Abstract
The natriuretic peptide receptor-A (NPR-A) is composed of an extracellular domain with a ligand binding site, a transmembrane-spanning domain, a kinase homology domain, and a guanylyl cyclase domain. In response to agonists (atrial natriuretic peptide (ANP) and brain natriuretic peptide), the kinase homology domain-mediated guanylate cyclase repression is removed, which allows the production of cyclic GMP. Previous work from our laboratory strongly indicated that agonists are exerting their effects through the induction of a juxtamembrane dimeric contact. However, a direct demonstration of this mechanism remains to be provided. As a tool, we are now using the properties of a new mutation, D435C. It introduces a cysteine at a position in NPR-A corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family (a disulfide-linked dimer). Although this D435C mutation only leads to trace levels of NPR-A disulfide-linked dimer at basal state, covalent dimerization can be induced by a treatment with rat ANP or with other agonists. The NPR-A(D435C) mutant has not been subjected to significant structural alterations, since it shares with the wild type receptor a similar dose-response pattern of cellular guanylyl cyclase activation. However, a persistent activation accompanies NPR-A(D435C) dimer formation after the removal of the inducer agonist. On the other hand, a construction where the intracellular domain of NPR-A(D435C) has been truncated (DeltaKC(D435C)) displays a spontaneous and complete covalent dimerization. In addition, the elimination of the intracellular domain in wild type DeltaKC and DeltaKC(D435C) is associated with an increase of agonist binding affinity, this effect being more pronounced with the weak agonist pBNP. Also, a D435C secreted extracellular domain remains unlinked even after incubation with rat ANP. In summary, these results demonstrate, in a dynamic fashion, the agonistic induction of a dimeric contact in the juxtamembrane domain of NPR-A. In addition, this process seems to require membrane attachment of the receptor. Finally, the intracellular domain represses this contact at the basal state, showing its potent influence on the outer juxtamembrane domain.
Highlights
The natriuretic peptide receptors (NPRs)1 are members of a family of single-transmembrane domain receptors that mediate their effects through the production of cyclic GMP [1]
We are using the properties of a new mutation, D435C. It introduces a cysteine at a position in natriuretic peptide receptor-A (NPR-A) corresponding to a supplementary cysteine found in NPR-C6, another receptor of this family. This D435C mutation only leads to trace levels of NPR-A disulfide-linked dimer at basal state, covalent dimerization can be induced by a treatment with rat ANP or with other agonists
The NPR-AD435C mutant has not been subjected to significant structural alterations, since it shares with the wild type receptor a similar dose-response pattern of cellular guanylyl cyclase activation
Summary
The natriuretic peptide receptors (NPRs) are members of a family of single-transmembrane domain receptors that mediate their effects through the production of cyclic GMP [1]. Three different NPRs have been identified, and two of these, NPR-A and NPR-B, respond to agonists by the activation of their guanylyl cyclase catalytic domain. A third receptor, called NPR-C or the clearance receptor, displays only 37 amino acids in its intracellular domain and is devoid of guanylyl cyclase activity. Several studies have demonstrated that this receptor is spontaneously preassociated in noncovalent dimers or oligomers (8 –10) Taken together, these studies have indicated that both extracellular and intracellular domains are involved in NPR-A dimerization. 4) ATP binding has two major effects in derepressing the guanylyl cyclase activity and increasing the off-rate of ANP from the receptor [12].
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