Abstract
Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress-responsive protein with neuroprotective effects in animal models of neurodegeneration, but the underlying mechanism is not understood. We constructed a set of lentiviral vectors that contain or lack the highly conserved final four amino acids of MANF ("RTDL"), which resemble the canonical ER retention signal ("KDEL"), to study MANF regulation in neuroblastoma cells and rat primary cortical neurons. The RTDL sequence was required for both ER retention and secretory response to ER stress. Overexpression of KDEL receptor paralogs (KDELRs) differentially reduced MANF secretion but had no effect on MANF lacking RTDL. MANF binding to the plasma membrane also required the RTDL sequence and was inhibited with a peptide known to interact with KDELRs, suggesting MANF binds KDELRs at the surface. We detected surface localization of FLAG-tagged KDELRs, with levels increasing following ER stress. Our study provides new insight into the regulation of MANF trafficking and has implications for other secreted proteins containing a KDEL-like retention signal.
Highlights
Mesencephalic astrocyte-derived neurotrophic factor is a secreted protein with an unknown mechanism of action
GFP-tagged Mesencephalic astrocyte-derived neurotrophic factor (MANF) Is Localized to the endoplasmic reticulum (ER)—The MANF protein is highly evolutionarily conserved, with 97.8% identity between the human and mouse protein sequences (Fig. 1A)
Based on the ER and Golgi localization of MANF [8, 9], we hypothesized that the Results: KDEL-like sequence of MANF (RTDL) sequence of human MANF functions as an ER retention signal critical to its trafficking
Summary
Mesencephalic astrocyte-derived neurotrophic factor is a secreted protein with an unknown mechanism of action. Results: KDEL-like sequence of MANF (“RTDL”) is required for ER retention, secretory responsiveness to ER stress, and surface binding. Significance: The plasma membrane localization of KDELRs has implications for MANF and other KDEL-containing proteins. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress-responsive protein with neuroprotective effects in animal models of neurodegeneration, but the underlying mechanism is not understood. The RTDL sequence was required for both ER retention and secretory response to ER stress. MANF binding to the plasma membrane required the RTDL sequence and was inhibited with a peptide known to interact with KDELRs, suggesting MANF binds KDELRs at the surface. Our study provides new insight into the regulation of MANF trafficking and has implications for other secreted proteins containing a KDEL-like retention signal
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