Close

Autoregulatory circuit regulating basolateral cargo export from the TGN: role of the orphan receptor GPRC5A in PKD signaling and cell polarity

Rosaria Di Martino, Anita Capalbo, Lucia Sticco, Alessandra Varavallo, Vidya Kunnathully, Valentina De Luca, Namrata Ravi Iyengar, Matteo Lo Monte, Petra Henklein, Jorge Cancino, Alberto Luini

Preprint posted on 27 May 2020 https://www.biorxiv.org/content/10.1101/2020.05.26.114710v1

Everything is in its right place: how a previously uncharacterized GPCR regulates self-control of trans-Golgi network (TGN) export.

Selected by Berrak Ugur

Categories: cell biology

This preLight is written in collaboration with Michael Hanna (@LifeCerealMikey)

Background

Cellular health depends on proper trafficking, modification and sorting of various cargo proteins. Trans-Golgi network (TGN) is the main hub for sorting newly synthesized proteins to their destinations as well as receiving recycled proteins (Bonifacino and Rojas, 2006; De Matteis and Luini, 2008). However, the mechanism by which TGN regulates sorting and trafficking of cargo is still not fully characterized.

Protein and lipid cargoes may be destined for different post-Golgi locations necessitating accurate sorting into carriers from the TGN. Much attention has recently been focused on the signaling mechanisms in place to ensure proper packaging and efflux of post-Golgi carriers. For example, one of the G protein-coupled receptor α subunits, G αo, acts as a regulator of trafficking by activating Rab1/3 at Golgi to promote cargo delivery to the plasma membrane (Solis et al., 2017). Sorting cargo toward the wrong destination may induce an ectopic effect. A such defect is observed in hepatocytes, where mis-sorting of E-cadherin away from the basolateral plasma membrane disrupts hepatic cell polarity (Konopka et al., 2007). However, we lack a clear understanding of the steps and signaling processes maintaining TGN efflux. Based on these observations, Di Martino et al., speculate that other autoregulatory mechanisms at the TGN are in place to ensure proper post-Golgi trafficking.

Key Findings:

 To test if such and autoregulatory mechanism exists, the authors focus on identifying signaling processes in response to cargoes destined to the basolateral plasma membrane. By utilizing the well characterized and temperature-sensitive cargo, vesicular stomatitis virus (VSVG), they are afforded stringent localization control to the ER, Golgi, or post-Golgi through temperature shifts. To assess if there is a signaling pathway activated in response to the TGN receiving specifically basolateral cargoes, the authors used a comparative phospho-antibody microarray to detect differences when VSVG retained in the ER versus when it is accumulated in the TGN. Their results indicated a significant increase in several signaling pathways, most notably the phosphorylation of protein kinase D (PKD) substrates.

To test if there is specificity of PKD activation, the authors tested if basolateral of apical cargoes trigger PKD phosphorylation and concluded that only basolateral cargoes stuck in the TGN results in PKD phosphorylation and recruitment. Moreover, the authors found that PKD depletion resulted in aberrant accumulation of VSVG cargo in the TGN indicating this kinase as necessary. At rest, the soluble PKD resides in the cytoplasm and upon activation by diacyl glycerol (DAG), undergoes autophosphorylation (Maeda et al., 2001; Matthews et al., 1999). A critical indicator of active PKD signaling is the local DAG production. Consistent with the previous observations, the authors also confirm that basolateral cargo in the TGN resulted in DAG enrichment at the TGN. Importantly, PKD signaling acts downstream DAG, PLCβ3 and Gβγ (Anitei et al., 2017; Díaz Añel, 2007; Sicart et al., 2015). The authors showed that each of these members were necessary for VSVG efflux from the TGN. The authors then reasoned that there may be a sensor at the TGN monitoring the presence of this specific cargo. To test this hypothesis, they immunoprecipitated VSVG when accumulated at varying points along the secretory pathway. Among the interactors, the orphan GPRC5A (The G protein-coupled receptor, class C, group 5, member A) stood out and it has already been shown to localize to the PM, Golgi and post Golgi vesicles (Zhou and Rigoutsos, 2014). Fitting with this data, GPRC5A depletion resulted in decreased PKD activation and PLCβ3 recruitment to the TGN.

As proper cargo trafficking is essential for cell polarity, the authors tested the role of the GPRC5 induced autoregulatory TGN control mechanism during cell polarization. For this purpose, they utilized HepG2 hepatocytes that rely on the coordinated trafficking of multiple cargo proteins from the TGN to undergo polarization (Müsch, 2014). The depletion of GPRC5C (highly expressed in hepatocytes) in HepG2 cells resulted in polarization defects as well as alterations in cargo secretion. Overall, the authors show that GPRC5 mediated PKD signaling pathway is important for mediating TGN cargo load and maintaining cell polarity (Fig 1).

Fig1. Model of GPRC5 mediated autoregulatory TGN signaling. See Di Martino et al., 2020 for details (https://doi.org/10.1101/2020.05.26.114710).

Take home messages:

  • Cargo arrival in the Trans-Golgi Network (TGN) activates a phosphorylation dependent signaling.
  • Accumulation of basolateral cargo triggers TGN resident orphan G-Protein Coupled Receptor, GPRC5A.
  • GPRC5A acts a cargo sensor to regulate Protein kinase D signaling.
  • PKD activation results in negative feedback signaling to regulate TGN cargo load.

What we liked about this story:

 Although we all learn about Golgi-complex as this huge hub for sorting, packaging and delivery of proteins, the regulatory measures that ensure proper Golgi function are not well-known. This story provides evidence of signaling pathways utilized in response to specific cargoes during post-Golgi trafficking. Moreover, the authors show that a previously orphaned GPRC5 family of Golgi receptors sits atop signaling cascade mediating Golgi trafficking. It remains to be seen whether GPRC5 family proteins have additional roles or if there are other proteins that regulate TGN cargo export.

Remaining Questions: 

  1. Does knockdown of GPRC5A in Hep2G cells also trigger the PLCb3-DAG-PKD signaling pathway?
  2. Do you think that GPRC5 has non-signaling related roles in cargo trafficking?
  3. You propose that GPRC5A and C may have redundant roles. Have you checked whether depletion of GPRC5C is triggering a similar signaling pathway as GPRC5A?
  4. Is the GPRC5A induced signaling pathway robust with endogenous cargoes?

References:

Anitei, M., Stange, C., Czupalla, C., Niehage, C., Schuhmann, K., Sala, P., Czogalla, A., Pursche, T., Coskun, Ü., Shevchenko, A., et al. (2017). Spatiotemporal Control of Lipid Conversion, Actin-Based Mechanical Forces, and Curvature Sensors during Clathrin/AP-1-Coated Vesicle Biogenesis. Cell Rep. 20, 2087–2099.

Bonifacino, J.S., and Rojas, R. (2006). Retrograde transport from endosomes to the trans -Golgi network. Nat. Rev. Mol. Cell Biol. 7, 568–579.

De Matteis, M.A., and Luini, A. (2008). Exiting the Golgi complex. Nat. Rev. Mol. Cell Biol. 9, 273–284.

Díaz Añel, A.M. (2007). Phospholipase C beta3 is a key component in the Gbetagamma/PKCeta/PKD-mediated regulation of trans-Golgi network to plasma membrane transport. Biochem. J. 406, 157–165.

Konopka, G., Tekiela, J., Iverson, M., Wells, C., and Duncan, S.A. (2007). Junctional adhesion molecule-A is critical for the formation of pseudocanaliculi and modulates E-cadherin expression in hepatic cells. J. Biol. Chem. 282, 28137–28148.

Maeda, Y., Beznoussenko, G.V., Van Lint, J., Mironov, A.A., and Malhotra, V. (2001). Recruitment of protein kinase D to the trans-Golgi network via the first cysteine-rich domain. EMBO J. 20, 5982–5990.

Matthews, S.A., Rozengurt, E., and Cantrell, D. (1999). Characterization of serine 916 as an in vivo autophosphorylation site for protein kinase D/Protein kinase Cmu. J. Biol. Chem. 274, 26543–26549.

Müsch, A. (2014). The unique polarity phenotype of hepatocytes. Exp. Cell Res. 328, 276–283.

Sicart, A., Katan, M., Egea, G., and Sarri, E. (2015). PLCγ1 participates in protein transport and diacylglycerol production triggered by cargo arrival at the Golgi. Traffic Cph. Den. 16, 250–266.

Solis, G.P., Bilousov, O., Koval, A., Lüchtenborg, A.-M., Lin, C., and Katanaev, V.L. (2017). Golgi-Resident Gαo Promotes Protrusive Membrane Dynamics. Cell 170, 939-955.e24.

Zhou, H., and Rigoutsos, I. (2014). The emerging roles of GPRC5A in diseases. Oncoscience 1, 765–776.

 

Tags: cell-polarity, golgi complex, pkd

Posted on: 30 June 2020

doi: https://doi.org/10.1242/prelights.22042

Read preprint (No Ratings Yet)

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.

 



List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.

 



List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage
Close