Forced homodimerization by site-directed mutagenesis alters guanylyl cyclase activity of natriuretic peptide receptor B.

Natriuretic peptides mediate their physiologic effects through activation of membrane-bound, guanylyl cyclase-coupled receptors (NPRs). Receptor dimerization is an important feature of signal transduction. This study was aimed at characterizing structurally important residues of the extracellular ligand-binding domain of NPR-B for receptor dimerization and cGMP generation. Deletion mutagenesis was used to replace cysteine residues at positions 53 (C53S), 417 (C417S), and 426 (C426S) by serine. Receptor expression, dimerization, whole-cell cGMP response, and guanylyl cyclase activity of membrane fractions were determined in stably transfected COS-7 cells. C53S, C417S, and C426S mutants were expressed and found to form disulfide-bridged covalent dimers. In contrast to NPR-B and C53S, C417S and C426S mutants displayed constitutive activity in whole cells (C417S, 146+/-12%, P<0.01; C426S, 153+/-7% of ligand-independent NPR-B cGMP generation, P<0.01). The cGMP response of C417S and C426S mutants in whole cells was dose dependent and approximately 4 times lower than that in NPR-B, whereas it was blunted in C53S-transfected cells (1 micromol/L CNP, NPR-B 2868+/-436%; C53S, 206+/-16% of control, P<0.001 vs NPR-B, C417S, and C426S). Guanylyl cyclase assay in transfected cells confirmed the constitutive activity of C417S and C426S mutants. These data suggest that receptor dimerization by covalent disulfide bridges alters ligand-independent as well as ligand-dependent receptor activity. Localization of the crosslink in relation to the cell membrane is important for configuration of the extracellular domain and the consecutive signal transduction.