Identification and subcellular distribution of muscarinic acetylcholine receptor-related proteins in rabbit corneal and Chinese hamster ovary cells

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Abstract

Purpose. The authors examined the muscarinic acetylcholine receptor (mAChR) subtypes in rabbit corneal epithelial and endothelial cells and in subcellular fractions of these cell types. A Chinese hamster ovary (CHO) cell line (nontransfected CHO K1), expected to be a negative control, also was investigated. Methods. Whole cell homogenate and subcellular fractions were labeled with the covalent-binding, mAChR-specific ligand [3H]propylbenzilylcholine mustard ([3H]PrBChM) and were analyzed by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or SDS-PAGE, and autoradiography. Results. A pattern of multiple PrBChM-binding proteins was detected in homogenates of corneal epithelial and endothelial cells and, surprisingly, in the CHO cells. Ligand binding to all of these proteins is inhibited by the mAChR antagonists atropine sulfate and quinuclidinyl benzilate. The sizes of four of the labeled protein bands are the same as the molecular masses deduced from mAChR sequence data for subtypes m3, m4, m5, and either ml or m2. One band of 47 kd, smaller than any reported sequence, was also observed. Two of the [3H]-PrBChM-binding proteins, one at 59 to 62 kd (corresponding to m5 in size) and another at 47 kd, clearly were present when highly purified nuclei were analyzed. Conclusions. The presence of multiple mAChR-like proteins at low concentrations in these disparate cell types suggests the possibility of a more general regulatory role for this type of receptor than was considered previously. Combined with other reports, the identification of proteins with the characteristics of mAChRs in purified nuclei adds support to data indicating the likelihood of G-protein-coupled signaling across the nuclear envelope.

Original languageEnglish (US)
Pages (from-to)1492-1507
Number of pages16
JournalInvestigative Ophthalmology and Visual Science
Volume36
Issue number8
StatePublished - 1995

Fingerprint

Muscarinic Receptors
Cricetulus
Ovary
Rabbits
Subcellular Fractions
Proteins
Propylbenzilylcholine Mustard
Polyacrylamide Gel Electrophoresis
Carrier Proteins
Endothelial Cells
Epithelial Cells
Quinuclidinyl Benzilate
Ligands
Nuclear Envelope
Cholinergic Antagonists
Autoradiography
Atropine
GTP-Binding Proteins
Sodium Dodecyl Sulfate
Cell Line

Keywords

  • Chinese hamster ovary (CHO)
  • corneal endothelium
  • corneal epithelium
  • muscarinic acetylcholine receptors (mAChRs)
  • nucleus

ASJC Scopus subject areas

  • Ophthalmology

Cite this

@article{2b86ff7b690d4935aa7b74b05a4d50c5,
title = "Identification and subcellular distribution of muscarinic acetylcholine receptor-related proteins in rabbit corneal and Chinese hamster ovary cells",
abstract = "Purpose. The authors examined the muscarinic acetylcholine receptor (mAChR) subtypes in rabbit corneal epithelial and endothelial cells and in subcellular fractions of these cell types. A Chinese hamster ovary (CHO) cell line (nontransfected CHO K1), expected to be a negative control, also was investigated. Methods. Whole cell homogenate and subcellular fractions were labeled with the covalent-binding, mAChR-specific ligand [3H]propylbenzilylcholine mustard ([3H]PrBChM) and were analyzed by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or SDS-PAGE, and autoradiography. Results. A pattern of multiple PrBChM-binding proteins was detected in homogenates of corneal epithelial and endothelial cells and, surprisingly, in the CHO cells. Ligand binding to all of these proteins is inhibited by the mAChR antagonists atropine sulfate and quinuclidinyl benzilate. The sizes of four of the labeled protein bands are the same as the molecular masses deduced from mAChR sequence data for subtypes m3, m4, m5, and either ml or m2. One band of 47 kd, smaller than any reported sequence, was also observed. Two of the [3H]-PrBChM-binding proteins, one at 59 to 62 kd (corresponding to m5 in size) and another at 47 kd, clearly were present when highly purified nuclei were analyzed. Conclusions. The presence of multiple mAChR-like proteins at low concentrations in these disparate cell types suggests the possibility of a more general regulatory role for this type of receptor than was considered previously. Combined with other reports, the identification of proteins with the characteristics of mAChRs in purified nuclei adds support to data indicating the likelihood of G-protein-coupled signaling across the nuclear envelope.",
keywords = "Chinese hamster ovary (CHO), corneal endothelium, corneal epithelium, muscarinic acetylcholine receptors (mAChRs), nucleus",
author = "Lind, {G. J.} and Cavanagh, {Harrison D}",
year = "1995",
language = "English (US)",
volume = "36",
pages = "1492--1507",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "8",

}

TY - JOUR

T1 - Identification and subcellular distribution of muscarinic acetylcholine receptor-related proteins in rabbit corneal and Chinese hamster ovary cells

AU - Lind, G. J.

AU - Cavanagh, Harrison D

PY - 1995

Y1 - 1995

N2 - Purpose. The authors examined the muscarinic acetylcholine receptor (mAChR) subtypes in rabbit corneal epithelial and endothelial cells and in subcellular fractions of these cell types. A Chinese hamster ovary (CHO) cell line (nontransfected CHO K1), expected to be a negative control, also was investigated. Methods. Whole cell homogenate and subcellular fractions were labeled with the covalent-binding, mAChR-specific ligand [3H]propylbenzilylcholine mustard ([3H]PrBChM) and were analyzed by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or SDS-PAGE, and autoradiography. Results. A pattern of multiple PrBChM-binding proteins was detected in homogenates of corneal epithelial and endothelial cells and, surprisingly, in the CHO cells. Ligand binding to all of these proteins is inhibited by the mAChR antagonists atropine sulfate and quinuclidinyl benzilate. The sizes of four of the labeled protein bands are the same as the molecular masses deduced from mAChR sequence data for subtypes m3, m4, m5, and either ml or m2. One band of 47 kd, smaller than any reported sequence, was also observed. Two of the [3H]-PrBChM-binding proteins, one at 59 to 62 kd (corresponding to m5 in size) and another at 47 kd, clearly were present when highly purified nuclei were analyzed. Conclusions. The presence of multiple mAChR-like proteins at low concentrations in these disparate cell types suggests the possibility of a more general regulatory role for this type of receptor than was considered previously. Combined with other reports, the identification of proteins with the characteristics of mAChRs in purified nuclei adds support to data indicating the likelihood of G-protein-coupled signaling across the nuclear envelope.

AB - Purpose. The authors examined the muscarinic acetylcholine receptor (mAChR) subtypes in rabbit corneal epithelial and endothelial cells and in subcellular fractions of these cell types. A Chinese hamster ovary (CHO) cell line (nontransfected CHO K1), expected to be a negative control, also was investigated. Methods. Whole cell homogenate and subcellular fractions were labeled with the covalent-binding, mAChR-specific ligand [3H]propylbenzilylcholine mustard ([3H]PrBChM) and were analyzed by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or SDS-PAGE, and autoradiography. Results. A pattern of multiple PrBChM-binding proteins was detected in homogenates of corneal epithelial and endothelial cells and, surprisingly, in the CHO cells. Ligand binding to all of these proteins is inhibited by the mAChR antagonists atropine sulfate and quinuclidinyl benzilate. The sizes of four of the labeled protein bands are the same as the molecular masses deduced from mAChR sequence data for subtypes m3, m4, m5, and either ml or m2. One band of 47 kd, smaller than any reported sequence, was also observed. Two of the [3H]-PrBChM-binding proteins, one at 59 to 62 kd (corresponding to m5 in size) and another at 47 kd, clearly were present when highly purified nuclei were analyzed. Conclusions. The presence of multiple mAChR-like proteins at low concentrations in these disparate cell types suggests the possibility of a more general regulatory role for this type of receptor than was considered previously. Combined with other reports, the identification of proteins with the characteristics of mAChRs in purified nuclei adds support to data indicating the likelihood of G-protein-coupled signaling across the nuclear envelope.

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KW - corneal endothelium

KW - corneal epithelium

KW - muscarinic acetylcholine receptors (mAChRs)

KW - nucleus

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