Cell-specific secretory granule sorting mechanisms: the role of MAGEL2 and retromer in hypothalamic regulated secretion

Abstract

Intracellular protein trafficking and sorting are extremely arduous in endocrine and neuroendocrine cells, which synthesize and secrete on-demand substantial quantities of proteins. To ensure that neuroendocrine secretion operates correctly, each step in the secretion pathways is tightly regulated and coordinated both spatially and temporally. At the trans-Golgi network (TGN), intrinsic structural features of proteins and several sorting mechanisms and distinct signals direct newly synthesized proteins into proper membrane vesicles that enter either constitutive or regulated secretion pathways. Furthermore, this anterograde transport is counterbalanced by retrograde transport, which not only maintains membrane homeostasis but also recycles various proteins that function in the sorting of secretory cargo, formation of transport intermediates, or retrieval of resident proteins of secretory organelles. The retromer complex recycles proteins from the endocytic pathway back to the plasma membrane or TGN and was recently identified as a critical player in regulated secretion in the hypothalamus. Furthermore, melanoma antigen protein L2 (MAGEL2) was discovered to act as a tissue-specific regulator of the retromer-dependent endosomal protein recycling pathway and, by doing so, ensures proper secretory granule formation and maturation. MAGEL2 is a mammalian-specific and maternally imprinted gene implicated in Prader-Willi and Schaaf-Yang neurodevelopmental syndromes. In this review, we will briefly discuss the current understanding of the regulated secretion pathway, encompassing anterograde and retrograde traffic. Although our understanding of the retrograde trafficking and sorting in regulated secretion is not yet complete, we will review recent insights into the molecular role of MAGEL2 in hypothalamic neuroendocrine secretion and how its dysregulation contributes to the symptoms of Prader-Willi and Schaaf-Yang patients. Given that the activation of many secreted proteins occurs after they enter secretory granules, modulation of the sorting efficiency in a tissue-specific manner may represent an evolutionary adaptation to environmental cues.

Keywords: MAGEL2; Prader-Willi and Schaaf-Yang syndromes; WASH complex; anterograde and retrograde protein sorting; hormones and neuropeptides; neuroendocrine cells; retromer; secretory granule.

FIGURE 1

Anterograde and retrograde transport pathways in secretory cells. After protein synthesis in the ER, secretory proteins are sorted in the TGN through import signals, post-translational modifications, and other oligomeric associations. In the regulated secretion pathway, SGs go through a maturation process that includes fusion with other immature SGs and condensation of cargo proteins, as well as the removal of excess membrane and missorted cargo through the budding of clathrin-coated constitutive-like vesicles that may be secreted. Mature SGs accumulate near the plasma membrane until receiving a signal to undergo exocytosis and release their contents. In contrast, secretory vesicles (SeVs) in the constitutive secretion pathway continuously release their contents through unregulated membrane fusion. In retrograde transport, endocytosed material (e.g., receptors) are brought to a sorting endosome that directs endosomal material either back to the membrane in a recycling endosome, to immature SGs, to the lysosome for degradation, or to the TGN.

FIGURE 2

Components of immature (A) and mature (B) secretory granules. Lipid-raft-associated proteins like CPE, CPD, and secretogranin III interact with aggregates of regulated secretory pathway proteins and granulogenic proteins (e.g., granins like ChgA and ChgB) that form the dense proteinaceous core of mature SGs. Proton pumps increasingly acidify the SG lumen during maturation, which activates proprotein convertases and carboxypeptidases that process prohormones. The budding of clathrin-coated constitutive-like vesicles from immature SGs removes missorted constitutively secreted proteins and many other proteins shown in brown, including the peptidase furin, M6P-lysosomal enzymes bound to mannose-6-phosphate receptors (CI-M6PR or CD-M6PR), sortilin, synaptotagmin IV, VAMP4, and syntaxin 6. Calcium binding to synaptotagmin 1 stimulates exocytosis, which is mediated by v-SNARE proteins and other complexes. Mature SG size ranges from 50 nm in the sympathetic nervous system to 1,000 nm in pituitary mammotrophs or neurohypophyseal cells.

FIGURE 3

Heatmap showing expression of constitutive components of SGs, retromer, MUST, WASH, and ARP2/3 complexes. Data was extracted from GTEx on 05/24/2023.

FIGURE 4

Proposed sorting models for secreted proteins. (A) In the “sorting for entry” model, secreted and lysosomal proteins are segregated by binding to specific receptors clustered in the TGN before granule formation. (B) In the “sorting by retention” model, secreted and lysosomal proteins enter nascent SGs, but the non-regulated secretory proteins are excluded from the maturing SG by budding, possibly mediated by clathrin.

FIGURE 5

Sorting of proteins destined for the regulated secretory pathway occurs through various mechanisms, motifs, and adaptor proteins. (A,B) Within the lumen of the TGN and ISGs, sorting motifs within RSPs and interactions with other proteins facilitate aggregation and association with the membrane. (C) On the cytosolic side of the TGN/SG membrane, adaptor proteins recognize specific motifs in RSPs to help with sorting, and phosphorylation of some RSPs, like furin, enhances sorting.

FIGURE 6

MAGEL2 functions in regulated secretion of the hypothalamus. (A) Within hypothalamic neurosecretory cells, MAGEL2 plays a critical role in the retromer-mediated transport of SG components (i.e., PC1/3, PC2, CHGA, CHGB, and CPE) and the lysosomal CI-MPR from the sorting endosome to the TGN. MAGEL2-TRIM27-mediated ubiquitination leads to WASH activation and actin nucleation. (B) The loss of MAGEL2 leads to decreased abundance of SGs, SG-resident proteins, and neuropeptides in the hypothalamus, thus impairing hypothalamic neuroendocrine function.