<b>Intracellular localization of GGA accessory protein p56 in cell lines and central nervous system neur</b><b>ons </b>

Abstract

Adaptor protein complex-1 (AP-1) and Golgi associated, γ-adaptin ear containing, Arf binding proteins (GGAs) are clathrin adaptors that regulate membrane trafficking between the trans-Golgi network (TGN) and endosomes. p56 is a clathrin adaptor accessory protein that may modulate the function of GGAs in mammalian cell lines. However, the precise relationship between p56 and the three GGAs (GGA1-3), as well as the physiological role of p56 in tissue cells, remain unknown. To this end, we generated an antibody against p56 and determined its cellular localization. In ARPE-19 cells and mouse embryonic fibroblasts, p56 was found to be localized as fine puncta in the TGN. Interestingly, the depletion of each clathrin adaptor by RNAi revealed that this localization was dependent on the expression of GGA1, but not that of GGA2, GGA3, or AP-1. Using immunohistofluorescence microscopy in the mouse central nervous system (CNS), p56 was clearly detected as scattered cytoplasmic puncta in spinal motor neurons, cerebellar Purkinje cells, and pyramidal neurons of the hippocampus and cerebral cortex. Moreover, double labeling with organelle markers revealed that the majority of these puncta were closely associated with the TGN; however, a small fraction was associated with endosomes or lysosomes in spinal motor neurons. Collectively, these results indicate a functional association of p56 with GGA1, suggesting an important role of p56 in larger CNS neurons.

Clathrin adaptor GGA1 modulates myogenesis of C2C12 myoblasts

“…Recently, it was shown that GGAs and the related clathrin adaptor AP-1 complex have functions not only in TGN-endosomal/lysosomal membrane trafficking, but also for the surface expression of a series of integral membrane proteins, including Notch and the epidermal growth factor receptor [ 13 , 21 ]. Moreover, Conejo et al reported that insulin and insulin-like growth factors signaling play significant roles in the maturation of myotubes [ 22 ].…”

“…The GGA family proteins are well known for their functions in recognition of cargo proteins that are sorted at the TGN; therefore, we initially speculated that GGA1 was involved in the sorting of fusion machinery to the plasma membrane. Indeed, a recent report showed that GGA2 has a crucial role in the cell surface expression of the epidermal growth factor receptor (EGFR), and the GGA-related clathrin adaptor AP-1 complex is also involved in targeting of the cell surface signaling molecule Notch and the secretion of certain soluble factors [ 13 , 21 , 24 , 25 ].…”

“…More recently, Uemura and coworkers showed that GGA2 regulates the cell surface expression of EGFR via direct recognition and binding to the cytoplasmic region of EGFR [ 13 ]. Notably, the binding region of EGFR did not contain the (D/E)xxLL-type, conventional GGA binding motif.…”

“…For example, the GAT domains of GGA1 and GGA3 have higher affinity for ubiquitin compared with that of GGA2 [ 11 ]. Recently, Uemura et al showed that p56, an accessory protein of GGAs, is localized at the TGN in a GGA1-dependent manner [ 12 , 13 ]. In addition, while single knock-out (KO) of Gga1 or Gga3 caused no obvious phenotypes in mice, the Gga1/Gga3 double KO or single Gga2 KO mice were embryonic lethal [ 9 , 10 ].…”

Inactivation of the three GGA genes in HeLa cells partially compromises lysosomal enzyme sorting

“…Over the last two decades, numerous publications of the GGA proteins in tissue culture cells have identified a myriad of binding partners and functions for these monomeric clathrin adaptors [2]. Employing siRNA targeting the individual GGAs, some studies have attributed distinct roles for each GGA [21][22][23], while other studies have found overlapping roles [24,25], or even antagonizing roles [26], depending on the pathway that was being studied. In addition, our analysis of Gga null mice revealed a non-redundant physiological role for the three orthologs in rodents [27].…”