Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

ARFRP1 (ADP-Ribosylation Factor Related Protein 1)

  • Deike Hesse
  • Alexander Jaschke
  • Annette Schürmann
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_177

Historical Background

Due to the remote similarity of the ADP-ribosylation factor related protein 1 (ARFRP1) to other members of the ARF family, it was designated as a distant ARF family member (Fig. 1a). Discovered by the screening of adipocytes with degenerated primers for ARF proteins, ARFRP1 was shown to be highly conserved among species (97% identical amino acids between rat and human, 79% identical to Saccharomyces cerevisiae, and 74% identical to Drosophila melanogaster) and closest related to ARL1 (33% identical) and ARL3 (39% identical) (Schürmann et al. 1995, 1999). In comparison to the membrane association motif of other members of the ARF family (myristoylation), membrane association of ARFRP1 is mediated by acetylation of the initial methionine and interaction with an integral membrane protein Sys1 (Behnia et al. 2004; Setty et al. 2003). The Arfrp1 gene consists of eight exons and is located on distal mouse chromosome 2 (human chromosome 20) with the transcriptional start codon in exon 2 (Mueller et al. 2002a). The gene product, ARFRP1, is a 25 kDa protein with a ubiquitous expression pattern and an intrinsic GTPase activity (Schürmann et al. 1995). In contrast, guanine nucleotide exchange is relatively slow in an in vitro system suggesting the existence of a so-far unidentified GEF (guanine nucleotide exchange factor) for ARFRP1 to act as a fast GTP-dependent molecular switch (Schürmann et al. 1995).
ARFRP1 (ADP-Ribosylation Factor Related Protein 1), Fig. 1

ARFRP1, a member of the ARF family is recruited to the trans-Golgi upon its activation. (a) The ARF family of small GTPases consists of ARF, ARF-like (ARL), and SAR proteins (Kahn et al. 2006). (b) Inactive, GDP-bound ARFRP1 is located in the cytosol, active, GTP-bound ARFRP1 associates with membranes of the trans-Golgi. Active ARFRP1 initiates recruitment of ARL1 and its effector Golgin-245 to this compartment (Zahn et al. 2006)

Molecular Function of ARFRP1

In the active GTP-bound form ARFRP1 is located at the trans-Golgi (Fig. 1b) (Zahn et al. 2006). The yeast homologue of ARFRP1, Arl3p, acts sequentially to recruit golgin proteins to the Golgi membranes. In yeast, Arl3p brings Arl1p, the yeast homolog of ARL1, to the Golgi apparatus which is then responsible for the recruitment of the yeast golgin Imh1p (Panic et al. 2003; Setty et al. 2003). Golgins are conserved proteins found in different parts of the Golgi stack, and they are typically anchored to the membrane at their carboxyl termini by a transmembrane domain or by binding a small GTPase (Rab and ARL1). They appear to have roles in membrane traffic and Golgi structure, but their precise function is in most cases unclear (Munro 2011). In cell culture as well as in murine embryos, ARFRP1 controls the targeting of ARL1 and its effector Golgin-245 to the trans-Golgi (Fig. 1b) (Zahn et al. 2006, 2008). Upon inhibition of the expression of Arfrp1 in cells or deletion of Arfrp1 in mice, the trans-Golgi structure appeared altered as several trans-Golgi markers (TGN38, ARL1, Syntaxin6, Golgin-245) showed a different distribution pattern or a dissociation from the Golgi membranes (Hommel et al. 2010; Zahn et al. 2006, 2008). However, other Golgi proteins located in the cis- and medial-region of the Golgi apparatus (giantin, GM130, 58 k) seemed less affected by the lack of ARFRP1 (Hommel et al. 2010; Zahn et al. 2006, 2008).

In mammalian cells, ARFRP1 seems to inhibit ARF1-regulated pathways such as the activation of phospholipase D (PLD) (Schürmann et al. 1999). ARFRP1 binds the Sec7 domain of the ARF-specific nucleotide exchange factor cytohesin in a GTP-dependent manner. This interaction does not modify the activity of ARFRP1 but results in the inhibition of the ARF/Sec7-dependent activation of PLD in a system of isolated membranes and in HEK-293 cells transfected with a constitutively active mutant of ARFRP1 (Schürmann et al. 1999).

Knockout Models Explaining the Physiological Role of ARFRP1

In order to characterize the function of ARFRP1 in a mammalian organism, its gene was disrupted by gene-targeting approaches. Homozygosity for the conventional transgene causes embryonic lethality, whereas tissue-specific deletion of Arfrp1 resulted in growth retardation according to lipid and glycogen storage defects.

Adhesion Defects Responsible for Embryonic Lethality of Conventional Arfrp1 Knockout Mice

Mueller et al. (2002b) showed that ARFRP1 is already important during early embryogenesis. The amount of Arfrp1 mRNA was detectable from embryonic day 4.5 and increases during gastrulation and neurulation (Mueller et al. 2002b). The conventional deletion of Arfrp1 in the mouse results in embryonic lethality during early gastrulation (Mueller et al. 2002b). Arfrp1-null mutant embryos seemed normal until embryonic day 5, but exhibited profound alterations of the distal part of the egg cylinder at day 6–6.5 due to a cell-adhesion defect (Mueller et al. 2002b; Zahn et al. 2008). Further investigations revealed that embryonic cells showed a mistargeting of E-cadherin to intracellular membranes which prevented epiblast cells to undergo an epithelial-to-mesenchymal transition, and resulted in a failure of mesoderm development (Mueller et al. 2002b; Zahn et al. 2008). This finding was confirmed in studies performed in intestinal epithelium of mice lacking Arfrp1 specifically in the intestine (see below). Here retention of E-cadherin in intracellular membranes was observed, it was co-localized with a cis-Golgi marker (GM130) in epithelial intestinal cells. Moreover, a direct interaction of ARFRP1 with the E-cadherin/catenin complex was demonstrated by co-immunoprecipitation experiments (Zahn et al. 2008) indicating that ARFRP1 is essential for the correct trafficking of E-cadherin through the Golgi and finally for the correct cell surface localization of the E-cadherin complex.

Adipocyte-Specific Deletion of Arfrp1 Resulting in Lipodystrophy and Reduced Survival

ARFRP1 is highly expressed in the adipose tissue of mice (Schürmann et al. 1995). Adipocyte-specific Arfrp1-deleted mice (Arfrp1 ad / ) were generated with the Cre/loxP recombination system using the Fatp4/aP2 promoter (He et al. 2003). Animals were born viable according to the expected Mendelian distribution but exhibited a markedly reduced survival with a mortality rate of 70% until weaning (Hommel et al. 2010). In addition, Arfrp1 ad / mice showed a postnatal growth retardation accompanied by a reduced surface body temperature which presumably is responsible for the impaired survival. The most obvious phenotype of the Arfrp1 ad / mice was a pronounced lipodystrophy indicated by the lack of subcutaneous and gonadal white adipose tissue depots as well as a significantly reduced amount of brown adipose tissue and an early hepatosteatosis. Oil-Red-O staining of brown adipose tissues of Arfrp1 ad / and control littermates indicated an altered lipid storage associated with smaller lipid droplets in the brown fat cells (Hommel et al. 2010). One reason for the impaired lipid storage of Arfrp1 ad / mice was shown to be a stimulation of lipolysis. The amount of phosphorylated hormone-sensitive lipase (HSL) was elevated, and the association of adipocyte triglyceride lipase (ATGL) with lipid droplets was enhanced in brown adipose tissue of Arfrp1 ad / mice indicating that lipolysis was activated. In fact, siRNA-induced knockdown of Arfrp1 in 3T3-L1 adipocytes increased basal lipolysis. A second cause for smaller lipid droplets in adipocytes lacking ARFRP1 was affiliated to a defective lipid droplet fusion. Electron microscopy showed that lipid droplets exhibited ultrastructural alterations such as a disturbed interaction of small lipid-loaded particles with larger lipid droplets (Fig. 2). The SNARE (soluble N-ethylmaleimide-sensitive-factor attachment receptor) protein SNAP23 (synaptosomal-associated protein) which is described to be involved in lipid droplet fusion (Boström et al. 2007) was predominantly located in the cytosol and plasma membrane in brown adipose tissue of Arfrp1 ad / mice, whereas it was associated with small lipid droplets in controls. This suggested that ARFRP1 mediates lipid droplet growth via sorting of SNAP23. Thus, disruption of ARFRP1 in the adipose tissue led to a lipodystrophic phenotype by activating lipolysis and preventing the normal enlargement of lipid droplets via fusion events.
ARFRP1 (ADP-Ribosylation Factor Related Protein 1), Fig. 2

In the absence of Arfrp1 in adipose tissues, the lipid droplets are much smaller as indicated in the ultrastructural analysis performed by electron microscopy (Hommel et al. 2010)

Since SNARE proteins (VAMP2, syntaxin-4, and SNAP23) have been implicated in the insulin-induced translocation of vesicles containing the glucose transporter GLUT4 to the plasma membrane of adipocytes (Hickson et al. 2000; Kawanishi et al. 2000; Bryant et al. 2002), subcellular distribution of GLUT4 in Arfrp1 ad / adipocytes was studied. GLUT4 accumulated at the plasma membrane rather than being sequestered into an intracellular insulin-sensitive compartment as in control adipocytes (Fig. 3) (Hesse et al. 2010). A similar missorting of GLUT4 was produced by siRNA-mediated knockdown of Arfrp1 in 3T3-L1 adipocytes which led to a significantly elevated glucose transport. Thus, ARFRP1 appears to be involved in the sorting of GLUT4.
ARFRP1 (ADP-Ribosylation Factor Related Protein 1), Fig. 3

Downregulation of Arfrp1 by siRNA results in a direct translocation of the glucose transporter GLUT4 to the cell surface without stimulation with insulin. (a) Immunocytochemical staining of GLUT4 in 3T3-L1 adipocytes that were transfected with scrambled or Arfrp1-specific siRNA (left panel). Glucose transport as detected by deoxyglucose uptake was significantly elevated in 3T3-L1 cells depleted for Arfrp1 (right panel). (b) Predicted model of how GLUT4 vesicles are mistargeted to the plasma membrane in the basal unstimulated state when ARFRP1 is deleted (Hesse et al. 2010)

Deletion of Arfrp1 in the Intestine Resulting in Fat Malabsorption

Conditional deletion of Arfrp1 in the intestinal epithelium of mice (Arfrp1 vil / ), as achieved by crossing Arfrp1 flox/flox mice with transgenic mice expressing the Cre-recombinase under the villin promoter, resulted in an early postnatal growth retardation according to an impaired maturation and lipidation of chylomicrons (Jaschke et al. in revision).

Arfrp vil / mice revealed decreased levels of triglyceride and free fatty acid concentrations in the plasma, indicating that their growth retardation is the consequence of a malabsorption. Actually, lipid uptake elucidated by oral fat tolerance tests was impaired in Arfrp1 vil / mice but fatty acids transport into the intestinal epithelium was normal and Arfrp1 vil / mice accumulated lipid droplets in epithelial cells after an oil bolus. However, the release of resynthesized triglycerides was massively decreased, the apolipoprotein ApoA-I accumulated in the Arfrp1 vil / epithelium, whereas its level in the plasma was reduced (Jaschke et al. in revision).

As stated above, ARFRP1 is required for recruitment of ARL1 and its effector, the golgin protein Golgin-245, to trans-Golgi membranes. Since several Rab proteins – involved in the regulation of vesicular trafficking – interact with Golgin-245, their subcellular distribution was studied in the Arfrp1 vil / epithelium. Indeed Rab2 revealed a modified distribution in Arfrp1 vil / epithelial cells as compared with Arfrp1 flox/flox cells. Whereas Rab2 was predominantly located in the cytosol and only partially associated with membranes of the Golgi in control cells, it was mainly detected at large vesicular structures adjacent to the nuclei and co-localized with ApoA-I in Arfrp1 vil / cells (Fig. 4). These data indicated that an ARFRP1-ARL1-golgin-Rab2 cascade is required for intestinal chylomicron maturation in the Golgi.
ARFRP1 (ADP-Ribosylation Factor Related Protein 1), Fig. 4

Co-localization of Rab2 with ApoA-I and its accumulation at Golgi membranes of intestinal Arfrp1 vil / cells. Immunohistochemical detection of Rab2 (left panels), ApoA-I (middle panels), and the merged picture (right panels) in sections of the small intestine of 4-weeks old Arfrp1 flox/flox and Arfrp1 vil / mice that had free access to their diet

Deletion of Arfrp1 in the Liver Impairing Glycogen Storage

The liver-specific deletion of Arfrp1 resulted in a postnatal growth retardation accompanied by a significantly lower absolute and relative liver weight. The discrepancy between liver and body weight observed in Arfrp1 liv / mice could at least partly be explained by the reduced glycogen storage which was reduced by 50% in knockout mice. This effect was referred to a reduced glucose uptake into the liver.

Immunohistochemical staining of the glucose transporter GLUT2 revealed a reduction of GLUT2 in the plasma membrane of Arfrp1 liv / hepatocytes. In addition, total GLUT2 protein in lysates from livers of Arfrp1 liv / mice was much lower compared to the controls. As the quantification of mRNA levels (Slc2a2) showed no alteration between the genotypes, it was speculated that a mistargeting of GLUT2 results in an advanced degradation of this transporter (Hesse et al. manuscript in preparation).

Suppression of ARFRP1 Expression in the Brain by Sleep Deprivation

ARFRP1 is not only expressed in peripheral tissues, it also shows a widespread distribution in the brain (Paratore et al. 2008). Highest expression levels of mRNA were determined by in situ hybridization and real-time PCR in the cerebral cortex, thalamic nuclei, colliculus, substantia nigra, and the granule cellular layer of the cerebellum. These brain areas show high levels of neurotransmitter release, synaptic remodeling, and neuronal plasticity and therefore require extensive synaptic vesicle trafficking. Sleep deprivation alters the expression of genes involved in neuronal plasticity and synapse-related genes. In cerebral cortex the expression of Arfrp1 was markedly reduced after sleep deprivation which could represent an adaptive response to the associated stress. Moderate levels of Arfrp1 were detected in some amygdaloid nuclei, CA2 area, and dentate gyrus of the hippocampus, endopiriform nuclei, globus pallidus, striatum, molecular layer of cerebellum, and locus coeruleus. No expression of Arfrp1 was observed in hypothalamic nuclei, CA1 and CA3 areas of the hippocampus and zona incerta.

Summary

ARFRP1 is a member of the family of ADP-ribosylation factors (ARFs) of GTPases which play a pivotal role in the regulation of membrane traffic. Activated, GTP-bound ARFRP1 associates with trans-Golgi membranes, and is required for the recruitment of ARF-like 1 (ARL1) and its effectors, specific golgin proteins to the trans-Golgi. ARFRP1 is essential for the correct targeting of several proteins (E-cadherin, GLUT4, GLUT2) but is also needed for the normal growth of lipid droplets (in adipose tissues) and the maturation of chylomicrons (in the small intestine) (Fig. 5). However, it is still not known how ARFRP1 is regulated, since no ARFRP1-specific GEF or GAP (GTPase-activating proteins) have been discovered so far. In addition, we did not solve the particular molecular action of ARFRP1 at the Golgi, how it initiates the ARL1-golgin-Rab cascade, and how this cascade finally modulates protein targeting, and lipid droplet and chylomicron maturation.
ARFRP1 (ADP-Ribosylation Factor Related Protein 1), Fig. 5

Proposed model of ARFRP1 action on lipid droplet formation and chylomicron maturation. ARFRP1 is necessary to recruit ARL1 to the Golgi, ARL1 binds to the scaffolding protein Golgin-245 which itself interacts with Rab proteins. Left part of the cartoon indicates that ARFRP1 is required for lipid droplet fusion and the regulation of lipases. Right part of the cartoon demonstrates the chylomicron formation in ER and Golgi. In the ER resynthesized triacylglycerol (TAG) is incorporated into ApoB48-containing pre-chylomicrons. Subsequently, ApoA-IV binds to the pre-chylomicrons which are then released to the cis-Golgi. ApoA-I is attached to the chylomicrons within the Golgi. ApoA-I loaded chylomicrons are then transported through the Golgi, released on the trans-site, and finally secreted into the lymph. Therefore, it is proposed that the ARFRP1-ARL1-golgin-Rab cascade is needed for an appropriate chylomicron assembly of ApoA-I and its transport through the Golgi

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Deike Hesse
    • 1
  • Alexander Jaschke
    • 1
  • Annette Schürmann
    • 1
  1. 1.Department of Experimental DiabetologyGerman Institute of Human Nutrition Potsdam-RehbrückeNuthetalGermany