Crosstalk between KDEL receptor and EGF receptor mediates cell proliferation and migration via STAT3 signaling.

Hostile microenvironment of cancer cells provoke a stressful condition for endoplasmic reticulum (ER) and stimulate the expression and secretion of ER chaperones, leading to tumorigenic effects. However, the molecular mechanism underlying these effects is largely unknown. In this study, we reveal that the last four residues of ER chaperones, which are recognized by KDEL receptor (KDELR), is required for cell proliferation and migration induced by secreted chaperones. By combining proximity-based mass spectrometry analysis, split venus imaging and membrane yeast two hybrid assay, we present that EGF receptor (EGFR) may be a co-receptor for KDELR on the surface. Prior to ligand addition, KDELR spontaneously oligomerizes and constantly undergoes recycling near the plasma membrane. Upon KDEL ligand binding, the interactions of KDELR with itself and with EGFR increase rapidly, leading to augmented internalization of KDELR and tyrosine phosphorylation in the C-terminus of EGFR. STAT3, which binds the phosphorylated tyrosine motif on EGFR, is subsequently activated by EGFR and mediates cell growth and migration. Taken together, our results suggest that KDELR serves as a bona fide cell surface receptor for secreted ER chaperones and transactivates EGFR-STAT3 signaling pathway.

KDEL Receptor Trafficking to the Plasma Membrane Is Regulated by ACBD3 and Rab4A-GTP.

KDEL receptor-1 maintains homeostasis in the early secretory pathway by capturing and retrieving ER chaperones to the ER during heavy secretory activity. Unexpectedly, a fraction of the receptor is also known to reside in the plasma membrane (PM), although it is largely unknown exactly how the KDEL receptor gets exported from the Golgi and travels to the PM. We have previously shown that a Golgi scaffolding protein (ACBD3) facilitates KDEL receptor localization at the Golgi via the regulating cargo wave-induced cAMP/PKA-dependent signaling pathway. Upon endocytosis, surface-expressed KDEL receptor undergoes highly complex itineraries through the Golgi and the endo-lysosomal compartments, where the endocytosed receptor utilizes Rab14A- and Rab11A-positive recycling endosomes and clathrin-decorated tubulovesicular carriers. In this study, we sought to investigate the mechanism through which the KDEL receptor gets exported from the Golgi en route to the PM. We report here that ACBD3 depletion results in greatly increased trafficking of KDEL receptor to the PM via Rab4A-positive tubular carriers emanating from the Golgi. Expression of constitutively activated Rab4A mutant (Q72L) increases the surface expression of KDEL receptor up to 2~3-fold, whereas Rab4A knockdown or the expression of GDP-locked Rab4A mutant (S27N) inhibits KDEL receptor targeting of the PM. Importantly, KDELR trafficking from the Golgi to the PM is independent of PKA- and Src kinase-mediated mechanisms. Taken together, these results reveal that ACBD3 and Rab4A play a key role in regulating KDEL receptor trafficking to the cell surface.

An A-kinase anchoring protein (ACBD3) coordinates traffic-induced PKA activation at the Golgi.

KDEL receptor (KDELR) is a key protein that recycles escaped endoplasmic reticulum (ER) resident proteins from the Golgi apparatus back to the ER and maintains a dynamic balance between these two organelles in the early secretory pathway. Studies have shown that this retrograde transport pathway is partly regulated by two KDELR-interacting proteins, acyl-CoA-binding domain-containing 3 (ACBD3), and cyclic AMP-dependent protein kinase A (PKA). However, whether Golgi-localized ACBD3, which was first discovered as a PKA-anchoring protein in mitochondria, directly interacts with PKA at the Golgi and coordinates its signaling in Golgi-to-ER traffic has remained unclear. In this study, we showed that the GOLD domain of ACBD3 directly interacts with the regulatory subunit II (RII) of PKA and effectively recruits PKA holoenzyme to the Golgi. Forward trafficking of proteins from the ER triggers activation of PKA by releasing the catalytic subunit from RII. Furthermore, we determined that depletion of ACBD3 reduces the Golgi fraction of RII, resulting in moderate, but constitutive activation of PKA and KDELR retrograde transport, independent of cargo influx from the ER. Taken together, these data demonstrate that ACBD3 coordinates the protein secretory pathway at the Golgi by facilitating KDELR/PKA-containing protein complex formation.

Retro-2 alters Golgi structure.

Retro-2 directly interacts with an ER exit site protein, Sec16A, inhibiting ER exit of a Golgi tSNARE, Syntaxin5, which results in rapid re-distribution of Syntaxin5 to the ER. Recently, it was shown that SARS-CoV-2 infection disrupts the Golgi apparatus within 6-12 h, while its replication was effectively inhibited by Retro-2 in cultured human lung cells. Yet, exactly how Retro-2 may influence ultrastructure of the Golgi apparatus have not been thoroughly investigated. In this study, we characterized the effect of Retro-2 treatment on ultrastructure of the Golgi apparatus using electron microscopy and EM tomography. Our initial results on protein secretion showed that Retro-2 treatment does not significantly influence secretion of either small or large cargos. Ultra-structural study of the Golgi, however, revealed rapid accumulation of COPI-like vesicular profiles in the perinuclear area and a partial disassembly of the Golgi stack under electron microscope within 3-5 h, suggesting altered Golgi organization in these cells. Retro-2 treatment in cells depleted of GRASP65/55, the two well-known Golgi structural proteins, induced complete and rapid disassembly of the Golgi into individual cisterna. Taken together, these results suggest that Retro-2 profoundly alters Golgi structure to a much greater extent than previously anticipated.

ACBD3 modulates KDEL receptor interaction with PKA for its trafficking via tubulovesicular carrier.

BACKGROUND: KDEL receptor helps establish cellular equilibrium in the early secretory pathway by recycling leaked ER-chaperones to the ER during secretion of newly synthesized proteins. Studies have also shown that KDEL receptor may function as a signaling protein that orchestrates membrane flux through the secretory pathway. We have recently shown that KDEL receptor is also a cell surface receptor, which undergoes highly complex itinerary between trans-Golgi network and the plasma membranes via clathrin-mediated transport carriers. Ironically, however, it is still largely unknown how KDEL receptor is distributed to the Golgi at steady state, since its initial discovery in late 1980s. RESULTS: We used a proximity-based in vivo tagging strategy to further dissect mechanisms of KDEL receptor trafficking. Our new results reveal that ACBD3 may be a key protein that regulates KDEL receptor trafficking via modulation of Arf1-dependent tubule formation. We demonstrate that ACBD3 directly interact with KDEL receptor and form a functionally distinct protein complex in ArfGAPs-independent manner. Depletion of ACBD3 results in re-localization of KDEL receptor to the ER by inducing accelerated retrograde trafficking of KDEL receptor. Importantly, this is caused by specifically altering KDEL receptor interaction with Protein Kinase A and Arf1/ArfGAP1, eventually leading to increased Arf1-GTP-dependent tubular carrier formation at the Golgi. CONCLUSIONS: These results suggest that ACBD3 may function as a negative regulator of PKA activity on KDEL receptor, thereby restricting its retrograde trafficking in the absence of KDEL ligand binding. Since ACBD3 was originally identified as PAP7, a PBR/PKA-interacting protein at the Golgi/mitochondria, we propose that Golgi-localization of KDEL receptor is likely to be controlled by its interaction with ACBD3/PKA complex at steady state, providing a novel insight for establishment of cellular homeostasis in the early secretory pathway.