Abstract
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 Trp(74) in the amino-terminal lobe of full-length NPRA. The predicted spacing between Trp(74) from both subunits drastically differs, depending on whether the V-shaped (84 A) or the A-shaped (8 A) 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.
Highlights
Proper signal transduction leading to cyclic GMP production and cellular response in the cardiovascular system [1]
These results indicate that the membrane-distal lobe of the NPRAM extracellular domains are dynamically interfacing in the unliganded state and that atrial natriuretic peptide (ANP) binding stabilizes the receptor dimer with more stringent spacing at the dimer interface
When the full-length NPRAW74C mutant transiently expressed in HEK293 cells is studied in SDS-PAGE under non-reducing conditions, we observe that the mutant is present almost exclusively as a dimer (Fig. 3, right panel)
Summary
Change occurring in the juxtamembrane region more than to the covalent dimerization process, because a double mutation, C423S/C432S, was shown to be constitutively active but not covalently dimeric [16] This interpretation is viable, the occurrence of a constitutive disulfide bridge still indicates the proximity of the extracellular juxtamembrane region of the receptor subunits. The contribution of the extracellular juxtamembrane region in mediating ANP-induced activation is well documented in a D435C mutant exposing a free cysteine three residues further toward the transmembrane region [19] This mutant is not covalently dimeric in the basal state, but ANP induces disulfide bridge formation through Cys435 upon receptor activation.
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