Evidence for α1-adrenergic receptor internalization in DDT1 MF-2 cells following exposure to agonists plus protein kinase C activators

M. S. Cowlen, Myron Lee Toews

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Agonist-induced sequestration and internalization of α1-adrenergic receptors were examined in DDT1 MF-2 cells. Pretreatment of cells with epinephrine or norepinephrine alone, but not with phorbol 12-myristate 13-acetate alone, resulted in a marked decrease in [3H]prazosin binding to intact cells at 4°. These pretreatments resulted in little or no change in the fraction of α1-adrenergic receptors exhibiting limited accessibility to the hydrophilic competing ligand epinephrine in short-time competition binding assays with intact cells and little or no change in the subcellular distribution of α1-adrenergic receptors between the plasma membrane and light vesicle compartments as assessed by sucrose density gradient centrifugation assays. Pretreatment with a combination of agonist plus phorbol 12-myristate 13-acetate resulted in a greater decrease in [3H]prazosin binding at 4° than was observed when cells were pretreated with agonist alone, induced the conversion of about half of cell surface α1-adrenergic receptors to a form exhibiting limited accessibility to epinephrine in short-term assays, and induced a shift of about half of α1-adrenergic receptors from the plasma membrane fraction to a light vesicle fraction on sucrose density gradients. A similar shift of α1-adrenergic receptors was observed on sucrose density gradients after exposure to agonist plus either mezerein or β-phorbol didecanoate, but not with agonist plus α-phorbol didecanoate, indicating involvement of protein kinase C. These results suggest that pretreatment with agonist alone induces the sequestration of α1-adrenergic receptors into a compartment that is inaccessible to [3H]prazosin at 4° but that is acccessible to hydrophilic ligands at 37° and remains associated with the plasma membrane. In contrast, α1-adrenergic receptors are apparently internalized from the plasma membrane to a separate compartment, presumably an intracellular vesicle, when cells are pretreated simultaneously with a combination of agonist plus a protein kinase C activator.

Original languageEnglish (US)
Pages (from-to)340-346
Number of pages7
JournalMolecular pharmacology
Volume34
Issue number3
StatePublished - Jan 1 1988

Fingerprint

Adrenergic Receptors
Protein Kinase C
Prazosin
Cell Membrane
Epinephrine
Sucrose
Acetates
Ligands
Light
Density Gradient Centrifugation
Norepinephrine

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

@article{66f33c34b8844dc58539a6347359cc07,
title = "Evidence for α1-adrenergic receptor internalization in DDT1 MF-2 cells following exposure to agonists plus protein kinase C activators",
abstract = "Agonist-induced sequestration and internalization of α1-adrenergic receptors were examined in DDT1 MF-2 cells. Pretreatment of cells with epinephrine or norepinephrine alone, but not with phorbol 12-myristate 13-acetate alone, resulted in a marked decrease in [3H]prazosin binding to intact cells at 4°. These pretreatments resulted in little or no change in the fraction of α1-adrenergic receptors exhibiting limited accessibility to the hydrophilic competing ligand epinephrine in short-time competition binding assays with intact cells and little or no change in the subcellular distribution of α1-adrenergic receptors between the plasma membrane and light vesicle compartments as assessed by sucrose density gradient centrifugation assays. Pretreatment with a combination of agonist plus phorbol 12-myristate 13-acetate resulted in a greater decrease in [3H]prazosin binding at 4° than was observed when cells were pretreated with agonist alone, induced the conversion of about half of cell surface α1-adrenergic receptors to a form exhibiting limited accessibility to epinephrine in short-term assays, and induced a shift of about half of α1-adrenergic receptors from the plasma membrane fraction to a light vesicle fraction on sucrose density gradients. A similar shift of α1-adrenergic receptors was observed on sucrose density gradients after exposure to agonist plus either mezerein or β-phorbol didecanoate, but not with agonist plus α-phorbol didecanoate, indicating involvement of protein kinase C. These results suggest that pretreatment with agonist alone induces the sequestration of α1-adrenergic receptors into a compartment that is inaccessible to [3H]prazosin at 4° but that is acccessible to hydrophilic ligands at 37° and remains associated with the plasma membrane. In contrast, α1-adrenergic receptors are apparently internalized from the plasma membrane to a separate compartment, presumably an intracellular vesicle, when cells are pretreated simultaneously with a combination of agonist plus a protein kinase C activator.",
author = "Cowlen, {M. S.} and Toews, {Myron Lee}",
year = "1988",
month = "1",
day = "1",
language = "English (US)",
volume = "34",
pages = "340--346",
journal = "Molecular Pharmacology",
issn = "0026-895X",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "3",

}

TY - JOUR

T1 - Evidence for α1-adrenergic receptor internalization in DDT1 MF-2 cells following exposure to agonists plus protein kinase C activators

AU - Cowlen, M. S.

AU - Toews, Myron Lee

PY - 1988/1/1

Y1 - 1988/1/1

N2 - Agonist-induced sequestration and internalization of α1-adrenergic receptors were examined in DDT1 MF-2 cells. Pretreatment of cells with epinephrine or norepinephrine alone, but not with phorbol 12-myristate 13-acetate alone, resulted in a marked decrease in [3H]prazosin binding to intact cells at 4°. These pretreatments resulted in little or no change in the fraction of α1-adrenergic receptors exhibiting limited accessibility to the hydrophilic competing ligand epinephrine in short-time competition binding assays with intact cells and little or no change in the subcellular distribution of α1-adrenergic receptors between the plasma membrane and light vesicle compartments as assessed by sucrose density gradient centrifugation assays. Pretreatment with a combination of agonist plus phorbol 12-myristate 13-acetate resulted in a greater decrease in [3H]prazosin binding at 4° than was observed when cells were pretreated with agonist alone, induced the conversion of about half of cell surface α1-adrenergic receptors to a form exhibiting limited accessibility to epinephrine in short-term assays, and induced a shift of about half of α1-adrenergic receptors from the plasma membrane fraction to a light vesicle fraction on sucrose density gradients. A similar shift of α1-adrenergic receptors was observed on sucrose density gradients after exposure to agonist plus either mezerein or β-phorbol didecanoate, but not with agonist plus α-phorbol didecanoate, indicating involvement of protein kinase C. These results suggest that pretreatment with agonist alone induces the sequestration of α1-adrenergic receptors into a compartment that is inaccessible to [3H]prazosin at 4° but that is acccessible to hydrophilic ligands at 37° and remains associated with the plasma membrane. In contrast, α1-adrenergic receptors are apparently internalized from the plasma membrane to a separate compartment, presumably an intracellular vesicle, when cells are pretreated simultaneously with a combination of agonist plus a protein kinase C activator.

AB - Agonist-induced sequestration and internalization of α1-adrenergic receptors were examined in DDT1 MF-2 cells. Pretreatment of cells with epinephrine or norepinephrine alone, but not with phorbol 12-myristate 13-acetate alone, resulted in a marked decrease in [3H]prazosin binding to intact cells at 4°. These pretreatments resulted in little or no change in the fraction of α1-adrenergic receptors exhibiting limited accessibility to the hydrophilic competing ligand epinephrine in short-time competition binding assays with intact cells and little or no change in the subcellular distribution of α1-adrenergic receptors between the plasma membrane and light vesicle compartments as assessed by sucrose density gradient centrifugation assays. Pretreatment with a combination of agonist plus phorbol 12-myristate 13-acetate resulted in a greater decrease in [3H]prazosin binding at 4° than was observed when cells were pretreated with agonist alone, induced the conversion of about half of cell surface α1-adrenergic receptors to a form exhibiting limited accessibility to epinephrine in short-term assays, and induced a shift of about half of α1-adrenergic receptors from the plasma membrane fraction to a light vesicle fraction on sucrose density gradients. A similar shift of α1-adrenergic receptors was observed on sucrose density gradients after exposure to agonist plus either mezerein or β-phorbol didecanoate, but not with agonist plus α-phorbol didecanoate, indicating involvement of protein kinase C. These results suggest that pretreatment with agonist alone induces the sequestration of α1-adrenergic receptors into a compartment that is inaccessible to [3H]prazosin at 4° but that is acccessible to hydrophilic ligands at 37° and remains associated with the plasma membrane. In contrast, α1-adrenergic receptors are apparently internalized from the plasma membrane to a separate compartment, presumably an intracellular vesicle, when cells are pretreated simultaneously with a combination of agonist plus a protein kinase C activator.

UR - http://www.scopus.com/inward/record.url?scp=0023813416&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023813416&partnerID=8YFLogxK

M3 - Article

VL - 34

SP - 340

EP - 346

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 3

ER -