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Research ArticleArticle

Antibody Tracking Demonstrates Cell Type-Specific and Ligand-Independent Internalization of Guanylyl Cyclase A and Natriuretic Peptide Receptor C

Deborah M. Dickey, Darcy R. Flora and Lincoln R. Potter
Molecular Pharmacology July 2011, 80 (1) 155-162; DOI: https://doi.org/10.1124/mol.110.070573
Deborah M. Dickey
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Darcy R. Flora
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Lincoln R. Potter
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This article has a correction. Please see:

  • Correction to “Antibody Tracking Demonstrates Cell Type-Specific and Ligand-Independent Internalization of Guanylyl Cyclase A and Natriuretic Peptide Receptor C” - February 01, 2017

Abstract

Atrial natriuretic peptide (ANP) binds guanylyl cyclase-A (GC-A) and natriuretic peptide receptor-C (NPR-C). Internalization of GC-A and NPR-C is poorly understood, in part, because previous studies used 125I-ANP binding to track these receptors, which are expressed in the same cell. Here, we evaluated GC-A and NPR-C internalization using traditional and novel approaches. Although HeLa cells endogenously express GC-A, 125I-ANP binding and cross-linking studies only detected NPR-C, raising the possibility that past studies ascribed NPR-C-mediated processes to GC-A. To specifically measure internalization of a single receptor, we developed an 125I-IgG-binding assay that tracks extracellular FLAG-tagged versions of GC-A and NPR-C independently of each other and ligand for the first time. FLAG-GC-A bound ANP identically with wild-type GC-A and was internalized slowly (0.5%/min), whereas FLAG-NPR-C was internalized rapidly (2.5%/min) in HeLa cells. In 293 cells, 125I-ANP and 125I-IgG uptake curves were superimposable because these cells only express a single ANP receptor. Basal internalization of both receptors was 8-fold higher in 293 compared with HeLa cells and ANP did not increase internalization of FLAG-GC-A. For FLAG-NPR-C, neither ANP, BNP, nor CNP increased its internalization in either cell line. Prolonged ANP exposure concomitantly reduced surface and total GC-A levels, consistent with rapid exchange of extracellular and intracellular receptor pools. We conclude that ligand binding does not stimulate natriuretic peptide receptor internalization and that cellular environment determines the rate of this process. We further deduce that NPR-C is internalized faster than GC-A and that increased internalization is not required for GC-A down-regulation.

Footnotes

  • This work was supported by the American Heart Association [Grants 0815607G, 0950053G].

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.110.070573.

  • ABBREVIATIONS:

    ANP
    atrial natriuretic peptide
    GC-A
    natriuretic peptide receptor-A
    BNP
    B-type natriuretic peptide
    PBS
    phosphate-buffered saline
    BSA
    bovine serum albumin
    FBS
    fetal bovine serum
    NPR-C
    natriuretic peptide clearance receptor
    tTA
    tetracycline transactivator
    DMEM
    Dulbecco's modified Eagle's medium
    GFP
    green fluorescent protein
    PAGE
    polyacrylamide gel electrophoresis.

  • Received December 14, 2010.
  • Accepted April 15, 2011.
  • Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 80 (1)
Molecular Pharmacology
Vol. 80, Issue 1
1 Jul 2011
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Research ArticleArticle

Antibody Tracking Demonstrates Cell Type-Specific and Ligand-Independent Internalization of Guanylyl Cyclase A and Natriuretic Peptide Receptor C

Deborah M. Dickey, Darcy R. Flora and Lincoln R. Potter
Molecular Pharmacology July 1, 2011, 80 (1) 155-162; DOI: https://doi.org/10.1124/mol.110.070573

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Research ArticleArticle

Antibody Tracking Demonstrates Cell Type-Specific and Ligand-Independent Internalization of Guanylyl Cyclase A and Natriuretic Peptide Receptor C

Deborah M. Dickey, Darcy R. Flora and Lincoln R. Potter
Molecular Pharmacology July 1, 2011, 80 (1) 155-162; DOI: https://doi.org/10.1124/mol.110.070573
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