Expression of the μ‐Opioid Receptor in CHO Cells

Ability of μ‐Opioid Ligands to Promote α‐Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Sumita Chakrabarti, Paul L. Prather, Lei Yu, Ping‐Yee ‐Y Law, Horace H Loh

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Abstract

Abstract: The identities of heterotrimeric G proteins that can interact with the μ‐opioid receptor were investigated by α‐azidoanilido[32P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)‐MORIVA3 cells, a CHO clone that stably expressed μ‐opioid receptor cDNA (MOR‐1). This clone expressed 1.01 × 106μ‐opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ‐opioid‐selective ligands [d‐Ala2,N‐MePhe4,Gly‐ol]‐enkephalin and [N‐MePhe3,d‐Pro4]‐morphiceptin, relative to the δ‐selective opioid agonist [d‐Pen2,d‐Pen5]‐enkephalin or the κ‐selective opioid agonist U‐50,488H. μ‐Opioid ligands induced an increase in α‐azidoanilido[32P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as Gi3α, Gi2α, and Go2α. The same pattern of simultaneous interaction of the μ‐opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10‐fold fewer receptors as those expressed in CHO‐MORIVA3 cells. The opioid‐induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist‐induced increase of α‐azidoanilido[32P]GTP incorporation into Gi2α (160–280%) and Go2α (110–220%) than for an unknown Gα (G?α) (60%) or Gi3α (40%) was produced by three different μ‐opioid ligands tested. In addition, slight differences were also found between the ability of various μ‐opioid agonists to produce half‐maximal labeling (ED50) of any given Gα subunit, with a rank order of Gi3α > Go2α > Gi2α = G?α. In any case, these results suggest that the activated μ‐opioid receptor couples to four distinct G protein α subunits simultaneously.

Original languageEnglish (US)
Pages (from-to)2534-2543
Number of pages10
JournalJournal of Neurochemistry
Volume64
Issue number6
DOIs
StatePublished - Jan 1 1995

Fingerprint

Protein Subunits
Opioid Receptors
Guanosine Triphosphate
Cricetulus
GTP-Binding Proteins
Labeling
Opioid Analgesics
Ovary
Ligands
Clone Cells
Enkephalins
Heterotrimeric GTP-Binding Proteins
Pertussis Toxin
Naloxone
Adenylyl Cyclases
Complementary DNA
Cells

Keywords

  • Adenylyl cyclase
  • Chinese hamster ovary cells
  • G proteins
  • Opioid
  • Pertussis toxin
  • μ‐Opioid receptor

Cite this

@article{937900077c014d34b7d7cbc337c00a0c,
title = "Expression of the μ‐Opioid Receptor in CHO Cells: Ability of μ‐Opioid Ligands to Promote α‐Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits",
abstract = "Abstract: The identities of heterotrimeric G proteins that can interact with the μ‐opioid receptor were investigated by α‐azidoanilido[32P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)‐MORIVA3 cells, a CHO clone that stably expressed μ‐opioid receptor cDNA (MOR‐1). This clone expressed 1.01 × 106μ‐opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ‐opioid‐selective ligands [d‐Ala2,N‐MePhe4,Gly‐ol]‐enkephalin and [N‐MePhe3,d‐Pro4]‐morphiceptin, relative to the δ‐selective opioid agonist [d‐Pen2,d‐Pen5]‐enkephalin or the κ‐selective opioid agonist U‐50,488H. μ‐Opioid ligands induced an increase in α‐azidoanilido[32P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as Gi3α, Gi2α, and Go2α. The same pattern of simultaneous interaction of the μ‐opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10‐fold fewer receptors as those expressed in CHO‐MORIVA3 cells. The opioid‐induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist‐induced increase of α‐azidoanilido[32P]GTP incorporation into Gi2α (160–280{\%}) and Go2α (110–220{\%}) than for an unknown Gα (G?α) (60{\%}) or Gi3α (40{\%}) was produced by three different μ‐opioid ligands tested. In addition, slight differences were also found between the ability of various μ‐opioid agonists to produce half‐maximal labeling (ED50) of any given Gα subunit, with a rank order of Gi3α > Go2α > Gi2α = G?α. In any case, these results suggest that the activated μ‐opioid receptor couples to four distinct G protein α subunits simultaneously.",
keywords = "Adenylyl cyclase, Chinese hamster ovary cells, G proteins, Opioid, Pertussis toxin, μ‐Opioid receptor",
author = "Sumita Chakrabarti and Prather, {Paul L.} and Lei Yu and Law, {Ping‐Yee ‐Y} and Loh, {Horace H}",
year = "1995",
month = "1",
day = "1",
doi = "10.1046/j.1471-4159.1995.64062534.x",
language = "English (US)",
volume = "64",
pages = "2534--2543",
journal = "Journal of Neurochemistry",
issn = "0022-3042",
publisher = "Wiley-Blackwell",
number = "6",

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TY - JOUR

T1 - Expression of the μ‐Opioid Receptor in CHO Cells

T2 - Ability of μ‐Opioid Ligands to Promote α‐Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

AU - Chakrabarti, Sumita

AU - Prather, Paul L.

AU - Yu, Lei

AU - Law, Ping‐Yee ‐Y

AU - Loh, Horace H

PY - 1995/1/1

Y1 - 1995/1/1

N2 - Abstract: The identities of heterotrimeric G proteins that can interact with the μ‐opioid receptor were investigated by α‐azidoanilido[32P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)‐MORIVA3 cells, a CHO clone that stably expressed μ‐opioid receptor cDNA (MOR‐1). This clone expressed 1.01 × 106μ‐opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ‐opioid‐selective ligands [d‐Ala2,N‐MePhe4,Gly‐ol]‐enkephalin and [N‐MePhe3,d‐Pro4]‐morphiceptin, relative to the δ‐selective opioid agonist [d‐Pen2,d‐Pen5]‐enkephalin or the κ‐selective opioid agonist U‐50,488H. μ‐Opioid ligands induced an increase in α‐azidoanilido[32P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as Gi3α, Gi2α, and Go2α. The same pattern of simultaneous interaction of the μ‐opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10‐fold fewer receptors as those expressed in CHO‐MORIVA3 cells. The opioid‐induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist‐induced increase of α‐azidoanilido[32P]GTP incorporation into Gi2α (160–280%) and Go2α (110–220%) than for an unknown Gα (G?α) (60%) or Gi3α (40%) was produced by three different μ‐opioid ligands tested. In addition, slight differences were also found between the ability of various μ‐opioid agonists to produce half‐maximal labeling (ED50) of any given Gα subunit, with a rank order of Gi3α > Go2α > Gi2α = G?α. In any case, these results suggest that the activated μ‐opioid receptor couples to four distinct G protein α subunits simultaneously.

AB - Abstract: The identities of heterotrimeric G proteins that can interact with the μ‐opioid receptor were investigated by α‐azidoanilido[32P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)‐MORIVA3 cells, a CHO clone that stably expressed μ‐opioid receptor cDNA (MOR‐1). This clone expressed 1.01 × 106μ‐opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ‐opioid‐selective ligands [d‐Ala2,N‐MePhe4,Gly‐ol]‐enkephalin and [N‐MePhe3,d‐Pro4]‐morphiceptin, relative to the δ‐selective opioid agonist [d‐Pen2,d‐Pen5]‐enkephalin or the κ‐selective opioid agonist U‐50,488H. μ‐Opioid ligands induced an increase in α‐azidoanilido[32P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as Gi3α, Gi2α, and Go2α. The same pattern of simultaneous interaction of the μ‐opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10‐fold fewer receptors as those expressed in CHO‐MORIVA3 cells. The opioid‐induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist‐induced increase of α‐azidoanilido[32P]GTP incorporation into Gi2α (160–280%) and Go2α (110–220%) than for an unknown Gα (G?α) (60%) or Gi3α (40%) was produced by three different μ‐opioid ligands tested. In addition, slight differences were also found between the ability of various μ‐opioid agonists to produce half‐maximal labeling (ED50) of any given Gα subunit, with a rank order of Gi3α > Go2α > Gi2α = G?α. In any case, these results suggest that the activated μ‐opioid receptor couples to four distinct G protein α subunits simultaneously.

KW - Adenylyl cyclase

KW - Chinese hamster ovary cells

KW - G proteins

KW - Opioid

KW - Pertussis toxin

KW - μ‐Opioid receptor

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U2 - 10.1046/j.1471-4159.1995.64062534.x

DO - 10.1046/j.1471-4159.1995.64062534.x

M3 - Article

VL - 64

SP - 2534

EP - 2543

JO - Journal of Neurochemistry

JF - Journal of Neurochemistry

SN - 0022-3042

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