Specific cross-linking of Lys233 and Cys235 in the mu opioid receptor by a reporter affinity label

Yan Zhang, Christopher R. McCurdy, Thomas G. Metzger, Philip S Portoghese

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The first example of the use of a reporter affinity label (NNA) that contains a fluorogenic naphthalene dialdehyde moiety to identify neighboring lysine and cysteine residues at a recognition site is described. The opioid receptors have served as the proof-of-concept because they contain multiple lysine and cysteine residues. The kinetics of isoindole formation resulting from covalent binding of NNA to wild-type and mutant opioid receptors were followed in cultured cells using flow cytometry. The finding that NNA bound to mutant mu opioid receptors (K233R and C235S) without producing specific fluorescence enhancement suggested that covalent bonding occurred at these positions to produce an isoindole fluorophore in the wild-type mu receptor. The similar kinetics of fluorophore formation for wild-type mu, delta, and kappa opioid receptors suggest that these conserved residues are the cross-linking sites in all three types of opioid receptors. The combined utilization of a reporter affinity label and site-directed mutagenesis offers a more expeditious method of identifying cross-linking at a recognition site when compared to classical procedures.

Original languageEnglish (US)
Pages (from-to)2271-2275
Number of pages5
JournalBiochemistry
Volume44
Issue number7
DOIs
StatePublished - Feb 22 2005

Fingerprint

Affinity Labels
mu Opioid Receptor
Opioid Receptors
Isoindoles
Fluorophores
Lysine
Cysteine
kappa Opioid Receptor
Mutagenesis
delta Opioid Receptor
Kinetics
Flow cytometry
Site-Directed Mutagenesis
Cultured Cells
Flow Cytometry
Fluorescence
Cells

Cite this

Specific cross-linking of Lys233 and Cys235 in the mu opioid receptor by a reporter affinity label. / Zhang, Yan; McCurdy, Christopher R.; Metzger, Thomas G.; Portoghese, Philip S.

In: Biochemistry, Vol. 44, No. 7, 22.02.2005, p. 2271-2275.

Research output: Contribution to journalArticle

Zhang, Yan ; McCurdy, Christopher R. ; Metzger, Thomas G. ; Portoghese, Philip S. / Specific cross-linking of Lys233 and Cys235 in the mu opioid receptor by a reporter affinity label. In: Biochemistry. 2005 ; Vol. 44, No. 7. pp. 2271-2275.
@article{4f90dc35a31447c69b8e71b5eef92ce4,
title = "Specific cross-linking of Lys233 and Cys235 in the mu opioid receptor by a reporter affinity label",
abstract = "The first example of the use of a reporter affinity label (NNA) that contains a fluorogenic naphthalene dialdehyde moiety to identify neighboring lysine and cysteine residues at a recognition site is described. The opioid receptors have served as the proof-of-concept because they contain multiple lysine and cysteine residues. The kinetics of isoindole formation resulting from covalent binding of NNA to wild-type and mutant opioid receptors were followed in cultured cells using flow cytometry. The finding that NNA bound to mutant mu opioid receptors (K233R and C235S) without producing specific fluorescence enhancement suggested that covalent bonding occurred at these positions to produce an isoindole fluorophore in the wild-type mu receptor. The similar kinetics of fluorophore formation for wild-type mu, delta, and kappa opioid receptors suggest that these conserved residues are the cross-linking sites in all three types of opioid receptors. The combined utilization of a reporter affinity label and site-directed mutagenesis offers a more expeditious method of identifying cross-linking at a recognition site when compared to classical procedures.",
author = "Yan Zhang and McCurdy, {Christopher R.} and Metzger, {Thomas G.} and Portoghese, {Philip S}",
year = "2005",
month = "2",
day = "22",
doi = "10.1021/bi048049v",
language = "English (US)",
volume = "44",
pages = "2271--2275",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "7",

}

TY - JOUR

T1 - Specific cross-linking of Lys233 and Cys235 in the mu opioid receptor by a reporter affinity label

AU - Zhang, Yan

AU - McCurdy, Christopher R.

AU - Metzger, Thomas G.

AU - Portoghese, Philip S

PY - 2005/2/22

Y1 - 2005/2/22

N2 - The first example of the use of a reporter affinity label (NNA) that contains a fluorogenic naphthalene dialdehyde moiety to identify neighboring lysine and cysteine residues at a recognition site is described. The opioid receptors have served as the proof-of-concept because they contain multiple lysine and cysteine residues. The kinetics of isoindole formation resulting from covalent binding of NNA to wild-type and mutant opioid receptors were followed in cultured cells using flow cytometry. The finding that NNA bound to mutant mu opioid receptors (K233R and C235S) without producing specific fluorescence enhancement suggested that covalent bonding occurred at these positions to produce an isoindole fluorophore in the wild-type mu receptor. The similar kinetics of fluorophore formation for wild-type mu, delta, and kappa opioid receptors suggest that these conserved residues are the cross-linking sites in all three types of opioid receptors. The combined utilization of a reporter affinity label and site-directed mutagenesis offers a more expeditious method of identifying cross-linking at a recognition site when compared to classical procedures.

AB - The first example of the use of a reporter affinity label (NNA) that contains a fluorogenic naphthalene dialdehyde moiety to identify neighboring lysine and cysteine residues at a recognition site is described. The opioid receptors have served as the proof-of-concept because they contain multiple lysine and cysteine residues. The kinetics of isoindole formation resulting from covalent binding of NNA to wild-type and mutant opioid receptors were followed in cultured cells using flow cytometry. The finding that NNA bound to mutant mu opioid receptors (K233R and C235S) without producing specific fluorescence enhancement suggested that covalent bonding occurred at these positions to produce an isoindole fluorophore in the wild-type mu receptor. The similar kinetics of fluorophore formation for wild-type mu, delta, and kappa opioid receptors suggest that these conserved residues are the cross-linking sites in all three types of opioid receptors. The combined utilization of a reporter affinity label and site-directed mutagenesis offers a more expeditious method of identifying cross-linking at a recognition site when compared to classical procedures.

UR - http://www.scopus.com/inward/record.url?scp=14044269530&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=14044269530&partnerID=8YFLogxK

U2 - 10.1021/bi048049v

DO - 10.1021/bi048049v

M3 - Article

VL - 44

SP - 2271

EP - 2275

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 7

ER -