Vasoactive intestinal peptide receptor/adenylate cyclase system: Differences between agonist- and protein kinase C-mediated desensitization and further evidence for receptor internalization

J. T. Turner, D. W. Bollinger, M. L. Toews

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

In this study we have characterized and compared the regulation of the HT29 cell vasoactive intestinal peptide receptor/adenylate cyclase system (VIP-R/AC) by the VIP-R agonist peptide histidineisoleucineamide (PHI) and by activators of protein kinase C (PKC) including phorbol 12-myristate, 13-acetate (PMA) and mezerein. Preincubation with either PHI or PKC activator decreased maximum VIP-stimulated AC activity and decreased the number of cell surface VIP-R. A [125I]VIP binding assay using solubilized VIP-R of the plasma membrane and light vesicle fractions from sucrose density step gradients was developed as a more direct measure of VIP-R internalization. Preincubation with PHI or PMA decreased plasma membrane fraction [125I]VIP binding and increased binding in the light vesicle fraction, thus providing the most direct evidence to date for translocation of VIP-R per se from the plasma membrane to another, presumably intracellular, compartment. Two experimental approaches differentiated between agonist and PKC activator regulation of VIP-R/AC. The protein kinase inhibitors H-7 and staurosporine blocked mezerein-, but not PHI-, induced losses of cell surface VIP-R. Also, down-regulation of PKC did not block PHI-induced loss of cell surface VIP-R. Thus, although both agonist and PKC activators can lead to desensitization and internalization of VIP-R, PKC is apparently not involved in the mechanisms of agonist-induced desensitization.

Original languageEnglish (US)
Pages (from-to)417-423
Number of pages7
JournalJournal of Pharmacology and Experimental Therapeutics
Volume247
Issue number2
StatePublished - Jan 1 1988

Fingerprint

Vasoactive Intestinal Peptide Receptors
Adenylyl Cyclases
Protein Kinase C
Peptides
Cell Membrane
Acetates
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
Light
HT29 Cells
Staurosporine
Protein Kinase Inhibitors
Sucrose
Down-Regulation
Cell Count

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

@article{93ff613215ea4917b7affc751f9ff6fa,
title = "Vasoactive intestinal peptide receptor/adenylate cyclase system: Differences between agonist- and protein kinase C-mediated desensitization and further evidence for receptor internalization",
abstract = "In this study we have characterized and compared the regulation of the HT29 cell vasoactive intestinal peptide receptor/adenylate cyclase system (VIP-R/AC) by the VIP-R agonist peptide histidineisoleucineamide (PHI) and by activators of protein kinase C (PKC) including phorbol 12-myristate, 13-acetate (PMA) and mezerein. Preincubation with either PHI or PKC activator decreased maximum VIP-stimulated AC activity and decreased the number of cell surface VIP-R. A [125I]VIP binding assay using solubilized VIP-R of the plasma membrane and light vesicle fractions from sucrose density step gradients was developed as a more direct measure of VIP-R internalization. Preincubation with PHI or PMA decreased plasma membrane fraction [125I]VIP binding and increased binding in the light vesicle fraction, thus providing the most direct evidence to date for translocation of VIP-R per se from the plasma membrane to another, presumably intracellular, compartment. Two experimental approaches differentiated between agonist and PKC activator regulation of VIP-R/AC. The protein kinase inhibitors H-7 and staurosporine blocked mezerein-, but not PHI-, induced losses of cell surface VIP-R. Also, down-regulation of PKC did not block PHI-induced loss of cell surface VIP-R. Thus, although both agonist and PKC activators can lead to desensitization and internalization of VIP-R, PKC is apparently not involved in the mechanisms of agonist-induced desensitization.",
author = "Turner, {J. T.} and Bollinger, {D. W.} and Toews, {M. L.}",
year = "1988",
month = "1",
day = "1",
language = "English (US)",
volume = "247",
pages = "417--423",
journal = "Journal of Pharmacology and Experimental Therapeutics",
issn = "0022-3565",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "2",

}

TY - JOUR

T1 - Vasoactive intestinal peptide receptor/adenylate cyclase system

T2 - Differences between agonist- and protein kinase C-mediated desensitization and further evidence for receptor internalization

AU - Turner, J. T.

AU - Bollinger, D. W.

AU - Toews, M. L.

PY - 1988/1/1

Y1 - 1988/1/1

N2 - In this study we have characterized and compared the regulation of the HT29 cell vasoactive intestinal peptide receptor/adenylate cyclase system (VIP-R/AC) by the VIP-R agonist peptide histidineisoleucineamide (PHI) and by activators of protein kinase C (PKC) including phorbol 12-myristate, 13-acetate (PMA) and mezerein. Preincubation with either PHI or PKC activator decreased maximum VIP-stimulated AC activity and decreased the number of cell surface VIP-R. A [125I]VIP binding assay using solubilized VIP-R of the plasma membrane and light vesicle fractions from sucrose density step gradients was developed as a more direct measure of VIP-R internalization. Preincubation with PHI or PMA decreased plasma membrane fraction [125I]VIP binding and increased binding in the light vesicle fraction, thus providing the most direct evidence to date for translocation of VIP-R per se from the plasma membrane to another, presumably intracellular, compartment. Two experimental approaches differentiated between agonist and PKC activator regulation of VIP-R/AC. The protein kinase inhibitors H-7 and staurosporine blocked mezerein-, but not PHI-, induced losses of cell surface VIP-R. Also, down-regulation of PKC did not block PHI-induced loss of cell surface VIP-R. Thus, although both agonist and PKC activators can lead to desensitization and internalization of VIP-R, PKC is apparently not involved in the mechanisms of agonist-induced desensitization.

AB - In this study we have characterized and compared the regulation of the HT29 cell vasoactive intestinal peptide receptor/adenylate cyclase system (VIP-R/AC) by the VIP-R agonist peptide histidineisoleucineamide (PHI) and by activators of protein kinase C (PKC) including phorbol 12-myristate, 13-acetate (PMA) and mezerein. Preincubation with either PHI or PKC activator decreased maximum VIP-stimulated AC activity and decreased the number of cell surface VIP-R. A [125I]VIP binding assay using solubilized VIP-R of the plasma membrane and light vesicle fractions from sucrose density step gradients was developed as a more direct measure of VIP-R internalization. Preincubation with PHI or PMA decreased plasma membrane fraction [125I]VIP binding and increased binding in the light vesicle fraction, thus providing the most direct evidence to date for translocation of VIP-R per se from the plasma membrane to another, presumably intracellular, compartment. Two experimental approaches differentiated between agonist and PKC activator regulation of VIP-R/AC. The protein kinase inhibitors H-7 and staurosporine blocked mezerein-, but not PHI-, induced losses of cell surface VIP-R. Also, down-regulation of PKC did not block PHI-induced loss of cell surface VIP-R. Thus, although both agonist and PKC activators can lead to desensitization and internalization of VIP-R, PKC is apparently not involved in the mechanisms of agonist-induced desensitization.

UR - http://www.scopus.com/inward/record.url?scp=0023776093&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0023776093&partnerID=8YFLogxK

M3 - Article

C2 - 2846820

AN - SCOPUS:0023776093

VL - 247

SP - 417

EP - 423

JO - Journal of Pharmacology and Experimental Therapeutics

JF - Journal of Pharmacology and Experimental Therapeutics

SN - 0022-3565

IS - 2

ER -