Characterization of receptors using cyanine 3-labeled neuropeptides

Nigel W. Bunnett; Paul F. Dazin; Donald G. Payan; Eileen F. Grady

doi:10.1016/0196-9781(95)00042-I

Characterization of receptors using cyanine 3-labeled neuropeptides

Nigel W. Bunnett, Paul F. Dazin, Donald G. Payan, Eileen F. Grady

Molecular Pathobiology

Research output: Contribution to journal › Article › peer-review

Abstract

We labeled substance P (SP), neurokinin A (NKA), and [Lys⁰]gastrin-releasing peptide-27 (GRP) with cyanine 3.18 (cy3). Cy3-peptides purified by HPLC were fully active, determined by [Ca²⁺]_i mobilization. Binding was specific because it was abolished by unlabeled peptides and receptor antagonists. Transfected cells yielded a log-fold greater cy3 intensity than control cells by FACS. Confocal microscopy of transfected cells and cultured enteric neurons showed that cy3-SP bound to surface receptors and was internalized. Internalization was observed in living cells by capture of sequential images. Recovery of surface binding sites was monitored by flow cytometry using cy3-SP. Thus, cy3 neuropeptides can be used to isolate and study receptor-bearing cells.

Original language	English (US)
Pages (from-to)	733-740
Number of pages	8
Journal	Peptides
Volume	16
Issue number	4
DOIs	https://doi.org/10.1016/0196-9781(95)00042-I
State	Published - 1995

Keywords

Endocytosis
Flow analysis
Fluorescent peptides
Gastrin-releasing peptide
Neurokinin A
Substance P

ASJC Scopus subject areas

Biochemistry
Physiology
Endocrinology
Cellular and Molecular Neuroscience

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10.1016/0196-9781(95)00042-I

Cite this

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title = "Characterization of receptors using cyanine 3-labeled neuropeptides",

abstract = "We labeled substance P (SP), neurokinin A (NKA), and [Lys0]gastrin-releasing peptide-27 (GRP) with cyanine 3.18 (cy3). Cy3-peptides purified by HPLC were fully active, determined by [Ca2+]i mobilization. Binding was specific because it was abolished by unlabeled peptides and receptor antagonists. Transfected cells yielded a log-fold greater cy3 intensity than control cells by FACS. Confocal microscopy of transfected cells and cultured enteric neurons showed that cy3-SP bound to surface receptors and was internalized. Internalization was observed in living cells by capture of sequential images. Recovery of surface binding sites was monitored by flow cytometry using cy3-SP. Thus, cy3 neuropeptides can be used to isolate and study receptor-bearing cells.",

keywords = "Endocytosis, Flow analysis, Fluorescent peptides, Gastrin-releasing peptide, Neurokinin A, Substance P",

author = "Bunnett, {Nigel W.} and Dazin, {Paul F.} and Payan, {Donald G.} and Grady, {Eileen F.}",

year = "1995",

doi = "10.1016/0196-9781(95)00042-I",

language = "English (US)",

volume = "16",

pages = "733--740",

journal = "Peptides",

issn = "0196-9781",

publisher = "Elsevier Inc.",

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T1 - Characterization of receptors using cyanine 3-labeled neuropeptides

AU - Bunnett, Nigel W.

AU - Dazin, Paul F.

AU - Payan, Donald G.

AU - Grady, Eileen F.

PY - 1995

Y1 - 1995

N2 - We labeled substance P (SP), neurokinin A (NKA), and [Lys0]gastrin-releasing peptide-27 (GRP) with cyanine 3.18 (cy3). Cy3-peptides purified by HPLC were fully active, determined by [Ca2+]i mobilization. Binding was specific because it was abolished by unlabeled peptides and receptor antagonists. Transfected cells yielded a log-fold greater cy3 intensity than control cells by FACS. Confocal microscopy of transfected cells and cultured enteric neurons showed that cy3-SP bound to surface receptors and was internalized. Internalization was observed in living cells by capture of sequential images. Recovery of surface binding sites was monitored by flow cytometry using cy3-SP. Thus, cy3 neuropeptides can be used to isolate and study receptor-bearing cells.

AB - We labeled substance P (SP), neurokinin A (NKA), and [Lys0]gastrin-releasing peptide-27 (GRP) with cyanine 3.18 (cy3). Cy3-peptides purified by HPLC were fully active, determined by [Ca2+]i mobilization. Binding was specific because it was abolished by unlabeled peptides and receptor antagonists. Transfected cells yielded a log-fold greater cy3 intensity than control cells by FACS. Confocal microscopy of transfected cells and cultured enteric neurons showed that cy3-SP bound to surface receptors and was internalized. Internalization was observed in living cells by capture of sequential images. Recovery of surface binding sites was monitored by flow cytometry using cy3-SP. Thus, cy3 neuropeptides can be used to isolate and study receptor-bearing cells.

KW - Endocytosis

KW - Flow analysis

KW - Fluorescent peptides

KW - Gastrin-releasing peptide

KW - Neurokinin A

KW - Substance P

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U2 - 10.1016/0196-9781(95)00042-I

DO - 10.1016/0196-9781(95)00042-I

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AN - SCOPUS:0029057994

SN - 0196-9781

VL - 16

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EP - 740

JO - Peptides

JF - Peptides

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ER -

Characterization of receptors using cyanine 3-labeled neuropeptides

Abstract

Keywords

ASJC Scopus subject areas

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