen

9 Citationer (Scopus)

Abstract

Biological activity of the neural cell adhesion molecule (NCAM) depends on both adhesion and activation of intra-cellular signaling. Based on in vitro experiments with truncated extra-cellular domains, several models describing homophilic NCAM trans- and cis-interactions have been proposed. However, cis-dimerization in living cells has not been shown directly and the role of the cytoplasmic part in NCAM dimerization is poorly understood. Here, we used the bioluminescence resonance energy transfer (BRET(2)) technique to directly demonstrate that full-length NCAM cis-homodimerizes in living cells. Based on BRET(2)50 values we suggest that the intra-cellular part of NCAM inhibits cis-dimerization, an effect mainly dependent on the palmitoylation sites.

Originalsprog	Engelsk
Tidsskrift	FEBS Letters
Vol/bind	585
Udgave nummer	1
Sider (fra-til)	58-64
Antal sider	7
DOI	https://doi.org/10.1016/j.febslet.2010.11.043
Status	Udgivet - 3 jan. 2011

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10.1016/j.febslet.2010.11.043

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title = "Direct demonstration of NCAM cis-dimerization and inhibitory effect of palmitoylation using the BRET2 technique",

abstract = "Biological activity of the neural cell adhesion molecule (NCAM) depends on both adhesion and activation of intra-cellular signaling. Based on in vitro experiments with truncated extra-cellular domains, several models describing homophilic NCAM trans- and cis-interactions have been proposed. However, cis-dimerization in living cells has not been shown directly and the role of the cytoplasmic part in NCAM dimerization is poorly understood. Here, we used the bioluminescence resonance energy transfer (BRET(2)) technique to directly demonstrate that full-length NCAM cis-homodimerizes in living cells. Based on BRET(2)50 values we suggest that the intra-cellular part of NCAM inhibits cis-dimerization, an effect mainly dependent on the palmitoylation sites.",

keywords = "Animals, COS Cells, Cercopithecus aethiops, Chemiluminescent Measurements, Energy Transfer, Green Fluorescent Proteins, Humans, Lipoylation, Neural Cell Adhesion Molecules, Protein Multimerization, Recombinant Fusion Proteins, Transfection",

author = "Nikolaj Kulahin and Grunnet, {Lars Groth} and Morten Lundh and Christensen, {Dan Ploug} and Rasmus Jorgensen and Anders Heding and Nils Billestrup and Vladimir Berezin and Elisabeth Bock and Thomas Mandrup-Poulsen",

year = "2011",

month = jan,

day = "3",

doi = "10.1016/j.febslet.2010.11.043",

language = "English",

volume = "585",

pages = "58--64",

journal = "FEBS Letters",

issn = "1873-3468",

publisher = "JohnWiley & Sons Ltd",

number = "1",

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TY - JOUR

T1 - Direct demonstration of NCAM cis-dimerization and inhibitory effect of palmitoylation using the BRET2 technique

AU - Kulahin, Nikolaj

AU - Grunnet, Lars Groth

AU - Lundh, Morten

AU - Christensen, Dan Ploug

AU - Jorgensen, Rasmus

AU - Heding, Anders

AU - Billestrup, Nils

AU - Berezin, Vladimir

AU - Bock, Elisabeth

AU - Mandrup-Poulsen, Thomas

PY - 2011/1/3

Y1 - 2011/1/3

N2 - Biological activity of the neural cell adhesion molecule (NCAM) depends on both adhesion and activation of intra-cellular signaling. Based on in vitro experiments with truncated extra-cellular domains, several models describing homophilic NCAM trans- and cis-interactions have been proposed. However, cis-dimerization in living cells has not been shown directly and the role of the cytoplasmic part in NCAM dimerization is poorly understood. Here, we used the bioluminescence resonance energy transfer (BRET(2)) technique to directly demonstrate that full-length NCAM cis-homodimerizes in living cells. Based on BRET(2)50 values we suggest that the intra-cellular part of NCAM inhibits cis-dimerization, an effect mainly dependent on the palmitoylation sites.

AB - Biological activity of the neural cell adhesion molecule (NCAM) depends on both adhesion and activation of intra-cellular signaling. Based on in vitro experiments with truncated extra-cellular domains, several models describing homophilic NCAM trans- and cis-interactions have been proposed. However, cis-dimerization in living cells has not been shown directly and the role of the cytoplasmic part in NCAM dimerization is poorly understood. Here, we used the bioluminescence resonance energy transfer (BRET(2)) technique to directly demonstrate that full-length NCAM cis-homodimerizes in living cells. Based on BRET(2)50 values we suggest that the intra-cellular part of NCAM inhibits cis-dimerization, an effect mainly dependent on the palmitoylation sites.

KW - Animals

KW - COS Cells

KW - Cercopithecus aethiops

KW - Chemiluminescent Measurements

KW - Energy Transfer

KW - Green Fluorescent Proteins

KW - Humans

KW - Lipoylation

KW - Neural Cell Adhesion Molecules

KW - Protein Multimerization

KW - Recombinant Fusion Proteins

KW - Transfection

U2 - 10.1016/j.febslet.2010.11.043

DO - 10.1016/j.febslet.2010.11.043

M3 - Journal article

C2 - 21115007

SN - 1873-3468

VL - 585

SP - 58

EP - 64

JO - FEBS Letters

JF - FEBS Letters

IS - 1

ER -

Direct demonstration of NCAM cis-dimerization and inhibitory effect of palmitoylation using the BRET2 technique

Abstract

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