Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism

Abstract

C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound 125 I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulummediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism.

Dephosphorylation is the mechanism of fibroblast growth factor inhibition of guanylyl cyclase-B

“…The extracellular domain of GC-B is glycosylated and terminal N-linked glycosylation is required for the formation of an active GC catalytic domain [15, 16]. AMDM dwarfism-causing missense mutations are most often associated with receptors lacking terminal N-linked glycosylation that markedly reduces or abolishes the ability of GC-B to form an active catalytic domain [16].…”

“…The extracellular domain of GC-B is glycosylated and terminal N-linked glycosylation is required for the formation of an active GC catalytic domain [15, 16]. AMDM dwarfism-causing missense mutations are most often associated with receptors lacking terminal N-linked glycosylation that markedly reduces or abolishes the ability of GC-B to form an active catalytic domain [16]. The intracellular portion of GC-B consists of a kinase homology domain that contains six chemically identified serine/threonine phosphorylation sites and one putative, functionally identified serine phosphorylation site [17–19], a short coiled-coiled dimerization region, and a carboxyl-terminal GC domain [14, 20].…”

Skeletal overgrowth-causing mutations mimic an allosterically activated conformation of guanylyl cyclase-B that is inhibited by 2,4,6,-trinitrophenyl ATP

“…1, inset). Furthermore, each mutant was completely processed to a hormonally active terminally glycosylated and phosphorylated protein represented by the upper, most slowly migrating diffuse band when purified by SDS-PAGE (18). Guanylyl cyclase assays conducted under basal conditions (without NP) indicated that the basal enzymatic activity of each mutant was markedly elevated compared with basal activity of the WT enzyme (Fig.…”

“…The membrane was blocked with Odyssey LI-COR blocking buffer (Li-Cor Biosciences, Lincoln, NE) for 1 h at room temperature and then incubated overnight at 4°C with rabbit polyclonal antiserum #6327 against the C terminus of rat GC-B at a dilution of 1/10,000 in 1:1 blocking buffer/PBS with 0.1% Tween. The specificity of this antiserum for human and rat GC-B has been previously demonstrated (18,26,32,33). The membrane was washed 4 ϫ 5 min with TBST and then incubated with IRDye 680 goat anti-rabbit antibody (Li-Cor Biosciences) with a dilution of 1/10,000 for 1 h at room temp in 1:1 blocking buffer/PBS with 0.1% Tween and 0.01% SDS.…”

“…More than 17 unique genetic mutations that inactivate both alleles of GC-B have been identified that cause acromesomelic dysplasia, type Maroteaux dwarfism (15)(16)(17). The majority of these mutations inactivate the enzyme by disrupting its normal secondary structure, which leads to improper folding and incomplete processing but normal plasma membrane targeting (18). Inactivation of single alleles was associated with reduced stature without disproportionality in one study (19), but the ability of an inactive allele to suppress a wild type (WT) allele through a dominant negative interaction raises the possibility that mutations in single alleles may cause disproportionate bone growth as well (20).…”