ER-associated degradation is required for vasopressin prohormone processing and systemic water homeostasis
Guojun Shi, … , Martin Spiess, Ling Qi
Guojun Shi, … , Martin Spiess, Ling Qi
Published September 18, 2017
Citation Information: J Clin Invest. 2017;127(10):3897-3912. https://doi.org/10.1172/JCI94771.
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Research Article Cell biology Endocrinology

ER-associated degradation is required for vasopressin prohormone processing and systemic water homeostasis

Abstract

Peptide hormones are crucial regulators of many aspects of human physiology. Mutations that alter these signaling peptides are associated with physiological imbalances that underlie diseases. However, the conformational maturation of peptide hormone precursors (prohormones) in the ER remains largely unexplored. Here, we report that conformational maturation of proAVP, the precursor for the antidiuretic hormone arginine-vasopressin, within the ER requires the ER-associated degradation (ERAD) activity of the Sel1L-Hrd1 protein complex. Serum hyperosmolality induces expression of both ERAD components and proAVP in AVP-producing neurons. Mice with global or AVP neuron–specific ablation of Se1L-Hrd1 ERAD progressively developed polyuria and polydipsia, characteristics of diabetes insipidus. Mechanistically, we found that ERAD deficiency causes marked ER retention and aggregation of a large proportion of all proAVP protein. Further, we show that proAVP is an endogenous substrate of Sel1L-Hrd1 ERAD. The inability to clear misfolded proAVP with highly reactive cysteine thiols in the absence of Sel1L-Hrd1 ERAD causes proAVP to accumulate and participate in inappropriate intermolecular disulfide–bonded aggregates, promoted by the enzymatic activity of protein disulfide isomerase (PDI). This study highlights a pathway linking ERAD to prohormone conformational maturation in neuroendocrine cells, expanding the role of ERAD in providing a conducive ER environment for nascent proteins to reach proper conformation.

Authors

Guojun Shi, Diane R.M. Somlo, Geun Hyang Kim, Cristina Prescianotto-Baschong, Shengyi Sun, Nicole Beuret, Qiaoming Long, Jonas Rutishauser, Peter Arvan, Martin Spiess, Ling Qi

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Figure 4

Sel1L deficiency causes intracellular ER retention of proAVP.

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Sel1L deficiency causes intracellular ER retention of proAVP. (A) Repres...
(A) Representative fluorescence staining of Hrd1 in the PVN of Sel1Lfl/fl and Sel1LERCre mice 6 days after tamoxifen injections. n = 3 mice each genotype. Dashed line outlines the PVN. (B) ISH of Avp mRNA in the PVN region of Sel1Lfl/fl and Sel1LAVP mice at 1 week, with quantification of probe hybridization intensity shown on the right. Dashed line traces the PVN. (C) Representative fluorescence images of c-Fos in AVP-positive neurons in the PVN of Sel1Lfl/fl and Sel1LERCre mice 6 days after tamoxifen administration. Higher-magnification images are shown below. 3V, third ventricle. (D) Quantification of the percentage of c-Fos–positive AVP neurons. n = 2 female mice/genotype. (E) Schematic structure of proAVP protein and its processed products (AVP, NPII, and GP). Eight disulfide bonds and areas recognized by the AVP- and NPII-specific antibodies are shown. Amino acid positions for each polypeptide are shown. (F) Representative fluorescence images of proAVP in the PVN recognized by 2 antibodies specific for different regions of proAVP (AVP and NPII) in 1-week-old Sel1Lfl/fl and Sel1LAVP mice, showing largely absent axonal distribution of AVP-containing transport vesicles (arrows) in Sel1LAVP mice. (G) Representative fluorescence images of proAVP staining showing the absence of AVP-positive transport vesicles (arrows) in the axons of 3-week-old Sel1LAVP mice, with zoomed-in images on the right. n = 3 mice/group. (H–J) Representative fluorescence images of proAVP and BiP costaining in 3-week-old mice (H). Quantification of colocalization and Pearson’s coefficient between AVP and BiP staining (I and J). n = 3 female mice/group. Values represent the mean ± SEM. NS, not significant. *P < 0.05, by Student’s t test. Data represent at least 2 independent experiments.