Catalytically active guanylyl cyclase-B requires glycosylation and mutations that inhibit this process cause dwarfism

Abstract

Background C-type natriuretic peptide (CNP) is a paracrine factor that stimulates long bone growth, axonal path finding and inhibits meiosis in the oocyte [1]. The biologic signaling receptor for CNP is guanylyl cyclase (GC)-B, also known at NPRB or NPR2 [2]. GC-B is a homo-oligomer, possibly a dimer, containing a glycosylated extracellular ligand-binding domain, a single membrane-spanning region, and intracellular kinase homology domain (KHD), dimerization domain and C-terminal GC catalytic domain. Phosphorylation of the region leading into and at the beginning of the kinase homology domain is required for CNP activation of GC-B and dephosphorylation inactivates the enzyme [3]. Homozygous inactivating mutations in GC-B result in Acromesomelic Dysplasia, Type Maroteaux (AMDM) dwarfism [4-6], and heterozygous inactivating mutations in GC-B cause non-pathological reductions in stature [7]. Conversely, genetic mutations that increase GC-B activity result in skeletal overgrowth [8-10]. More than fifteen inactivating missense mutations in GC-B have been identified in humans. These mutations are randomly distributed from the N-terminus (P32) to the C-terminus (G959A) of the enzyme, consistent with two potential mechanisms. The first involves multiple processes like disruption of CNP or GTP binding to the extracellular or catalytic domains, respectively. The second more general mechanism involves conformational changes in secondary, tertiary or quaternary structure that preclude catalytic domain formation or activation. Previous investigators reported that 11 out of 12 [1], 2 out of 3 [2] or 1 out of 2 [3] missense mutations inhibited the transport of GC-B to the cell surface due to defective cellular trafficking and retention in the ER as indicated by reduced immunofluorescence imaging. Thus, the current hypothesis is that AMDM mutations inactivate GCB by disrupting intracellular trafficking. Here, we report that four intracellular GC-B mutants known to cause AMDM dwarfism bind CNP on the cell surface but have dramatically reduced catalytic activity.