ARFRP1 (ADP-Ribosylation Factor Related Protein 1)
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
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).
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.
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