tissue- and development-specific glycosylation states of the natriuretic peptide receptors guanylyl...

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POSTER PRESENTATION Open Access Tissue- and development-specific glycosylation states of the natriuretic peptide receptors guanylyl cyclase-A (GC-A) and GC-B Dieter Müller * , Mirjam Hildebrand, Jörn Lübberstedt, Ralf Middendorff From 5th International Conference on cGMP: Generators, Effectors and Therapeutic Implications Halle, Germany. 24-26 June 2011 Background The natriuretic peptides (NPs) atrial NP (ANP), B-type NP (BNP) and C-type NP (CNP) elicit cellular effects by binding to transmembrane proteins with guanylyl cyclase (GC) activity, referred to as GC-A (representing the com- mon receptor for ANP and BNP) and GC-B (the CNP receptor). GC-A and GC-B are structurally closely related proteins with very similar cDNA-deduced molecular masses in the range of 115 kDa, but their native molecu- lar weights are significantly (by up to 20 kDa) higher due to glycosylation. Differential glycosylation of cell surface receptors can affect folding, trafficking, ligand binding and/or agonist- induced signal transduction. Moreover, specific temporal expression patterns of carbohydrate structures are fre- quently involved in developmental processes. Hitherto, however, the glycosylation states of GC-A and GC-B in vivo are largely unknown, since currently available data are based to a great extent on studies with receptor-trans- fected cell lines. This investigation aimed to characterize the sizes and posttranslational modifications of native GC-A and GC- B in different tissues of adult rats and mice. Based on increasing evidence for specific activities of NPs and their receptors in the developing brain (1-3), we exam- ined the glycosylation of GC-A and GC-B during post- natal brain development. Methods and results Western blot (as well as receptor/ligand crosslinking) experiments and treatments with carbohydrate-digest- ing enzymes served to investigate the sizes and posttranslational modifications of the two receptors. Due to marked tissue-specific differences in N-linked glycosylation, mean apparent molecular masses of both rat and mouse GC-A range from 119 kDa in brain to values between 124 (lung) and 132 (heart) kDa in per- ipheral tissues. The reduced glycosylation in brain seems to be central nervous system (CNS)-specific, since the pituitary, which contains both CNS and CNS-unrelated tissue, co-expresses the CNS-typical (119 kDa, reference: olfactory bulb) and the peripheral (up to 130 Da) forms of GC-A. GC-B sizes differed in the same manner between periph- eral organs, indicating equal tissue-dependent influences on oligosaccharide processing at GC-A and GC-B. A higher (by 3 kDa) total amount of N-linked carbohydrates suggests usage of an additional consensus N-glycosylation site (7 instead of 6 in the case of GC-A). The existence of region-specific GC-B size variants in cerebellum and olfac- tory bulb discriminates this receptor from GC-A in the adult brain. Both GC-A and GC-B (but not two other glycosylated membrane proteins examined) are hyperglycosylated at N-linked sites during early postnatal brain development. At postnatal day 1, the vast majority of GC-B (but not GC-A) contain additionally an O-linked carbohydrate modification. Affinity labelling and membrane GC assays did not reveal marked glycoform-associated effects on ligand binding or agonist-induced cGMP production. Conclusion The data uncover tissue-, development- and receptor- specific glycosylation states. Functional consequences remain to be elucidated. * Correspondence: [email protected] Institute of Anatomy and Cell Biology, University of Giessen, Germany Müller et al. BMC Pharmacology 2011, 11(Suppl 1):P52 http://www.biomedcentral.com/1471-2210/11/S1/P52 © 2011 Müller et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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POSTER PRESENTATION Open Access

Tissue- and development-specific glycosylationstates of the natriuretic peptide receptorsguanylyl cyclase-A (GC-A) and GC-BDieter Müller*, Mirjam Hildebrand, Jörn Lübberstedt, Ralf Middendorff

From 5th International Conference on cGMP: Generators, Effectors and Therapeutic ImplicationsHalle, Germany. 24-26 June 2011

BackgroundThe natriuretic peptides (NPs) atrial NP (ANP), B-typeNP (BNP) and C-type NP (CNP) elicit cellular effects bybinding to transmembrane proteins with guanylyl cyclase(GC) activity, referred to as GC-A (representing the com-mon receptor for ANP and BNP) and GC-B (the CNPreceptor). GC-A and GC-B are structurally closely relatedproteins with very similar cDNA-deduced molecularmasses in the range of 115 kDa, but their native molecu-lar weights are significantly (by up to 20 kDa) higher dueto glycosylation.Differential glycosylation of cell surface receptors can

affect folding, trafficking, ligand binding and/or agonist-induced signal transduction. Moreover, specific temporalexpression patterns of carbohydrate structures are fre-quently involved in developmental processes. Hitherto,however, the glycosylation states of GC-A and GC-B invivo are largely unknown, since currently available dataare based to a great extent on studies with receptor-trans-fected cell lines.This investigation aimed to characterize the sizes and

posttranslational modifications of native GC-A and GC-B in different tissues of adult rats and mice. Based onincreasing evidence for specific activities of NPs andtheir receptors in the developing brain (1-3), we exam-ined the glycosylation of GC-A and GC-B during post-natal brain development.

Methods and resultsWestern blot (as well as receptor/ligand crosslinking)experiments and treatments with carbohydrate-digest-ing enzymes served to investigate the sizes and

posttranslational modifications of the two receptors.Due to marked tissue-specific differences in N-linkedglycosylation, mean apparent molecular masses of bothrat and mouse GC-A range from 119 kDa in brain tovalues between 124 (lung) and 132 (heart) kDa in per-ipheral tissues. The reduced glycosylation in brainseems to be central nervous system (CNS)-specific,since the pituitary, which contains both CNS andCNS-unrelated tissue, co-expresses the CNS-typical(119 kDa, reference: olfactory bulb) and the peripheral(up to 130 Da) forms of GC-A.GC-B sizes differed in the same manner between periph-

eral organs, indicating equal tissue-dependent influenceson oligosaccharide processing at GC-A and GC-B. Ahigher (by 3 kDa) total amount of N-linked carbohydratessuggests usage of an additional consensus N-glycosylationsite (7 instead of 6 in the case of GC-A). The existence ofregion-specific GC-B size variants in cerebellum and olfac-tory bulb discriminates this receptor from GC-A in theadult brain.Both GC-A and GC-B (but not two other glycosylated

membrane proteins examined) are hyperglycosylated atN-linked sites during early postnatal brain development.At postnatal day 1, the vast majority of GC-B (but notGC-A) contain additionally an O-linked carbohydratemodification.Affinity labelling and membrane GC assays did not

reveal marked glycoform-associated effects on ligandbinding or agonist-induced cGMP production.

ConclusionThe data uncover tissue-, development- and receptor-specific glycosylation states. Functional consequencesremain to be elucidated.* Correspondence: [email protected]

Institute of Anatomy and Cell Biology, University of Giessen, Germany

Müller et al. BMC Pharmacology 2011, 11(Suppl 1):P52http://www.biomedcentral.com/1471-2210/11/S1/P52

© 2011 Müller et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

Published: 1 August 2011

References1. DiCicco-Bloom E, Lelièvre V, Zhou X, Rodriguez W, Tam J, Waschek JA:

Embryonic expression and multifunctional actions of the natriureticpeptides and receptors in the developing nervous system. Dev Biol 2004,271:161-175.

2. Cao L-H, Yang XL: Natriuretic peptides and their receptors in the centralnervous system. Prog Neurobiol 2008, 84:234-248.

3. Müller D, Hida B, Guidone G, Speth RC, Michurina TV, Enikolopov G,Middendorff R: Expression of guanylyl cyclase (GC)-A and GC-B duringbrain development: Evidence for a role of GC-B in perinatalneurogenesis. Endocrinology 2009, 150:5520-5529.

doi:10.1186/1471-2210-11-S1-P52Cite this article as: Müller et al.: Tissue- and development-specificglycosylation states of the natriuretic peptide receptors guanylylcyclase-A (GC-A) and GC-B. BMC Pharmacology 2011 11(Suppl 1):P52.

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Müller et al. BMC Pharmacology 2011, 11(Suppl 1):P52http://www.biomedcentral.com/1471-2210/11/S1/P52

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