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Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling

Abstract

The Type-I bone morphogenetic protein receptors (BMPRs), BMPR1A and BMPR1B, present the highest sequence homology among BMPRs, suggestive of functional similitude. However, sequence elements within their extracellular domain, such as signal sequence or N-glycosylation motifs, may result in differential regulation of biosynthetic processing and trafficking and in alterations to receptor function. We show that (i) BMPR1A and the ubiquitous isoform of BMPR1B differed in mode of translocation into the endoplasmic reticulum; and (ii) BMPR1A was N-glycosylated while BMPR1B was not, resulting in greater efficiency of processing and plasma membrane expression of BMPR1A. We further demonstrated the importance of BMPR1A expression and glycosylation in ES-2 ovarian cancer cells, where (i) CRISPR/Cas9-mediated knockout of BMPR1A abrogated BMP2-induced Smad1/5/8 phosphorylation and reduced proliferation of ES-2 cells and (ii) inhibition of N-glycosylation by site-directed mutagenesis, or by tunicamycin or 2-deoxy-d-glucose treatments, reduced biosynthetic processing and plasma membrane expression of BMPR1A and BMP2-induced Smad1/5/8 phosphorylation.

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Abbreviations

2-DG:

2-Deoxy-d-glucose

ACVR1/2:

Type-I/II activin receptor

AMH:

Anti-müllerian hormone

AMHR2:

Type-II AMH receptor

BMP:

Bone morphogenetic protein

BMPR1A or R1A:

Type-IA BMP receptor

BMPR1B or R1B:

Type-IB BMP receptor

CRISPR:

Clustered regularly interspaced short palindromic repeats

CRISPR/Cas9:

CRISPR-associated nuclease 9

ECD:

Extracellular domain

EndoH:

Endoglucosydase H

ER:

Endoplasmic reticulum

ICD:

Intracellular (cytosolic) domain

IS1/2:

Isoform½

MAPK:

Mitogen-activated protein kinase

PCC:

Pearson’s correlation coefficient

p-Smad1/5/8:

Phosphorylated Smad1/5/8

PNGaseF:

Peptide-N-glycosidase F

SAPK:

Stress-activated protein kinase

s.e.m.:

Standard error of the mean

sgRNA:

Small guide RNA

sgαGFP:

SgRNA targeting GFP

sgαR1A:

SgRNA targeting BMPR1A

TGFβ:

Transforming growth factor beta

TGFβ_SF:

TGFβ superfamily

TM:

Transmembrane domain

t-Smad1/5/8:

Total Smad1/5/8

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Acknowledgements

We would like to thank Prof. Eran Bacharach and Dr. Rachel Zamostiano for their assistance in planning and cloning of CRISPR/Cas9 vectors for knockout of R1A and the control vector directed against GFP.

Author contributions

TH, NIS, and ME planned the study. TH, ML, and OD carried out experiments. TH and ME wrote the manuscript.

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Correspondence to Marcelo Ehrlich.

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Hirschhorn, T., Levi-Hofman, M., Danziger, O. et al. Differential molecular regulation of processing and membrane expression of Type-I BMP receptors: implications for signaling. Cell. Mol. Life Sci. 74, 2645–2662 (2017) doi:10.1007/s00018-017-2488-y

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Keywords

  • Bone morphogenetic protein
  • ER translocation
  • N-Glycosylation
  • Disulfide bond formation
  • Intracellular localization
  • Ovarian cancer