Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi


Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_137


Historical Background

In 1975, coated vesicles, which transport many membrane proteins, were purified and shown to include an ~180-kDa protein named clathrin (Pearse 1975). Later, other molecules involved in clathrin binding to the vesicles were also identified (Keen et al. 1979). In 1987, Keen et al. showed that these molecules formed heterotetrameric complexes, which were termed as adaptor protein complex (AP)-1 and AP-2 (Keen 1987). AP-1 and AP-2 are composed of two large subunits (γ and β1 for AP-1 and α and β2 for AP-2), one medium subunit (μ1 for AP-1 and μ2 for AP-2), and one small subunit (σ1 for AP-1 and σ2 for AP-2). Later, another AP complex  AP-3 was identified on the basis of its structural homology to AP-1 and AP-2 (Pevsner et al. 1994; Newman et al. 1995). The  AP-3 complex, which consists of one molecule each of δ, β3, μ3, and σ3 subunits, is involved in protein trafficking and organelle biogenesis within the endosomal–lysosomal system. In 1997, Wang et al. identified a novel human protein termed μ-ARP2, which was homologous to the μ subunits of AP complexes (Wang and Kilimann 1997) and later renamed μ4. Later studies identified three additional proteins, ε, β4, σ4 and μ-ARP2 (renamed μ4), which form a novel adaptor protein complex AP-4 (Dell’Angelica et al. 1999; Hirst et al. 1999). More recently, the fifth adaptor protein complex AP-5, consisting of ζ, β5, σ5 and μ5, has been identified (Hirst et al. 2011).

Basic Features

AP-4 is a heterotetrameric protein complex composed of two large subunits (β4 and ε), one medium subunit (μ4), and one small subunit (σ4). Because of the extensive similarities among the four AP complexes, AP-4 is likely to form a structure similar to AP-2 (Fig. 1). The C-terminal domain of two large subunits forms “ear domains,” and the “head domain” is composed of the N-terminal domain (trunk domain) of two large subunits and medium and small subunits. The ear and head domains are connected by the flexible “hinge domains” of large subunits. The cargo proteins are thought to be recognized by the μ4 subunit.
AP-4, Fig. 1

Schematic drawing of the AP-4 complex. AP-4 consists of two large subunits (ε and β4), one medium subunit (μ4), and one small subunit (σ4). The C-terminal domain of large subunits forms ear domains. Head domains are composed of two N-terminal trunk domains of large subunits, medium subunit and small subunit. Ear and head domain are connected by hinge domains of large subunits. The interactions between ε subunit and σ subunit, β4 subunit and μ4 subunit, and ε subunit and β4 subunit have been shown by yeast two-hybrid experiments

Interestingly, AP-4 is found only in the mammals and plants, but the reason why other organisms have lost AP-4 during evolution is unknown. The all four subunits of AP-4 are expressed ubiquitously in almost all mammalian tissues. Immunocytochemical analysis using a Golgi stack maker mannosidase II and trans-Golgi network (TGN) markers TGN38 and furin indicates that the AP-4 complex is associated with the TGN (Dell’Angelica et al. 1999; Hirst et al. 1999; Simmen et al. 2002). Brefeldin A treatment caused dissociation of AP-4 from the TGN, suggesting that the recruitment of AP-4 to TGN membranes is regulated by a member of ADP ribosylation factor family protein (Dell’Angelica et al. 1999; Hirst et al. 1999). Immunoelectron microscopic analysis further indicated that AP-4 localized on the nonclathrin-coated vesicles in TGN (Hirst et al. 1999). Indeed, the hinge domains of the ε and β4 subunits lack clathrin-binding motifs conserved in other adaptor protein complexes. These results suggest that AP-4 is likely to be involved in sorting of nonclathrin-coated vesicles in post-Golgi compartments.

Recently, proteomic analysis of AP-4-enriched vesicle fraction has identified tepsin (tetra-epsin) as an associated cytosolic protein. Tepsin contains an ENTH/VHS domain and is structurally related to the AP-1 binding protein EpsinR (Borner et al. 2012). Tepsin is shown to bind to the C-terminal ear domains of the β4 and ε4 (Mattera et al. 2015). Such dual interactions may contribute to the assembly of the AP-4 coat.

Function of AP-4: Transport to Lysosomes

The YXXØ (where Ø indicates a bulky hydrophobic residue) motif mediates targeting of vesicles at various intracellular transport steps, such as endocytosis, targeting from the plasma membrane to the TGN, targeting to lysosomal compartments, and basolateral sorting in polarized cells. Like μ subunits in other AP complexes, the μ4 subunit of AP-4 binds to the YXXØ motif of several proteins, such as lysosomal membrane proteins Lamp-1 (Igp120) and CD-63 (Lamp-3) (Hirst et al. 1999; Stephens and Banting 1998). Screening of a combinatorial peptide library has shown that μ4 prefers aspartic acid at position Y+1, proline or arginine at Y+2, and phenylalanine at positions Y−1 and Y+3 (Aguilar et al. 2001). A signal that fits this preference is found in Lamp-2. Indeed, Tac chimeral proteins bearing a μ4-specific tyrosine-based sorting signal were targeted to the endosomal–lysosomal system. Nevertheless, knockdown of μ4 has no effect on the localization of these AP-4-interacting proteins to lysosomes (Janvier and Bonifacino 2005). This may be because the interaction between μ4 and the YXXØ motif is very weak (Stephens and Banting 1998), and Lamps are mainly trafficked to lysosomes by endocytosis from the plasma membrane via AP-2 (Janvier and Bonifacino 2005). However, approximately half of the Lamps still reach lysosomes in cells depleted with AP-2, indicating that AP-4 (and other APs) may also be involved in direct transport of Lamps from TGN to lysosomes under certain conditions (Fig. 2).
AP-4, Fig. 2

Schematic drawing of cargo protein recognition at the trans-Golgi network (TGN) and transport to lysosome and endosomes. AP-4 binds to the tyrosine-based sorting motifs (YXXΦ) in certain lysosomal membrane proteins (Lamps) and the sequence YKFFE located in the cytoplasmic region of the Alzheimer’s disease amyloid precursor protein (APP) via the μ4 subunit. This association is essential for the transport of these cargo proteins from TGN to lysosome and endosomes

Function of AP-4: Endosomal Targeting of APP

Recently, the μ4 subunit has been reported to interact with the sequence YKFFE located in the cytoplasmic region of the Alzheimer’s disease amyloid precursor protein (APP) (Burgos et al. 2010). The YKFFE motif encompasses a YXXØ motif, but it does not bind to μ subunits of other AP complexes. Interestingly, X-ray crystallographic structure determination shows that the YKFFE sequence binds to a site on μ4 that is different from the YXXØ-binding site on the μ2 subunit of AP-2. A mutation in the YKFFE sequence or depletion of μ4 shifted the distribution of APP from endosomes to the TGN, suggesting that AP-4 mediates transport of APP from TGN to endosomes. Moreover, overexpression of mutant μ4 subunits containing F225A or R283D mutation in the YKFFE motif also shifted APP from endosome to the TGN (Ross et al. 2014). Impaired transport of APP to endosomes enhanced the generation of pathogenic amyloid β peptide by γ-secretase-catalyzed cleavage of APP. These findings indicate that the interaction between AP-4 and APP regulates APP trafficking at TGN or in the late secretory pathway. Furthermore, they suggest that defects in AP-4 could be a potential risk factor of Alzheimer’s disease by producing excessive amyloid β peptide.

Function of AP-4: Polarized Sorting

The plasma membrane of epithelial cells is polarized: They are differentiated into apical and basolateral domains, containing distinctive sets of proteins and lipids. When AP-4 mRNA is disrupted, basolaterally transported proteins, such as the low-density lipoprotein receptor (LDLR) and 46 K cation-dependent mannose-6-phosphate receptor, were distributed nonselectively to both basolateral and apical domains (Simmen et al. 2002). Similarly, Tac chimeric proteins bearing a cytosolic domain of furin were missorted to apical domains. These results indicate that AP-4 mediates basolateral sorting of membrane proteins in epithelial cells (Fig. 3). However, basolateral sorting is also regulated by the AP-1 adaptor protein complex containing the μ1B subunit, which is specifically expressed in most epithelial cells (Ohno et al. 1999). Indeed, many basolateral proteins, such as LDLR and transferrin receptor, are missorted to the apical domain of the epithelial cell line lacking μ1B. On the other hand, it was reported that basolateral sorting of human kidney anion exchanger 1 (kAE1) was affected by the knockdown of μ4, but not μ1B (Junking et al. 2014). Therefore, AP-1B and AP-4 may play complementary and sometimes redundant roles.
AP-4, Fig. 3

Polarized sorting in neuronal cells and epithelial cells by AP-4. α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors indirectly bind to the μ4 subunit of AP-4 via transmembrane AMPA receptor regulatory proteins (TARPs). LDL receptors (LDLR) and the δ2 glutamate receptor (GluD2) directly bind to μ4. These proteins are sorted to the somatodentritic domain at TGN (left panel). In epithelial cells, AP-4 binds to cargo proteins, such as LDLR and 46 K cation-dependent mannose-6-phosphate receptor (MPR46), via the μ4 subunit and regulates basolateral sorting at the TGN (right panel)

Neurons are also highly polarized cells composed of the somatodendritic domain and the axonal domain. The somatodendritic domain receives inputs from other neurons, and the axonal domain transports signals to other cells. Certain neurotransmitter receptors localize in the somatodendritic domain to receive signals from other neurons. For example, the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptor is mainly transported to the dendrites where it mediates the majority of excitatory synaptic transmission. Disruption of the gene encoding the β subunit of AP-4 resulted in nonselective distribution of AMPA receptors in axons of hippocampal neurons and cerebellar Purkinje cells both in vitro and in vivo (Matsuda et al. 2008). Similarly, the δ2 subtype of glutamate receptor ( GluD2) and LDLR were missorted to axons in AP-4β −/− neurons. In contrast, the GluN1 subunit of N-methyl-D-aspartate (NMDA) receptors and metabotropic glutamate receptor (mGluR) 1α proteins was selectively targeted to the somatodendritic domain even in the AP-4β −/− neurons. These results indicate that AP-4 mediates the somatodendritic transport of AMPA receptors,  GluD2, and LDLR. In addition, because neurons lack AP-1B, certain somatodendritic cargos, such as mGluR1 and GluN1, are likely recognized by unknown adaptor proteins other than AP-4 and AP-1B.

A motif containing FR and FTF in the C-terminus of the δ2 subtype of glutamate receptors was shown to bind to the μ4 subunit of AP-4 (Yap et al. 2003). In contrast, μ4 does not directly bind to AMPA receptors but associates with transmembrane AMPA receptor regulatory proteins (TARPs), which tightly bind to all subunits of AMPA receptors (Nicoll et al. 2006). An unconventional motif containing phenylalanine and tyrosine (YRYRF) was shown to be essential for the binding of TARP γ3 to μ4 (Matsuda et al. 2008). When TARPs are expressed in hippocampal neurons, they are excluded from axons in wild-type neurons, whereas they are missorted to the axons in AP-4β −/− hippocampal neurons. Furthermore, the specific disruption of the interaction between AP-4 and TARPs caused the mislocalization of endogenous AMPA receptors in the axons of wild-type neurons (Matsuda et al. 2008). These results indicate that AP-4 regulates proper somatodendritic-specific distribution of AMPA receptors by binding to TARPs in neurons (Fig. 3).

Human Diseases: AP-4 Deficiency Syndrome

Recently, autosomal recessive loss-of-function mutations in any one of the four subunits of AP-4 are shown to result in similar neurodevelopmental human disorders (Verkerk et al. 2009; Moreno-De-Luca et al. 2011; Abou Jamra et al. 2011). These include five patients in one family lacking the μ4 subunit, four patients in two families with disrupted ε, three patients in one family lacking σ4, and three patients in one family lacking β4. Commonly observed symptoms include an infantile muscular hypotonia that progresses to spastic tetraplegia and hypertonia, leading to inability to walk, severe intellectual disability, absent or markedly delayed speech, stereotypic laughter, and growth retardation. The disruption of any subunit generally destabilizes the entire AP complexes. Indeed, knockdown of μ4 or knockout of β4 results in depletion of the entire AP-4 complex in mice (Matsuda et al. 2008). Thus, these human disorders are now referred to as “AP-4 deficiency syndrome.” More recently, the second family with null mutations in genes encoding σ4 has been reported. Two siblings showed infantile onset seizure, severe developmental delay, and spastic paraplegia, phenotypes consistent with the AP-4 deficiency syndrome (Hardies et al. 2015). Furthermore, next-generation sequencing revealed four cases of mutations in the σ4 gene in 110 patients who previously received a diagnosis of cerebral palsy (Tessa et al. 2016). It was also reported that the identical twins with homozygous nonsense mutation in the ε1 gene presented with lymphadenitis caused by the live Bacillus Calmette-Guerin (BCG) vaccine as well as neurodevelopmental human disorders, suggesting that AP-4 may also play a role in the immunological systems (Kong et al. 2013).

As observed in AP-4β −/− mice (Matsuda et al. 2008), postmortem brain histology showed irregular thickening of Purkinje cell axons and aberrant localization of  GluD2 in a patient lacking the μ4 subunit (Verkerk et al. 2009). Nevertheless, symptoms of AP-4 deficiency syndrome are much severer than those observed in AP-4β−/− mice. For example, although the mice exhibited a significantly poorer rotarod performance than wild-type mice, they could walk along a straight line with regular steps throughout their lives. No significant differences in body weight or grip power were observed between the wild-type and AP-4β −/− mice. In addition, there seems to be a certain variation in the severity of symptoms in human patients. In a family lacking β4, some patient showed normal speech and could walk independently until the age of 2 (Abou Jamra et al. 2011). Thus, it remains to be determined what causes each symptom in human and what makes difference in phenotypes between human and mice.


AP-4 is the most recently identified AP complex, whose function has just started to be clarified. It mainly localizes in the TGN and mediates the trafficking of various membrane proteins. It binds to the certain types of tyrosine-based lysosomal trafficking motifs. It also mediates APP trafficking at TGN or in the late secretory pathway. Moreover, in polarized cells, such as the epithelial cells and neurons, AP-4 regulates the polarized sorting of several membrane proteins, such as AMPA receptors and  GluD2. The loss-of-function mutations in any one of the four subunits of AP-4 result in similar neurodevelopmental human disorders called AP-4 deficiency syndrome. It remains to be determined how AP-4-mediated trafficking plays role in the brain development and symptoms in these disorders.


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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of NeurophysiologySchool of Medicine, Keio UniversityShinjuku-ku, TokyoJapan
  2. 2.Department of Engineering ScienceGraduate School of Informatics and Engineering, University of Electro-CommunicationsTokyoJapan