Netrin binds discrete subdomains of DCC and UNC5 and mediates interactions between DCC and heparin

Brian V. Geisbrecht, Kimberly A. Dowd, Ronald W. Barfield, Patti A. Longo, Daniel J. Leahy

Research output: Contribution to journalArticle

Abstract

Netrins are secreted proteins that elicit both attractive and repulsive responses in migrating cells in the central and peripheral nervous systems. Netrins interact with members of two distinct families of transmembrane receptors, represented by DCC (deleted in colorectal cancer) and UNC5. A human netrin fragment (soluble netrin; sNetrin) was purified from an engineered Chinese hamster ovary cell line and used in a pull-down assay to map the interactions between netrin and its receptors. We find that sNetrin binds exclusively to the fifth fibronectin type III repeat of DCC and to each immunoglobulin repeat of UNC5. Both DCC and UNC5 bind to sNetrin with 1:1 stoichiometry in solution, and the minimal receptor fragments behave similarly to larger fragments in cross-linking experiments with purified sNetrin. We find no evidence for formation of a ternary complex between sNetrin and soluble forms of DCC and UNC5. We also find no evidence for an interaction between DCC and heparin and instead demonstrate that a loop on the fifth fibronectin type III repeat of DCC previously implicated in mediating interactions with heparin is important for sNetrin binding. Since netrin binds heparin, our results suggest that interactions between DCC and heparin are probably mediated by netrin.

Original languageEnglish (US)
Pages (from-to)32561-32568
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number35
DOIs
StatePublished - Aug 29 2003

Fingerprint

Heparin
Colorectal Neoplasms
Fibronectins
Neurology
Stoichiometry
Immunoglobulins
Assays
Cells
Peripheral Nervous System
Cricetulus
Ovary
Central Nervous System
Proteins
Experiments
Cell Line

ASJC Scopus subject areas

  • Biochemistry

Cite this

Geisbrecht, B. V., Dowd, K. A., Barfield, R. W., Longo, P. A., & Leahy, D. J. (2003). Netrin binds discrete subdomains of DCC and UNC5 and mediates interactions between DCC and heparin. Journal of Biological Chemistry, 278(35), 32561-32568. https://doi.org/10.1074/jbc.M302943200

Netrin binds discrete subdomains of DCC and UNC5 and mediates interactions between DCC and heparin. / Geisbrecht, Brian V.; Dowd, Kimberly A.; Barfield, Ronald W.; Longo, Patti A.; Leahy, Daniel J.

In: Journal of Biological Chemistry, Vol. 278, No. 35, 29.08.2003, p. 32561-32568.

Research output: Contribution to journalArticle

Geisbrecht, Brian V. ; Dowd, Kimberly A. ; Barfield, Ronald W. ; Longo, Patti A. ; Leahy, Daniel J. / Netrin binds discrete subdomains of DCC and UNC5 and mediates interactions between DCC and heparin. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 35. pp. 32561-32568.
@article{c5ecc9630cb24a78b160ff576824c5e7,
title = "Netrin binds discrete subdomains of DCC and UNC5 and mediates interactions between DCC and heparin",
abstract = "Netrins are secreted proteins that elicit both attractive and repulsive responses in migrating cells in the central and peripheral nervous systems. Netrins interact with members of two distinct families of transmembrane receptors, represented by DCC (deleted in colorectal cancer) and UNC5. A human netrin fragment (soluble netrin; sNetrin) was purified from an engineered Chinese hamster ovary cell line and used in a pull-down assay to map the interactions between netrin and its receptors. We find that sNetrin binds exclusively to the fifth fibronectin type III repeat of DCC and to each immunoglobulin repeat of UNC5. Both DCC and UNC5 bind to sNetrin with 1:1 stoichiometry in solution, and the minimal receptor fragments behave similarly to larger fragments in cross-linking experiments with purified sNetrin. We find no evidence for formation of a ternary complex between sNetrin and soluble forms of DCC and UNC5. We also find no evidence for an interaction between DCC and heparin and instead demonstrate that a loop on the fifth fibronectin type III repeat of DCC previously implicated in mediating interactions with heparin is important for sNetrin binding. Since netrin binds heparin, our results suggest that interactions between DCC and heparin are probably mediated by netrin.",
author = "Geisbrecht, {Brian V.} and Dowd, {Kimberly A.} and Barfield, {Ronald W.} and Longo, {Patti A.} and Leahy, {Daniel J.}",
year = "2003",
month = "8",
day = "29",
doi = "10.1074/jbc.M302943200",
language = "English (US)",
volume = "278",
pages = "32561--32568",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "35",

}

TY - JOUR

T1 - Netrin binds discrete subdomains of DCC and UNC5 and mediates interactions between DCC and heparin

AU - Geisbrecht, Brian V.

AU - Dowd, Kimberly A.

AU - Barfield, Ronald W.

AU - Longo, Patti A.

AU - Leahy, Daniel J.

PY - 2003/8/29

Y1 - 2003/8/29

N2 - Netrins are secreted proteins that elicit both attractive and repulsive responses in migrating cells in the central and peripheral nervous systems. Netrins interact with members of two distinct families of transmembrane receptors, represented by DCC (deleted in colorectal cancer) and UNC5. A human netrin fragment (soluble netrin; sNetrin) was purified from an engineered Chinese hamster ovary cell line and used in a pull-down assay to map the interactions between netrin and its receptors. We find that sNetrin binds exclusively to the fifth fibronectin type III repeat of DCC and to each immunoglobulin repeat of UNC5. Both DCC and UNC5 bind to sNetrin with 1:1 stoichiometry in solution, and the minimal receptor fragments behave similarly to larger fragments in cross-linking experiments with purified sNetrin. We find no evidence for formation of a ternary complex between sNetrin and soluble forms of DCC and UNC5. We also find no evidence for an interaction between DCC and heparin and instead demonstrate that a loop on the fifth fibronectin type III repeat of DCC previously implicated in mediating interactions with heparin is important for sNetrin binding. Since netrin binds heparin, our results suggest that interactions between DCC and heparin are probably mediated by netrin.

AB - Netrins are secreted proteins that elicit both attractive and repulsive responses in migrating cells in the central and peripheral nervous systems. Netrins interact with members of two distinct families of transmembrane receptors, represented by DCC (deleted in colorectal cancer) and UNC5. A human netrin fragment (soluble netrin; sNetrin) was purified from an engineered Chinese hamster ovary cell line and used in a pull-down assay to map the interactions between netrin and its receptors. We find that sNetrin binds exclusively to the fifth fibronectin type III repeat of DCC and to each immunoglobulin repeat of UNC5. Both DCC and UNC5 bind to sNetrin with 1:1 stoichiometry in solution, and the minimal receptor fragments behave similarly to larger fragments in cross-linking experiments with purified sNetrin. We find no evidence for formation of a ternary complex between sNetrin and soluble forms of DCC and UNC5. We also find no evidence for an interaction between DCC and heparin and instead demonstrate that a loop on the fifth fibronectin type III repeat of DCC previously implicated in mediating interactions with heparin is important for sNetrin binding. Since netrin binds heparin, our results suggest that interactions between DCC and heparin are probably mediated by netrin.

UR - http://www.scopus.com/inward/record.url?scp=0042357532&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0042357532&partnerID=8YFLogxK

U2 - 10.1074/jbc.M302943200

DO - 10.1074/jbc.M302943200

M3 - Article

C2 - 12810718

AN - SCOPUS:0042357532

VL - 278

SP - 32561

EP - 32568

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 35

ER -