Trafficking of hormones and trophic factors to secretory and extracellular vesicles: a historical perspective and new hypothesis

Abstract

It is well known that peptide hormones and neurotrophic factors are intercellular messengers that are packaged into secretory vesicles in endocrine cells and neurons and released by exocytosis upon the stimulation of the cells in a calcium-dependent manner. These secreted molecules bind to membrane receptors, which then activate signal transduction pathways to mediate various endocrine/trophic functions. Recently, there is evidence that these molecules are also in extracellular vesicles, including small extracellular vesicles (sEVs), which appear to be taken up by recipient cells. This finding raised the hypothesis that they may have functions differentiated from their classical secretory hormone/neurotrophic factor actions. In this article, the historical perspective and updated mechanisms for the sorting and packaging of hormones and neurotrophic factors into secretory vesicles and their transport in these organelles for release at the plasma membrane are reviewed. In contrast, little is known about the packaging of hormones and neurotrophic factors into extracellular vesicles. One proposal is that these molecules could be sorted at the trans-Golgi network, which then buds to form Golgi-derived vesicles that can fuse to endosomes and subsequently form intraluminal vesicles. They are then taken up by multivesicular bodies to form extracellular vesicles, which are subsequently released. Other possible mechanisms for packaging RSP proteins into sEVs are discussed. We highlight some studies in the literature that suggest the dual vesicular pathways for the release of hormones and neurotrophic factors from the cell may have some physiological significance in intercellular communication.

Keywords: Hormone trafficking; endocrine cells; exosomes; extracellular vesicles; neurons; sEV; trophic factor.

Figure 1

Secretory pathways in (neuro)endocrine cells. In endocrine cells and neurons, peptide hormones, neuropeptides, trophic factors, and granins are sorted at the TGN into immature vesicles that then mature to become DCV. Their content is released via the RSP upon stimulation. Other proteins in the Golgi complex are packaged into constitutive vesicles and secreted via the CSP, a default pathway. sEVs originate from ILVs formed through either endocytic pathway or possibly ER/Golgi secretory pathway. They are then taken up by MVBs. When the MVBs fuse with the plasma membrane, the sEVs are released. TGN: trans-Golgi network; DCV: dense core vesicles; RSP: regulated secretory pathway; CSP: constitutive secretory pathway; sEVs: small extracellular vesicles; ILVs: intraluminal vesicles; ER: endoplasmic reticulum; MVBs: multivesicular bodies.

Figure 2

TGN lumenal sorting mechanisms. Motifs that are required for the sorting of RSP proteins to DCVs. These include (A) the interaction between vasopressin and neurophysin domains in their precursor form; (B) disulfide bond; (C) charged α-helices; (D) the sorting signal motif of POMC that is conformation-dependent and comprises of two acidic residues, Asp10 and Glu14, and the two hydrophobic residues, Leu11 and Leu18. (Figure reproduced from Cawley et al. with permission)^[27]; (E) The sorting mechanism for POMC, pro-enkephalin, and BDNF use similar sorting motifs comprising of a pair of acidic amino acids binding to a pair of basic amino acids in a sorting receptor, membrane CPE which associates specifically with cholesterol-sphingolipid-rich lipid raft domains at the TGN membrane prior to budding off to form a DCV. (F) RSP proteins can also be sorted to the RSP by aggregation at pH 5-6 and 1-10 mM Ca²⁺ inside the TGN lumen. TGN: trans-Golgi network; POMC: pro-opiomelanocortin; BDNF: brain-derived neurotrophic factor; CPE: carboxypeptidase E; DCV: dense core vesicles.

Figure 3

Cytoplasmic contribution to DCV formation (I). (A) HID-1 promotes the acidification of TGN lumen by increasing H-ATPase activity, subsequently decreasing pH value and facilitating protein aggregation; (B) The µ1A subunit of AP-1A complex is critical for the sorting of PAM-1 to immature DCVs from TGN. Proteins (yellow), Protein Aggregates (orange), PAM1 (red). HID-1: high-temperature-induced dauer formation protein 1; AP-1A: adaptor protein 1A; PAM-1: peptidylglycine α-amidating monooxygenase-1; TGN: trans-Golgi network; DCV: dense core vesicles.

Figure 4

Cytoplasmic contribution to DCV formation (II). SNARE and t-SNAREs 1A/1B (Vti1a/1b) are involved in the formation of DCV. Vti1a also plays a role in DCV generation and Ca²⁺ channel trafficking. CAPS1 regulates the exocytosis of DCVs. Arf4/Arf5 is involved in CAPS1-mediated DCV formation. Snx19 is required for the formation of DCVs. Rab2, along with ICA69 and TBC-8, are involved in the early DCV formation. PICK1 is involved in the ICA69-mediated DCV formation from endosomal origin. Proteins (blue); SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; CAPS1: Ca²⁺-dependent activator protein for secretion 1; Arf4: ADP-ribosylation factor 4; Snx19: sorting nexin 19; ICA69: islet cell autoantigen of 69 kD; TBC-8: Tre-2/Bub2/Cdc16 protein 8; PICK1: protein interacting with C-kinase 1.

Figure 5

Sorting of RSP proteins into DCV by retention and constitutive-like secretion. RSP proteins can be packaged into DCVs by a “sorting-by-retention” mechanism: C-terminal disulfide bond is necessary for proTRH to remain in RSP vesicles (A); Non-RSP proteins in immature DCVs are removed by constitutive-like secretory pathways. AP-1 binds to the cytoplasmic tails of furin and M6PR and removes furin and M6PR from immature DCV via clathrin-mediated constitutive-like secretion (B). Golgi-localized, γ-ear containing ADP-ribosylation factor binding (GGA) mediates the removal of VAMP4 from immature DCVs (B); APS1 increases the activity of H-ATPase on DCVs to facilitate vesicle acidification. Rbcn3 promotes the translocation of CAPS1 to DCVs from the cytoplasm (C). proTRH: prothyrotropin-releasing hormone; M6PR: mannose-6-phosphate receptor; Rbcn3: rabconnectin 3; DCV: dense core vesicles; RSP: regulated secretory pathway.

Figure 6

Microtubule-based bidirectional DCV transport. The anterograde axonal transport of DCVs on microtubules is mediated mainly by kinesin-3 and some by other kinesins. Rab2 is required for bidirectional DCV transport in the axon. The Arf-like GTPase, Arl8, is an adaptor for kinesin-3-mediated DCV movement. CPE cytoplasmic tail on DCVs recruits dynactin and KIF1A (kinesin-3) and KIF3A (kinesin-2). KIF1A and KIF3A mediate the anterograde transport of these vesicles on microtubules. Cytoplasmic dynein, a minus-end directed motor, binds dynactin and mediates the return of DCVs from the neurite terminus back to the cell body under non-stimulated conditions for vesicle homeostasis. Dynactin, a microtubule anchor protein complex for cytoplasmic dynein and kinesins, mediates the bidirectional movement of DCVs. CPE: carboxypeptidase E; DCV: dense core vesicles.

Figure 7

F-actin network controls DCV tethering, transport and secretion at the plasma membrane. (A) F-actins block the access of DCVs to the PM; (B) Actin-severing proteins such as scinderin and gelsolin cut F actins into small filaments, thus facilitating the release of DCV. PI3K causes the depolymerization of F-actins to facilitate the docking of DCVs to the PM; (C) Myosin Va is activated by increased Ca²⁺ levels during stimulated secretion and interacts with Rab27a and MyRIP on DCVs to facilitate the mobilization of DCVs to the PM. PM: plasma membrane; PI3K: phosphatidylinositol 3 kinase; DCV: dense core vesicles.

Figure 8

Different biological activities of DCV-derived soluble CPE and sEV-CPE. Soluble CPE secreted from high metastatic HCCH cells inds HTR1E receptors on recipient HCCH cells and activates ERK-BCL2 signaling pathway to promote survival under hypoxic stress. HCCH-derived sEVs that contain CPE are taken up by recipient low metastatic HCCL cells to enhance proliferation and invasion. HCCH: hepatocellular carcinoma; DCV: dense core vesicles; sEVs: small extracellular vesicles; CPE: carboxypeptidase E.