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J. Biol. Chem., Vol. 278, Issue 13, 11159-11166, March 28, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/13/11159    most recent M212862200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by De Léan, A. Articles by Labrecque, J. Search for Related Content PubMed PubMed Citation Articles by De Léan, A. Articles by Labrecque, J. Social Bookmarking                      What's this?

Natriuretic Peptide Receptor A Activation Stabilizes a Membrane-distal Dimer Interface^*

André De Léan, Normand McNicoll, and Jean Labrecque

From the Department of Pharmacology, Faculty of Medicine, Université de Montréal, Montréal, Québec H3T 1J4, Canada

We have shown previously (Rondeau, J.-J., McNicoll, N., Gagnon, J., Bouchard, N., Ong, H., and De Léan, A. (1995) Biochemistry 34, 2130-2136) that atrial natriuretic peptide (ANP) stabilizes a dimeric form of the natriuretic peptide receptor A (NPRA) by simultaneously interacting with both receptor subunits. However, the first crystallographic study of unliganded NPRA extracellular domain documented a V-shaped dimer involving a membrane-proximal dimer interface and separate binding sites for ANP on each monomer. We explored the possibility of an alternative A-shaped dimer involving a membrane-distal dimer interface by substituting an unpaired solvent-exposed cysteine for Trp⁷⁴ in the amino-terminal lobe of full-length NPRA. The predicted spacing between Trp⁷⁴ from both subunits drastically differs, depending on whether the V-shaped (84 Å) or the A-shaped (8 Å) dimer model is considered. In contrast with the expected results for the reported V-shaped dimer, the NPRA^W74C mutant was constitutively covalently dimeric. Also, the subunits spontaneously reassociated following transient disulfide reduction by dithiothreitol and reoxidation. However, ANP could neither bind to nor activate NPRA^W74C. Permanent disulfide opening by reduction with dithiothreitol and alkylation with N-ethylmaleimide rescued ANP binding to NPRA^W74C. The NPRA mutant could be maintained as a covalent dimer while preserving its function by crosslinking with the bifunctional alkylating agent phenylenedimaleimides (PDM), the ortho-substituted oPDM being more efficient than mPDM or pPDM. These results indicate that the membrane-distal lobe of the NPRAM extracellular domains are dynamically interfacing in the unliganded state and that ANP binding stabilizes the receptor dimer with more stringent spacing at the dimer interface.

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