Central Nervous System-Specific Glycosylation of the Type A Natriuretic Peptide Receptor

Abstract

Physiological effects of atrial natriuretic peptide (ANP) are thought to be mediated by binding to and activation of a widely expressed membrane receptor, termed guanylyl cyclase (GC)-A. During comparative analyses of ANP receptor expression in various rat and bovine tissues, by UV light-induced affinity cross-linking to (125)I-ANP, we uncovered a size heterogeneity of GC-A, detectable as a 130-kDa protein in peripheral and as a 122-kDa protein in central nervous system tissues. This heterogeneity could be explained by differences in N-linked glycosylation, because treatments with N-glycosidase F reduced the apparent molecular weights of both receptor variants to the same value of 116,000. As judged by displacement experiments, the two receptor glycoforms did not differ in their binding affinities for natriuretic peptides. Assessments of GC activities did not reveal any difference in ANP-induced generation of the second messenger, cyclic GMP. The examination of GC-A expression in brain during ontogeny revealed an alteration of the apparent molecular mass during early postnatal development from the 130-kDa to the 122-kDa form, suggesting a change in oligosaccharide processing. This study provides a reliable method for characterizing GC-A expression and identifies, for the first time, a differential glycosylation of this receptor in vivo.

The membrane receptors guanylyl cyclase‐A and ‐B undergo distinctive changes in post‐translational modification during brain development

“…Frozen tissues were pulverized in a mortar, suspended in 10 mL/g tissue of ice-cold homogenization buffer (Müller et al 2002) and homogenized by 10 strokes in a Potter-Elvehjem homogenizer (Wheaton, Millville, NJ, USA). After centrifugation at 3000 g for 8 min at 4°C to remove cell debris and nuclei, the supernatant fractions were centrifuged for 30 min at 100 000 g at 4°C.…”

“…Regarding structural features of NPRs, the brain is of particular interest. Previous studies demonstrated a CNSspecific N-linked glycosylation of GC-A in adult rats, resulting in an 8 kDa lower molecular mass than in peripheral tissues (Müller et al 2002). The same investigation, however, discovered that the size of GC-A in the early postnatal brain is similar to that in peripheral tissues, indicating marked but not yet molecularly characterized alterations during brain development.…”

“…Guanylyl cyclase-A and GC-B are structurally closely related proteins with very similar cDNA-deduced molecular masses in the range of 115 kDa (SwissProt). Their native molecular weights are significantly higher (by about 5-15 kDa) because of glycosylation (Lowe and Fendly 1992;Fenrick et al 1997;Miyagi et al 2000;Müller et al 2002). The rat proteins contain six (GC-A) and seven (GC-B) consensus N-glycosylation sites, respectively (SwissProt), and N-linked glycosylation appears to represent the major size-affecting post-translational modification (Fenrick et al 1997;Miyagi et al 2000;Müller et al 2002).…”

“…Their native molecular weights are significantly higher (by about 5-15 kDa) because of glycosylation (Lowe and Fendly 1992;Fenrick et al 1997;Miyagi et al 2000;Müller et al 2002). The rat proteins contain six (GC-A) and seven (GC-B) consensus N-glycosylation sites, respectively (SwissProt), and N-linked glycosylation appears to represent the major size-affecting post-translational modification (Fenrick et al 1997;Miyagi et al 2000;Müller et al 2002). However, the physiological consequences of glycosylation, including potential effects on ligand binding and cell surface expression, are still poorly understood (Kuhn 2009).…”

Natriuretic peptide guanylyl cyclase receptors in the kidney of the Japanese eel, Anguilla japonica

“…Western blotting showed that both antibodies bound to gill proteins of approximately 120 kDa in size, the predicted size of NPR-GC. Western blots for mammalian NPR-A exhibited three immunoreactive bands corresponding to 116, 122 and 130 kDa because of differential glycosylation (Müller et al 2002). In the eel, four additional bands were observed in the NPR-A immunoblot, some of which may have represented differential glycosylation.…”