Adaptor Proteins: Inter-Organelle Traffic Controllers

INTERORGANELLAR COMMUNICATION: COMPONENTS

Contents Adaptor Proteins: Inter-Organelle Traffic Controllers SNAREs: Membrane Fusion and Beyond ESCRTing around the Cell The Retromer Complex Vesicle Tethers BAR Domains and BAR Domain Superfamily Proteins

Adaptor Proteins: Inter-Organelle Traffic Controllers K Madhivanan, W-C Hsieh, and RC Aguilar, Purdue University Center for Cancer Research, West Lafayette, IN, USA r 2016 Elsevier Inc. All rights reserved.

Adaptor Proteins Adaptors were originally defined as proteins able to link transmembrane cargo proteins to the membrane coat. This definition was implicitly expanded to also require binding to phospholipids and other elements of the trafficking machinery to incorporate cargo into and to facilitate the assembly of transport carriers. Whereas some adaptors have the ability to bind membrane, cargo, and coat proteins, others indirectly fulfill these requirements by interacting with bridging elements and consolidating the protein network (Table 1).

Heterotetrameric Adaptor Complexes Assembly polypeptides (APs) or adaptins are a family of protein complexes involved in vesicle trafficking with five members (AP1–5) found in most eukaryotes (Bonifacino, 2004; Hirst et al., 2013). APs are heterotetramers composed of two large subunits (B100 kDa, β1/γ, β2/α, β3/δ, β4/ε, β5/ζ in AP1–5 respectively), one medium (B50 kDa, μ1–5), and one small subunit (B20 kDa, s1–5) (Figure 1(a)). In addition, AP1–3 exhibit cell type-specific isoforms. While vertebrates and plants have all the 5 members of this family, only AP1–3 are present in flies, worms, and yeast (Hirst et al., 2013). Surprisingly, while in multicellular organisms several APs are required for normal embryonic development, they are dispensable for survival and clathrin-mediated endocytosis (CME) in yeast (Mitsunari et al., 2005; Huang et al., 1999). The large subunits consist of an N-terminal trunk domain, followed by an unstructured hinge domain and a C-terminal appendage domain (Collins et al., 2002; Figure 1(a)). The N-terminal trunks together with the μ and s subunits form the core of the AP complex. While the trunk domains of α, γ, δ, ε,

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and ζ subunits are involved in binding to target membranes, the μ and s subunits are involved in cargo recognition. AP recruitment to membranes is facilitated by ADP-ribosylation factor (ARF) family of proteins in their membrane-bound, GTP-loaded state. While, Arf-6 primarily fulfils this function at the plasma membrane, Arf-1 does it at the trans-Golgi network (TGN) (Ren et al., 2013). The hinge domains of the β subunits in AP1–3 have one or more LФXФ[D/E] (L represents Leucine, X represents any amino acid, and Ф represents bulky hydrophobic amino acids, such as leucine, isoleucine, methionine, valine, and phenylalanine) motifs responsible for binding to clathrin (Owen et al., 2004). However, β4 and β5 lack this sequence and thus AP4 and AP5 complexes were suggested to mediate clathrin-independent vesicle trafficking (Hirst et al., 2013). The appendage domains serve as a platform for interacting with other adaptor and accessory proteins (Figure 1(b); Owen et al., 2004). While the N-terminal domain of μ subunits contributes to the core, the C-terminal domain recognizes tyrosine-based sorting signals within cargoes. AP complexes mediate the sorting of various transmembrane proteins, including type I, type II, and multi-span proteins. To recruit cargoes to vesicles, APs recognize short sorting signal sequences that reside in the cytosolic region, usually at 4–10 residues from the transmembrane domain. There are two well-characterized AP-targeting sorting signal sequences: tyrosine-based and leucine-based signals (Nakatsu and Ohno, 2003; Traub, 2009). The tyrosine signal consists of four amino acids fitting the YXXФ consensus, where Y stand for tyrosine. The medium μ subunits of AP complexes have been identified to play a central role in YXXФ sequence recognition. The leucine-based signals recognized by APs fit into the [D/E]XXXL[L/Ф] consensus, where the -4 position from the first leucine is either aspartic acid or glutamic acid, then followed by three nonconserved amino acids. This sequence motif precedes two

Encyclopedia of Cell Biology, Volume 2

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Interorganellar Communication: Components: Adaptor Proteins: Inter-Organelle Traffic Controllers

Table 1

Adaptor properties

Adaptor name

Clathrin (CBM)

AP2 (ABM)

Lipid – Adaptor binding domain

Example of cargo (signal) – Adaptor Other domains – binding interacting domain/subunit partners

Classical heterotetrameric adaptor proteins recognizing YxxФ and dill signals AP1 þ PI4P – γ,β1 CI-M6PR (YXXФ)-μ1, (diLL)-[γ/s1] TfR (YXXФ)-μ2, CD4 (diLL)-[α/s2] AP2 þ PI(4,5)P2, PI(3,4,5)P3 – α,m2 AP3 þ ? Lamp-1(YXXФ)-μ3, Tyrosinase (diLL)-[δ/s3] AP4 – ? APP (YKFFE)-m4 AP5 – ? ?

451

Localization

TGN Plasma membrane Endosome TGN Late endosome

Adaptors for cargo carrying acidic DXXLL peptide GGA1,2,3 þ PI4P – GAT

M6PR(DXXLL)-VHS

GAT – Ub

TGN

Adaptors for ubiquitinated-cargo Epsin þ þ

PI(4,5)P2 – ENTH

VEGFR2 – UIM

NPF – Eps15

PI(3,5)P2 – EH

EGFR – UIM

ESFGDGFADFSTLS – AP1

Plasma membrane Plasma membrane

PI4P – ENTH

c-Met receptor – coiled coil Vtib-?

DFxD[F/W] – AP1

Eps15

–

Eps15R

þ

þ

Adaptors for cargo carrying NPxY peptide Dab1 – þ PI(4,5)P2 – PTB

ApoER2 (NPxY) – PTB

Dab2

þ

þ

PI(4,5)P2 – PTB

LDLR (NPxY) – PTB

ARH

þ

þ

PI(4,5)P2 – PTB

LDLR (NPxY) – PTB

Numb

þ

þ

PI(4,5)P2 – PTB

APP (NPxY) – PTB

NPF – Eps15

NPF – Eps15

TGN Plasma membrane Plasma membrane Plasma membrane Plasma membrane

Adaptors for GPCRs (Ser and Thr clusters) β-Arr þ þ PI(4,5)P2

GPCRs – Arrestin fold

Plasma membrane

Adaptors for VAMPs AP180 þ

Plasma membrane Plasma membrane

CALM

þ

þ

PI(4,5)P2 – ANTH

Synaptobrevin2 – ANTH

þ

PI(4,5)P2 – ANTH

Synaptobrevin2 – ANTH

NPF – Eps15

Notes: þ , Motif present;  , Motif absent; ?, Not known; Ф, Hydrophobic amino acid; x, Any amino acid, single letter abbreviations of amino acids are used in the table above. Abbreviations: ANTH, AP180 N-terminal homology domain; ApoER2, Apolipoprotein E receptor 2; APP, Amyloid b precursor protein; CI-M6PR, Cation-independent mannose phosphate receptor; EGFR, Epidermal growth factor receptor; EH, Eps15 Homology; ENTH, Epsin N-terminal homology domain; GAT, GGA and Tom; GPCR, G-Protein coupled receptor; LDLR, Low-density lipoprotein 2; PIP, Phosphotidyl inositol phosphate; PTB, Phosphotyrosine binding domain; TfR, Transferrin receptor; TGN, Trans-Golgi network; Ub, ubiquitin; VEGFR, Vascular endothelial growth factor; VHS − Vps27, Hrs and STAM.

leucines that lead to its ‘dileucine signal’ denomination. The first leucine cannot be replaced by any other amino acid, while the second can be substituted with a hydrophobic residue. In some proteins, the acidic residue at -4 position in the consensus can be replaced by a serine that upon phosphorylation acquires a negative charge and becomes functionally equivalent to the D/E residues. The dileucine signal is recognized by γ-s1, α-s2, and δ-s3 hemicomplexes of AP1–3 adaptors (Mattera et al., 2011).

AP1 This adaptor is expressed ubiquitously and is essential during mammalian development (Meyer et al., 2000). The AP1

gamma subunit interacts with the Golgi apparatus-enriched lipid phosphatidylinositol-4-phosphate (PI(4)P) and it is involved in cargo transport between the TGN and endosomal/ lysosomal compartments (Wang et al., 2003; Lefkir et al., 2003). The localization of AP1 is also regulated by phosphorylation of its subunits, namely AP1 associates with membranes when the μ1 subunit is phosphorylated, whereas it detaches when β1 is phosphorylated (Ghosh and Kornfeld, 2003). The cargoes of AP1 include furin, mannose-6-phospate receptors (M6PR), and other proteins of the secretory pathway that are processed at the Golgi apparatus (Bonnemaison et al., 2013; Table 1). It has also been suggested that AP1 and Golgilocalized gamma-adaptin ear-containing Arf-binding proteins (GGAs) cooperate in cargo recruitment to clathrin-coated vesicles (CCVs) by recognition of sorting motifs on the

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Interorganellar Communication: Components: Adaptor Proteins: Inter-Organelle Traffic Controllers









AP1

AP2

AP3

AP4

AP5

1

2

3

4

5

1

2

3

4

5

1

2

3

4

5











(a)

GFP-EPsin2

AP2

Merge

(b)

Figure 1 Heterotetrameric adaptor complexes. (a). Left panel: Cartoon representing the subunit organization of the AP1 complex as example of a heterotetrameric adaptin. Right panel: Subunit composition of known AP complexes. (b) Colocalization of AP2 with the monomeric adaptor Epsin2. HeLa cells expressing GFP-Epsin2 (green) were fixed and the presence of AP2 was revealed by indirect immunofluorescence using an anti-AP2 antibody (red). Scale bar: 20 mm.

cytoplasmic tail of the cargoes (Bonnemaison et al., 2013). In fact, AP1 is believed to be a central player in CCV formation at the TGN affecting a wide variety of transmembrane proteins (Hirst et al., 2012). Additionally, AP1 has also been linked to retrograde transport wherein several receptors were enriched in peripheral compartments upon AP1 depletion (Hirst et al., 2012). The μ1B subunit isoform specifically expresses in epithelial cells forming the AP1B complex which is involved in basolateral sorting of cargo such as the low-density lipoprotein receptor (LDLR) (Gonzalez and Rodriguez-Boulan, 2009). Mutations in AP1 have been linked to X-linked mental retardation (Tarpey et al., 2006), MEDNIK syndrome (Montpetit et al., 2008), and Pettigrew syndrome (Cacciagli et al., 2014).

AP2 This adaptor is expressed ubiquitously and is essential for mammalian development (Mitsunari et al., 2005). AP2 is capable of binding the plasma membrane-enriched lipids PI (4,5)P2 and/or PI(3,4,5)P3 and is a key element for CME. Upon recruitment to the plasma membrane, AP2 undergoes a conformational change from a ‘lock’ state to an ‘open’ state that exposes the clathrin-binding motif on the β2 hinge (Collins et al., 2002). The interaction of AP2 with cargo

stabilizes the open state, and thus facilitating formation and cargo inclusion in clathrin-coated pits (CCPs) (Kelly et al., 2014). The α and β appendage domains interact with other clathrin adaptors and accessory proteins, such as Epsin, ARH, and AP180 to consolidate CCVs. The initial presumption that AP2 was essential for CME was challenged by the observation that although depletion of AP2 reduced CCV formation and specifically blocked transferrin-receptor internalization, epidermal growth factor receptor (EGFR), and LDLR internalization still progressed via CME (Motley et al., 2003).

AP3 Organisms deficient in this adaptor survive to adulthood; however, they have defects in lysosome and lysosomal-related organelle biosynthesis (Yang et al., 2000). AP3 membrane localization is similar to AP1, but with different degree of colocalization with clathrin, suggesting localization/functional differences (Peden et al., 2004). Selective depletion of AP3 subunits resulted in indirect trafficking of lysosomal membrane proteins namely Lamp-1, Lamp-2, and CD63 via a plasma membrane-to-endosome pathway (Dell’Angelica et al., 1999). The β3 and μ3 subunits of the AP3 complex in metazoans have two isoforms: the isoform A is ubiquitously present, while the

Interorganellar Communication: Components: Adaptor Proteins: Inter-Organelle Traffic Controllers

isoform B is specifically expressed in neuronal and neuroendocrine tissues resulting in two AP3 complexes. The ubiquitous AP3A sorts its cargo (Lamp-1, Lamp-2, CD63) to the lysosome, while neuronal AP3B sorts its cargo (Chloride Channel-3, Vesicular glutamate transporter 1, vesicular GABA transporter, and many more synaptic proteins) to synaptic vesicles (Nakatsu and Ohno, 2003). Mutations in the ubiquitous AP3A complex results in Hermansky–Pudlak syndrome type-2, while mutations in the neuronal AP3B complex results in neurological symptoms such as epilepsy and hyperactivity (Badolato and Parolini, 2007; Nakatsu and Ohno, 2003).

VHS

GGA1

GAT

GAE

UIM CBM

Epsin1

ENTH

EpsinR

ENTH

Eps15

E H

Dab2

PTB

ARH

PTB

-Arrestin1

Arr-N

639

NPF

ABM(8)

551

 Appendage binding site

E H

453

625 E H

Coiled-coil

12

897

5

1

770

308

AP4 This adaptor is also ubiquitously expressed in all tissues, but is not essential for development as evidenced by the existence of healthy knockout mice (Matsuda et al., 2008). AP4 localizes to the TGN, but does not colocalize with AP1 perhaps due to the lack of CBMs (Hirst et al., 2013). Notably, perturbation of CCVassembly increased AP4 vesicle formation without changing the expression levels of AP4, suggesting a dynamic compensatory mechanism for TGN trafficking (Hirst et al., 2012). Hence, AP4 is speculated to traffic at least some cargo also transported by CCV. Surprisingly, AP4 recognizes various types of sorting motifs on its cargoes, including canonical tyrosine motif on LDLR, YX[F/Y/L][F/L]E motifs on amyloid precursor protein (APP), FI (Phenylalanine-Isoleucine) motif on Furin, and unconventional basolateral sorting signal (EQFPHLAFWQDLG(Bresciani et al., 1997)) on M6PR (Simmen et al., 2002). AP4 complex was required for the basolateral trafficking of these cargoes. Additionally, AP4 is also involved in basolateral trafficking in neurons where its cargoes are LDLR, AMPA (α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), and δ2 glutamate receptors (Matsuda et al., 2008). AP4-deletion showed aberrant accumulation of APP in TGN rather than in the endosomes implying a role for the AP4 complex in TGN-toendosomes trafficking (Burgos et al., 2010). Deficiency of AP4 in humans has been shown to cause intellectual disability and progressive spasticity that gradually develops to hereditary spastic paraplegia (HSP), suggesting that AP4 participates in human brain development (Hirst et al., 2013).

AP5 This adaptor is the most recently identified, ubiquitous, heterotetrameric adaptor protein complex. Despite having less than 10% protein sequence identity with other APs, the secondary structure prediction of AP5 subunits is similar to the counterparts in other APs (Hirst et al., 2013). AP5 does not associate with clathrin and was shown to localize on late endosomal compartments. Surprisingly, RNAi knock-down of AP5 resulted in cation-independent M6PR trapping in swollen early endosomes, albeit AP5 localization in late endosomes (Hirst et al., 2013). AP5 endosomal trafficking route and cargoes are yet to be described. Mutations in AP5 ζ subunits, similar to AP4 subunits, lead to HSP. It is possible that the same cargo is transported by AP4 and AP5 at different points along the vesicle trafficking pathway.

AP180

ANTH

CALM

ANTH

Arr-C

1

418 12

1

916 652

Figure 2 Monomeric adaptors. Domain/motif organization of several monomeric adaptor complexes is shown. ABM, AP2-Binding Motif; ANTH, AP180 N-terminal Homology domain; Arr, Arrestin fold; CBM, Clathrin-binding motif; EH, Eps15 homology domain; ENTH, Epsin Nterminal Homology domain; GAE, Gamma-adaptin ear homology domain; GAT, GGA and Tom; NPF, Asparagine (N)-Proline (P)Phenylalanine (F) peptide; PTB, Phosphotyrosine binding domain; UIM: Ubiquitin interacting motif; VHS, Vps27, Hrs, and STAM.

GGAs GGAs were identified in early 2000s, and as their name indicates, they are localized at the Golgi apparatus (TGN), but they can also be found on endosomes (Ghosh and Kornfeld, 2004). GGAs are ubiquitously expressed and evolutionarily conserved, with three GGAs in human and mouse, two in yeast, and one in nematode and fruit fly. The existence of distinct GGA functions of the three GGAs has been demonstrated in mouse models. GGA2 null mice are embryonic or neonatal lethal; double mutant of GGA1 and GGA3 result in neonatal lethal, indicating that GGAs are required for developmental process and not fully redundant (Govero et al., 2012). There are four folding modules in GGAs, from N-terminal to C-terminal: VHS (Vps27, Hrs, and STAM), GAT (GGA and Tom), hinge, and GAE (gamma-adaptin ear homology) domain (Figure 2). The GAT domain has an N-terminal α-helical hook subdomain which interacts with Arf-1 and consequently is recruited to the TGN (Collins et al., 2003; Shiba et al., 2003). Like the hinge region of β subunits in AP1–3, the hinge region of GGAs also contains an LФXФ[D/E] CBM, that allows the GGAs to bind clathrin (Bonifacino, 2004). This indicated that GGAs are involved in CM-trafficking from the TGN. The C-terminus of the GAT domain binds ubiquitin and has been implicated in sorting of ubiquitinated-(Ub)-cargo from TGN to the vacuole in yeast (Prag et al., 2005). The role of GGAs in trafficking Ub-cargo in mammals has not been fully explored. GGAs also recognize cargo via the VHS domain which binds a DXXLL motif located in the cytosolic tail of the transported proteins, called acidic-cluster-dileucine signal. The motif consists of an aspartate residue followed by two leucines

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Interorganellar Communication: Components: Adaptor Proteins: Inter-Organelle Traffic Controllers

at the third downstream position (Puertollano et al., 2001; Doray et al., 2002). The D and dileucine bind to the sixth to eighth α-helices of VHS where an electropositive pocket and two hydrophobic pockets reside (Misra et al., 2000; Shiba et al., 2002). In addition, acidic-cluster-dileucine signals also have been found in the hinge region of human GGA1 and GGA3, suggesting the formation of intramolecular complexes competing with cargo recognition; i.e., with auto-inhibitory properties (Bonifacino, 2004). The cargoes trafficked by GGAs include M6PRs and Sortilin (Puertollano et al., 2001; Hirst et al., 2012). Notably GGAs and AP1 cooperate for the trafficking of M6PR from the TGN to the lysosome. The GAE domain homologous to the γ-adaptin ear of AP1, also serves as a platform for binding to other endocytic accessory proteins with a DFGXФ motif, namely γ-synergin, p56 , Rabaptin 5 and Clint/EpsinR in mammals (Bonifacino, 2004). In addition, evidence showed the involvement of GGA1 in the retrograde trafficking of β-site APP-cleaving enzyme 1 from endosomes to the TGN while GGA3 plays a role in the targeting of BACE1 to the lysosomes (Tan and Evin, 2012).

The Epsin and Eps15 Families The Epsin Family These endocytic adaptors including Epsin and EpsinR are highly conserved in yeast and most vertebrates (Sen et al., 2012). There are three Epsin paralogs in humans (Rosenthal et al., 1999; Chen et al., 1999; Spradling et al., 2001). While Epsin-1 and 2 are ubiquitously expressed, Epsin3 has a more restricted expression pattern being confined to migratory keratinocytes, stomach cells, and cancer cells (Rosenthal et al., 1999; Chen et al., 1999; Coon et al., 2011; Spradling et al., 2001). The N-terminus of Epsins has a highly conserved Epsin N-terminal homology domain (ENTH) (Figure 2) that binds PI(4,5)P2, a lipid enriched at endocytic and signaling sites (Sen et al., 2012). This interaction triggers a conformational change in the ENTH domain resulting in the formation of an additional α-helix, helix 0 which inserts into the plasma membrane inducing membrane curvature (Ford et al., 2002). This structural transformation of the ENTH domain is believed to constitute the initial step of membrane invagination required for the formation of CCPs (Horvath et al., 2007; LegendreGuillemin et al., 2004). C-terminal to the ENTH domain is an unstructured region containing multiple motifs required for engaging cargo and endocytic machinery. Each Epsin has two or three UIMs (ubiquitin interacting motifs) able to bind ubiquitinated-cargo (Madshus, 2006). Epsins also bind clathrin via 1 (e.g., yeast) or 2 (e.g., mammalian) clathrin-bindng motifs (CBMs) with the consensus LФZФZ, where L is leucine, Ф is any hydrophobic residue, and Z is any polar residue. In higher eukaryotes, the CBMs flank 3–8 DP[W/F] (aspartate, proline, and tryptophan/phenylalanine) repeats that bind to AP2 (Figure 1B). Lastly, the C-terminus houses 2–3 NPFs (asparagine, proline, phenylalanine) which bind to Eps15 homology (EH) domain containing proteins, namely Eps15 and REPS1/2. In fact, Eps15 and Epsin together have been implicated in EGFR internalization (Sigismund et al., 2005).

EGFR is a receptor tyrosine kinase belonging to the ErbB family which plays a major role in proliferation and differentiation. Interestingly, two different routes have been proposed for Epsin-bound activated EGFR. Classical route entails EGFR ubiquitination and recruitment to CCPs for internalization (Bertelsen et al., 2011; Hawryluk et al., 2006). However, at high EGF concentrations, a clathrin-independent, Eps15- and Epsin-dependent internalization was also found (Sigismund et al., 2005). Interestingly, it has been reported that Epsin binding to ubiquitin negatively regulates clathrin binding (Chen and De Camilli, 2005), supporting the existence of an Epsin-mediated clathrin-independent route. Notably, the fate of EGFR differs based on the internalization route, wherein the clathrin-dependent route and the clathrinindependent route favor recycling and degradation of the EGFR receptor, respectively (Sigismund et al., 2005). While Epsin can contribute to EGFR signaling termination, it is required for activation of the notch signaling pathway (Wang and Struhl, 2004). Elegant studies done in flies to study the juxtacrine notch signaling pathway has shown that Epsindependent Delta endocytosis in the signal-sending cell is required for signaling (Wang and Struhl, 2004). Supporting evidence comes from Epsin-deficient mutants in flies, worms, and mice where defects in notch signaling pathway such as cardiovascular development, germline, and neural tube differentiation were observed (Tian et al., 2004; Chen et al., 2009). Other Epsin-specific cargoes that undergo UIM-mediated internalization are the epithelial sodium channel ENaC (Wang et al., 2006) and the Vascular Endothelial Growth Factor Receptor-2 (Tessneer et al., 2013). Progression from an initiated pit into a vesicle depends on reaching a critical mass of cargo and endocytic components. Epsin-1 contributes to this checkpoint which when unsatisfied will result in the destabilization and abortion of pits (Sen et al., 2012). In addition to serving as an endocytic adaptor protein, Epsins are proposed to function as stabilizers of the endocytic network. Yeast Epsins have also shown to play an analogous role in expanding and sustaining the endocytic network by mediating protein–protein interaction via their NPFs and UIMs (Dores et al., 2010). The Epsin family of adaptor proteins has been recognized as regulators of cancer progression (Tessneer et al., 2013). Epsins are upregulated in skin, breast, prostate, pancreatic, and lung cancers (Tessneer et al., 2013; Sen et al., 2012). The enhanced invasion potential of Epsin-overexpressing cells is attributed to the nonclassical Epsin function in RhoGTPase signaling regulation that leads to enhanced cell migration and invasion (Coon et al., 2010). In addition, Epsin’s role in angiogenesis by regulating VEGFR2 levels has been demonstrated, and considered as a potential treatment of cancer. Knockout of Epsin in Lewis Lung Cancer model effectively reduced tumorigenesis by misregulation of VEGFR2 and angiogenesis (Tessneer et al., 2013).

The EpsinR Family These adaptors bear an ENTH domain similar to Epsin (Figure 2), but differs in lipid specificity as it binds PI(4)P and

Interorganellar Communication: Components: Adaptor Proteins: Inter-Organelle Traffic Controllers

therefore, localizes to the TGN. EpsinR (epsin-related protein) has an AP1-binding motif [D/E]FxD[F/W] and four identified CBM of the consensus DФF. Some of the cargo proteins under control of EpsinR include M6PR and the SNARE protein Vtib (Hirst et al., 2004; Mills et al., 2003). This adaptor is believed to play a role in cargo recycling to the TGN.

Eps15 Family The presence of UIMs in Eps15 (Figure 2) indicates that, similar to Epsin, this protein is capable of ubiquitinated-cargo recognition (although lacks coat-binding determinants). Eps15 contains NPF-binding, EH domains in the N-terminus which also interacts with PI(3,5)P2 (Naslavsky et al., 2007). The Eps15 coiled-coiled region following the EH domain is involved in protein dimerization (van Bergen En Henegouwen, 2009) and along with the UIMs have been shown to define distinct cargo-specificity (c-MET and EGFR) (Parachoniak and Park, 2009; Sigismund et al., 2005; De Melker et al., 2004; Polo et al., 2002). The C-terminus also houses an array of AP2-interacting motifs, and an AP1-interacting motif. Other members of this family include Eps15R which is structurally similar to Eps15 and Eps15b, but lacks the N-terminal EH-domains (van Bergen En Henegouwen, 2009). In addition to its involvement in endocytosis, Eps15 cooperates with AP1 in sorting cargo at the Golgi apparatus. Indeed, mutation of the AP1 binding site on Eps15 impaired the secretion of nascent secretory proteins and TGN-to-endosomal transport of mannose-6-phosphate receptor (van Bergen En Henegouwen, 2009).

AP180 and Clathrin Assembly Lymphoid Myeloid Leukemia The ubiquitous clathrin assembly lymphoid myeloid leukemia (CALM) protein and its neuronal counterpart the assembly protein 180 (AP180) have a characteristic ANTH (AP180 N-terminal homology) domain (structurally related to the ENTH domain) (Figure 2). AP180/CALM binds to PI(4,5)P2, but does not induce membrane deformation (Stahelin et al., 2003). The C-terminus of AP180/CALM has a subtype of CBMs (DL[L/F] – Aspartate, Leucine[Leucine/Phenylalanine]), and several NPF motifs. Except for the yeast homologs, AP180s contain multiple AP2-binding motifs. AP180s are found conserved in yeast (Yap1801, Yap1802), Caenorhabditis elegans (unc-11), and in Drosophila melanogaster (Lap) while CALM is only present in mammalian cells. CALM and AP180 play a conserved role in the endocytosis of SNAREs, specifically vesicle-associated membrane proteins (VAMP)/ R-SNAREs (Moshkanbaryans et al., 2014). Depletion of AP180 and CALM in hippocampal neurons resulted in aberrant retention of synaptobrevin2/VAMP at the neuronal surface, while the abundant synaptic vesicular protein vGLUT1 was unaffected. CALM also plays a role in VAMP3 and VAMP8 internalization in non-neuronal cells. Study of the CALM– SNARE interface by X-Ray crystallography revealed that the N-terminal half of the SNARE domain binds the ANTH

455

domain in a manner similar to the SNARE complex formation (Moshkanbaryans et al., 2014). The involvement of CALM and AP180 in neurodegenerative diseases has gained enormous support in recent years. Genome wide-association study identified alterations in the PICALM gene encoding CALM as a genetic risk factor in the onset of Alzheimer's disease (AD) (Moshkanbaryans et al., 2014). It has also been implicated in affecting cognitive function with aging. A recent study directly correlated the levels of CALM to the levels of pathogenic Aβ complexes in AD, by affecting the endocytosis and localization s-secretase involved in the production of toxic Aβ (Kanatsu et al., 2014). On the contrary, cytotoxicity of Aβ peptide was reduced in yeast, worms, and in mice cortical neurons by overexpressing AP180 family of proteins (Moshkanbaryans et al., 2014). Although these studies are contradictory, they emphasize the role of CALM in APP processing highlighting its therapeutic potential for treatment of AD.

PTB Domain Containing Family (DAB, ARH, and Numb) This family of adaptor proteins is comprised of neuronal disabled-1 (Dab1), ubiquitous Dab2, autosomal recessive hypercholesterolemia (ARH), and numb which share a common phosphotyrosine binding domain (PTB) (Figure 2). This fold is commonly found in signaling molecules and binds to phosphorylated-tyrosines. However, the PTB domain of this adaptor family specifically recognizes the tyrosine-based signal (FX)NPXY on the cytoplasmic tail of receptor proteins (Mishra et al., 2002). The PTB domain structure has been solved and shows simultaneous binding of cargo and PI(4,5)P2 (Dvir et al., 2012; Stolt et al., 2003) and hence regulates vesicle budding from the plasma membrane. These adaptors have been linked to CME via their interaction with AP2 and/or clathrin directly. Dab2 and ARH interact with AP2 and clathrin, while numb and Dab1 only interact with AP2. Additionally, Dab2 has multiple NPF motifs capable of binding to EH domain containing proteins such as Eps15 (Figure 2). Dab2 and ARH have been reported to be functionally redundant for CME of LDLR (Keyel et al., 2006; Maurer and Cooper, 2006). However, while ARH-mediated LDLR internalization required AP2, Dab2-mediated LDLR internalization does not (Keyel et al., 2006; Maurer and Cooper, 2006). The AP2-binding mechanism is very distinct for ARH as it engages AP2 by binding the β2 appendage while the others bind to the α-appendage of AP2 (Mishra et al., 2005). Dab2 was also required for albumin internalization (Maurer and Cooper, 2005) by recognizing the (ФX)NPXY motif on the cytoplasmic tail of the albumin receptor, megalin (Gallagher et al., 2004). PTB domain containing adaptor proteins bind to the amyloid precursor protein family as a (YX)NPXY motif is conserved among members (Howell et al., 1999; Homayouni et al., 1999). Both numb and Dab2 have been implicated in the endocytosis of integrins which also contains NPXY motifs in their cytoplasmic tails (Bridgewater et al., 2012). Mutations in ARH lead to defective endocytosis of LDLR and in consequence to hypercholesterolemia. Dab2 is unable to compensate for the loss of ARH, due to the low levels of

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Interorganellar Communication: Components: Adaptor Proteins: Inter-Organelle Traffic Controllers

expression of Dab2 in hepatocytes. The role of Dab and Numb in APP processing and production of Aβ has been exploited and depletion of these adaptor proteins was shown to reduce the levels of Aβ production (Xie et al., 2012).

Arrestin Family The arrestin family of adaptor proteins shares a common arrestin-fold domain and consists of α-arrestins, a group of vacuolar protein sorting (Vps) 26 related proteins, visual and nonvisual arrestins or β-arrestins. The members of this family show evolutionary conservation from humans, flies, and worms to yeasts (Aubry et al., 2009). Arrestin family in humans has 14 members: 6 α-arrestins, 4 visual and β-arrestins, and 4 Vps26 genes. Phylogenetic analysis suggests that βarrestins, the best-studied members of this clan, branched from the α-arrestins and coevolved with G-protein coupled receptors (GPCRs) to mediate their internalization (Alvarez, 2008). The Arrestins were named for their scaffolding function in binding and terminating G-protein coupled signaling. However, many more functions of this protein family in assisting the internalization of cargoes including GPCRs have been described since. In addition to functioning in signaling regulation of GPCRs by binding to their cytoplasmic tails, β-arrestins’ ability to act as adaptor proteins became clear as they can bind clathrin and AP2 via motifs in their C-termini (Owen et al., 2004; Figure 2). Also, arrestin interaction with phosphoinosities at the plasma membrane is required for recruitment of the cargo into endocytic vesicles (Gaidarov et al., 1999). Structural studies have shed light on the mechanism of action of β-arrestins. In an inactive phosphorylated state, intramolecular interactions between the N- and C-domains of arrestins lead to the shielding of a core of polar residues involved in the recognition of cargo and binding other elements of the endocytic machinery. Arrestin binding to an activated phosphorylated GPCR and subsequent arrestin dephosphorylation (by an unknown phosphatase) disrupts the intramolecular interactions in arrestins and allows binding to the endocytic machinery (Kang et al., 2014). Arrestins internalize cargoes from the plasma membrane and traffic them to the early endosomes by binding phosphorylated Ser/Thr clusters in their C-termini (Kang et al., 2014). The rates of receptor recycling and degradation depend on the strength of receptor–arrestin interaction. For example, Class-A receptors (β2 Adrenergic receptor, vasopressin 1a) with weak interaction are recycled back quickly, whereas Class-B receptors (angiotensin II type 1A receptor, vasopressin 2 receptor) with strong interaction are recycled slowly (Kang et al., 2014). Arrestins get ubiquitinated by the E3 ubiquitin-ligase Mdm2 and their ability to recruit deubiqutinases (DUBs) and undergo deubiquitination also leads to differential consequences. While Class-A receptor–arrestin complex recruits DUBs leading to receptor recycling, Class-B receptors do not have a conformation that supports DUB recruitment and hence leads to stable ubiqutination and degradation (Kang et al., 2014). Subsequently, arrestin–GPCR interaction strength also affects signaling. Specifically Class-B receptors result in the formation of stable signalosomes due to a strong interaction.

Opsin/rhodopsin family of GPCRs serve as cargoes for the visual arrestins. In addition to classic GPCRs, β-arrestins also interact with activated/phosphorylated unconventional GPCRs (Disheveled and Smoothened), Receptor Tyrosine Kinases (IGF-1R, TGFβIIIR), and ion channels (NHE1/5, Ca(v)1, TRPV4, Nicotinic cholinergic receptor) to mediate their internalization. The vps26-related proteins are structurally more similar to β-arrestins than to other members of the family. Vps26A/B, 29, and 35 are four mammalian members of the Vps family part of the so-called retromer complex. The retromer is involved in endosomes-to-TGN cargo transport. However, Vps26 is not directly involved in cargo recognition and therefore, cannot be considered an adaptor. α-Arrestins lack the helix 1 that forms the polar core of inactive β-arrestins. Another major difference is that the C-terminus of α-arrestins contains PPXY motifs instead of the clathrin- and AP2-binding sites found in β-arrestins. Members of the α-arrestin family include the 10 yeast ARTS (Arrestin related trafficking adaptors) and the 6 α-arrestin (ARRDC 1–5; Arrestin domain containing proteins and TXNIP Thioredoxininteracting protein) mammalian proteins. ARTS in yeast have been shown to function as cargo adaptors for the HECT ubiquitin-ligase Rsp5, i.e., facilitating cargo ubiquitination and internalization (Lin et al., 2008). Similar to ARTS in yeast, the role for ARRDCs in mammalian cells as E3 ligase adaptors has been well-established linking GPCR to E3 HECT ubiquitin ligases Nedd4, AIP4, WWP1, and WWP2. In addition, ARTS have been shown to function as secondary adaptors to recruit cargo, β-arrestin-E3 ligase complex on early endosomes (Nabhan et al., 2010). Further, they interact with components of the ESCRT complex and hence are implicated in multivesicular body/vacuolar sorting. GPCRs represent the largest family of receptors (B1000 members) that localize on the cell surface and serve as target for about 40% of the drugs available. Therefore, arrestins are considered valuable targets to control GPCR signaling regulation. Further, arrestin involvement in pathological conditions has been substantially documented. Mutation of visual arrestins is associated with blindness following photoreceptor death due to impaired receptor desensitization. An alternative mechanism leading to blindness by stable association of visual arrestin to the photoreceptor rhodopsin has been suggested (Chen et al., 2006). Overexpression of β-arrestins in AD contributes to the formation of extracellular neuritic plaques containing the amyloid-β (Aβ) peptide. This key pathogenic component of AD was reduced by disrupting the interaction between β-arrestin and β2-adrenergic receptor which in turn lowers Aβ production (Jiang et al., 2013). β-Arrestins participate in signaling pathways mediated by Smoothened and Frizzled are required for the maintenance and onset of chronic myeloid leukemia (CML). Recently, targeting β-arrestin by RNA-aptamers was effective in reducing tumorigenic growth in CML models and patient samples (Kotula et al., 2014).

The Adaptor Protein Network As described above, adaptors bear multiple-binding motifs that mediate several types of protein–protein interactions. This

Interorganellar Communication: Components: Adaptor Proteins: Inter-Organelle Traffic Controllers

promiscuous binding behavior has two general consequences: (1) it allows low-affinity (micromolar range) protein–protein interactions to sustain stable, but dynamic, multi-protein structures, and (2) it confers these massive complexes with classical network properties. For example, it is clear that AP2 at the plasma membrane (or AP1 at the TGN/endosomal level) along with clathrin constitute the ‘hubs’ (maximal connectivity centers) of the adaptor network. This observation explains why these key components are regulatory targets allowing the fast assembly or disassembly of the adaptor network.

See also: Interorganellar Communication: Interplay and Processes: Clathrin and Clathrin-Dependent Endocytosis

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