arf gaps and their interacting proteins

# 2007 The Authors

Journal compilation # 2007 Blackwell Publishing Ltd

doi: 10.1111/j.1600-0854.2007.00624.xTraffic 2007; 8: 1465–1475Blackwell Munksgaard

Review

Arf GAPs and Their Interacting Proteins

Hiroki Inoue and Paul A. Randazzo*

Laboratory of Cellular and Molecular Biology, Center forCancer Research, National Cancer Institute, Bethesda,MD 20892, USA*Corresponding author: Paul A. Randazzo,[email protected]

Membrane trafficking and remodeling of the actin cyto-

skeleton are critical activities contributing to cellular

events that include cell growth, migration and tumor

invasion. ADP-ribosylation factor (Arf)-directed GTPase

activating proteins (GAPs) have crucial roles in these

processes. The Arf GAPs function in part by regulating

hydrolysis of GTP bound to Arf proteins. The Arf GAPs,

which have multiple functional domains, also affect the

actin cytoskeleton and membranes by specific interac-

tions with lipids and proteins. A description of these

interactions provides insights into the molecular mecha-

nisms by which Arf GAPs regulate physiological and

pathological cellular events. Here we describe the Arf

GAP family and summarize the currently identified

protein interactors in the context of known Arf GAP

functions.

Key words: actin, Arf1, Arf6, ASAP1, circular dorsal

ruffles, cortactin, EGF receptor, focal adhesion, invado-

podia, podosome

Received 10 May 2007, revised and accepted for publica-

tion 6 July 2007, uncorrected manuscript published

online 12 July 2007, published online 31 July 2007

The ADP-ribosylation factor (Arf) GTPase activating pro-

teins (GAPs) are a family of multidomain proteins ex-

pressed in eukaryotes. The proteins were first identified

as regulators of Arf proteins. The Arfs are GTP-binding

proteins that control membrane traffic and remodeling of

the actin cytoskeleton. By regulating Arfs, Arf GAPs affect

bothmembranes and the actin cytoskeleton. However, the

Arf GAPs have multiple domains that can function both

dependently and independently of Arf proteins to elicit

structural changes and to transduce signals in cells.

The Arf family of GTP-binding proteins is a subfamily of the

Ras superfamily. There are six Arf genes in the mammalian

genome (five in the human genome). The six Arf proteins

are divided into three classes on the basis of amino acid

sequence (1). Class I includes Arf1, Arf2 and Arf3; class II

includes Arf4 and Arf5; and class III includes Arf6. Of

these, Arf1 and Arf6 are the most extensively studied

(2–4). Arf1 has been implicated in Golgi–endoplasmic

reticulum (ER) retrograde transport, intra-Golgi transport,

trafficking from the trans Golgi network (TGN), transport in

the endocytic pathway and recruitment of paxillin to focal

adhesions (FAs). Arf6 has been found to affect endocyto-

sis, phagocytosis, receptor recycling and the formation of

actin-rich protrusions and ruffles (4,5).

The function of Arf proteins is dependent on binding and

hydrolyzing GTP, thereby cycling between GTP-bound

(Arf�GTP) and GDP-bound (Arf�GDP) forms of the protein.

The nucleotide exchange rate intrinsic to Arf is less than

0.01/min and the intrinsic GTPase rate is not detectable (6).

Consequently, the cycle requires the action of accessory

proteins called guanine nucleotide exchange factors and

GAPs. ArfGAP1 was the first Arf GAP purified and cloned.

Examination of ArfGAP1 in vivo yielded data that were

consistent with function as an Arf1 regulator at the Golgi

apparatus (7). The Arf GAPs that were subsequently

identified are structurally complex proteins with molecular

weights between 80 and 200 kDa. In addition to regulating

membrane traffic, several Arf GAPs have been found to be

regulators of the actin cytoskeleton and to be elements of

signal transduction pathways.

The actin structures affected by Arf GAPs are integrated

with membranes and involved in changes in cell shape or

cell movement such as migration and cancer cell invasion.

The actin structures include FAs, invadopodia (and highly

related podosomes), peripheral membrane ruffles and

circular dorsal ruffles (CDRs). Focal adhesions, invadopo-

dia and podosomes (8,9) attach the actin cytoskeleton to

the extracellular matrix (ECM), mediate signal transduction

and are sites of active endo- and exocytosis. Peripheral

membrane ruffles are actin-rich extensions from the cell

edge that are also sites of active endo- and exocytosis and

signaling (10). Circular dorsal ruffles are ring-like structures

projecting from the dorsal surface of the cell. Circular

dorsal ruffles are comprised of labyrinths of tubulated

membranes linked to polymerized actin and are sites of

endocytosis (8,9,11,12).

Part of the effect of the Arf GAPs on cytoskeletal struc-

tures is mediated by the regulation of Arfs. Arf6 has been

implicated in the formation of invadopodia and peripheral

membrane ruffles (5,13). Arf1 has been implicated in FA

dynamics (14). However, the molecular basis for Arf

function at each of these sites is not well defined.

Furthermore, some of the effects of Arf GAPs on the

cytoskeleton have been found to be independent or only

partly dependent on GAP activity; in these cases, the

www.traffic.dk 1465

dominant determinant of the effect of the Arf GAP protein

is association with proteins that contribute to the regula-

tion or structure of the cytoskeletal element. These con-

siderations together with examination of Arf GAPs as Arf

effectors have provided the basis for models explaining the

regulation of cell movement and new insights into the

regulation of the cytoskeleton and associated signaling. In

this review, we describe the Arf GAP family, catalog

proteins that interact with Arf GAPs and discuss hypoth-

eses related to the significance of the associated proteins

to cell signaling and regulation of cell structure.

The Arf GAP Family

The Arf GAPs have a common domain, the Arf GAP

domain, comprising a zinc-binding motif. At least 24 genes

that encode proteins with Arf GAP domains have been

found in the human genome. Most of them have several

synonyms, which can be a source of confusion in the Arf

GAP literature. In Figure 1A, the synonyms are summa-

rized. We have classified the Arf GAPs into two major

types, ArfGAP1 and AZAP types, according to the overall

domain structure (15) (Figure 1). The former type of Arf

GAPs have an Arf GAP domain at the extreme N-terminus

of the protein and the latter contain an Arf GAP domain

between the PH and ankyrin (ANK) repeat domains. Each

Arf GAP group is further subdivided based on additional

domains (Figure 1B).

The six genes grouped as ArfGAP1 type are divided into

three subtypes – ArfGAP, SMAP and GIT. ArfGAP1 and

ArfGAP3 belong to the Arf GAP subtype. In addition to an

N-terminal GAP domain, ArfGAP1 has two ArfGAP1 lipid-

packing sensor (ALPS) domains, which are short stretches

of amino acids that can sense membrane curvature (16).

The GAP activity of ArfGAP1 is stimulated by diacylglycerol

and by increases in membrane curvature (16,17). The

primary function of ArfGAP1 is regulation of the Golgi

apparatus. Initially, ArfGAP1 was proposed to function in

coat disassembly of transport vesicles from the Golgi

through inactivation of Arf1 (7,18,19). Consistent with this

model, the ALPS domains of ArfGAP1 provide a mecha-

nism by which GAP activity increases with changes in

membrane curvature associated with forming transport

vesicles (20,21). Recent evidence indicates that ArfGAP1

may also be a component of a vesicle coat complex that

promotes cargo sorting and that drives vesicle formation

(22–26).

SMAP1 and SMAP2 have been recently characterized

(27,28). Each contains a clathrin box for clathrin binding in

addition to an N-terminal Arf GAP domain. SMAP1 and

SMAP2 function as GAPs for Arf6 and Arf1, respectively.

Evidence for this specificity includes colocalization with

Arf6 in HeLa cells treated with aluminum fluoride (AlF4)

(27,28), which is thought to stabilize an Arf–GDP–AlF4–GAP

complex (29). SMAP2 colocalized with the clathrin adaptor

protein-1 (AP1) and EpsinR (an AP1-binding protein involved

in TGN38 trafficking), on early endosomes and TGN, which

is an Arf1-dependent pathway (28). The primary function of

SMAPs is thought to be as regulators of Arfs.

Two members of the GIT subfamily, GIT1 (Cat1/p95APP1)

and GIT2 (Pkl/Cat2/p95APP2), have three ANK repeats,

a Spa-homology domain (SHD), a coiled-coil domain and

a C-terminal paxillin-binding site (PBS). The GITs have been

proposed to function as regulators of both membrane

traffic and FAs. Although GITs do not show a preference

for particular Arf isozymes in vitro (30), they colocalize with

Arf6 in the cell periphery in vivo. Moreover, functional

analyses of GITs on endocytic events, including G-protein-

coupled receptor internalization, suggest GITs function

with Arf6 (31,32). It has also been reported that over-

expression of GIT2-short, a splice variant of GIT2 lacking

the C-terminal PBS, induced redistribution of b-COP,

which is consistent with function as an Arf1 GAP (33).

AZAP-type Arf GAPs are characterized by a PH, Arf GAP,

ANK repeat domain structural motif. Twelve genes for

AZAPs are subdivided into four subtypes (ASAPs, ACAPs,

ARAPs and AGAPs). ASAP-subtype GAPs comprise a Bin/

amphiphysin/Rvs (BAR) domain, a PH domain, an Arf GAP

domain, ANK repeats, a proline-rich (Pro) domain and an

SH3 domain; ASAP3 (UPLC1/DDEFL1/ACAP4) does not

have the C-terminal SH3 domain. ASAP1 (DEF1/DDEF1/

centaurin b4/AMAP1/PAG2) has been implicated in the

regulation of FAs, CDRs, invadopodia and podosomes

(12,34,35). ASAP1 and ASAP2 (Papa/DDEF2/centaurin

b3/AMAP2/PAG3) prefer Arf1 and Arf5 to Arf6 in vitro

(36,37). Consistent with the in vitro results, overexpres-

sion of ASAP1 in vivo has been reported to decrease

Arf1�GTP levels and to increase Arf6�GTP levels (38,39).

Furthermore, ASAP1 does not inhibit Arf6-dependent

membrane protrusions (40). Some evidence supports the

idea that ASAP1 and ASAP2 also function with Arf6

through direct binding and slow catalysis (41,42).

ASAP1 GAP activity is more extensively analyzed than

those of other Arf GAPs. The GAP activity is stimulated

about 10 000-fold by PI(4,5)P2 and phosphatidic acid (PA)

(36,43). This stimulation depends on a conformational

change that is caused by PI(4,5)P2 binding to the PH

domain (43,44). Kinetic studies support the proposal that

ASAP1 functions in binary complex with Arf1 to induce

GTP hydrolysis; however, a description of the details

concerning the contribution of specific amino acids from

ASAP1 and Arf to catalysis will require further structural

studies (29,44,45).

ACAPs are similar to ASAPs in structure. They are com-

posed of BAR, PH, Arf GAP and ANK repeats domains.

ACAP1 (centaurin b1) and ACAP2 (centaurin b2) were

biochemically characterized and found to have a preference

for Arf6 over Arf1 and Arf5 in vitro and in vivo (40). Both are

activated by PI(4,5)P2 and PI(3,5)P2 (40).

1466 Traffic 2007; 8: 1465–1475

Inoue and Randazzo

A

ArfGAP1

ArfGAP3

SMAP1

SMAP2

GIT1

GIT2

ASAP1

ASAP2

ASAP3

ACAP1

ACAP2ACAP3ARAP1

ARAP2

ARAP3

AGAP1

AGAP2

AGAP3

Subtype SynonymNCBI Gene ID

(human/mouse)Arf GAPs

55738 / 228998

26286 / 66251

60682 / 98366

10902 / 69780

28964 / 216963

9815 / 26431

50807 / 13196

8853 / 211914

55616 / 230837

9744 / 216859

23527 / 78618116983 / 140500116985 / 69710

116984 / 212285

64411 / 106952

116987 / 347722

116986 / 216439

116988 / 213990

ArfGAP

SMAP

GIT

ASAP(AZAP-type)

ACAP

ARAP

AGAP

(ArfGAP1-type)

ARF1GAP / MGC39924 / HRIHFB2281

FLJ13159 / FLJ42245

BRD8 / p120 / Smap1l

Cat-1 / p95-APP1 / KIAA0148 / MGC760

Cat-2 / p95-APP2 / PKL

AMAP1 / DDEF1 / DEF1 / PAG2 / Centaurin beta4 / Shag1 / KIAA1249PAP / AMAP2 / DDEF2 / PAG3 / Centaurin beta3 / FLJ42910 / Gm1523 /

DDEFL1 / UPLC1 / ACAP4 / FLJ20199 /

Centaurin beta1 / KIAA0050 / MGC25782 /

Centaurin beta2 / KIAA0041Centaurin beta5 / KIAA1716Centaurin delta2 / KIAA0782Centaurin delta1 / FLJ13675 / FLJ44916 / KIAA0580 / PARX

MGC47358

Gm140 / MGC102639

Gm592 / MGC90837

Centaurin delta3 / DRAG1 / FLJ21065 / KIAA4097Centaurin gamma2 / GGAP1 / KIAA1099 / MGC71657Centaurin gamma1 / FLJ16430 / GGAP2 / KIAA0167 / PIKECentaurin gamma3 / CRAG / FLJ16146 / MRIP-1 / MGC37541

ARFGAP1

ANKhARAP1

1 1450 aa

ArfGAPSAM PH RARhoGAPPH PHPH PH

mASAP11 1147 aa

ANKArf GAPBAR PH Pro(PxxP)3

SH3Pro(D/ELPPKP)8

100 aa

B

rArfGAP11 415 aa

Arf GAP ALPS1/2

mSMAP21 428 aa

Arf GAP CALMBD

CB

mGIT11 770 aa

ANKArf GAP PBSSHD CC

hACAP11 740 aa

ANKArf GAPBAR PH

hAGAP11 804 aa

ANKArf GAPGLD PH

Figure 1: Synonyms and representa-

tive domain structures of Arf GAPs.

A) Synonyms of Arf GAPs. Arf GAPs

were classified into two large types and

subdivided into seven subtypes based

on their overall domain structures. Syn-

onyms that have been used in literature

and databases are listed with their NCBI

Gene ID. B) Domain structures of repre-

sentative Arf GAPs. One from each sub-

type was drawn to scale. Arf GAP, Arf

GAP domain; CALM BD, CALM binding

domain; CB, clathrin box; CC, coiled-coil

domain; h, human; m, mouse; Pro (D/

ELPPKP)8, 8 tandem proline-rich (D/

ELPPKP) motifs; Pro (PxxP)3, 3 proline-

rich (PxxP) motifs; r, rat; Rho GAP, Rho

GAP domain.

Traffic 2007; 8: 1465–1475 1467

Arf GAP Binding Proteins

ASAPs and ACAPs contain BAR domains. The structure of

the BAR domain of Drosophila amphiphysin has recently

been solved. It was found to be a crescent-shaped dimer.

The BAR domain of amphiphysin as well as those from

other proteins including ACAP1 bind synthetic liposomes.

The efficiency of binding was inversely related to the radii of

the vesicles and directly related to the curvature. Based on

this property, the BAR domain was proposed to be a curva-

ture sensor. Recombinant BAR domains also cause defor-

mation of the liposomes, resulting in tubular structures (46).

The BAR–PH domain of ACAP1 was found to function

primarily as a curvature sensor. Similarly, a recombinant

protein comprising the BAR and PH domain of ASAP1

sensed membrane curvature. On the other hand, a recom-

binant protein comprising the BAR, PH, Arf GAP and ANK

repeat domains of ASAP1 poorly sensed membrane curva-

ture but efficiently induced tubulation of liposomes. The

tubulating activity was regulated by Arf1�GTP and the GAP

domain of ASAP1 (47). A functional relationship with the

geometry of membrane surfaces may be a common char-

acteristic of ASAPs and ACAPs, which contain BAR do-

mains, and ArfGAP1, which contains ALPS domains.

ARAPs are the largest proteins in the Arf GAP family. GAPs

in this subtype have a Rho GAP domain in addition to an Arf

GAP domain. They also contain a sterile a-motif (SAM),

five PH domains and a Ras association (RA) domain. Two

of the five PH domains contain a PI(3,4,5)P3-binding con-

sensus sequence. PI(3,4,5)P3 more potently stimulates

the Arf GAP activity of ARAPs than do other phosphoinosi-

tides (48–51). The three members of the ARAP subtype of

Arf GAPs have different Arf specificities. ARAP1 (centaurin

d1) functions with Arf1 and Arf5 (48). ARAP2 (centaurin d1/

PARX) preferentially uses Arf6, as compared with Arf1 and

Arf5, as a substrate in vitro and in vivo (50). ARAP3 (DRAG/

centaurin d3) has been reported to function as an Arf6 or

Arf5 GAP in vitro (49,51) and to regulate Arf6-dependent

events including membrane protrusions and ruffling in vivo

(49,52).

AGAPs have a GTP-binding protein-like domain (GLD) at

the N-terminus. The PH domains of AGAPs are split with

an 80-amino-acid insert between b strands 5 and 6. AGAP1

(GGAP1/centaurin g2) and AGAP2 (PIKE/GGAP2/centaurin

g1) prefer Arf1 and Arf5 to Arf6 in vitro, and function at

endosomes with Arf1 in vivo. The GAP activity of the

AGAPs is stimulated by PI(4,5)P2 and PA (53–55).

Proteins That Interact with Arf GAPs

As described, Arf GAPs are multidomain proteins. Through

these domains, Arf GAPs are able to interact with a variety

of proteins (Table 1) that we have categorized into three

groups based on function in membrane traffic, signaling or

regulation of the actin cytoskeleton. Selected interactions

are mapped in Figure 2 together with the proposed Arf

specificity of the Arf GAPs.

Membrane traffic

Transmembrane cargo proteins

Two Arf GAPs, ArfGAP1 and ACAP1, have been found to

bind directly to transmembrane proteins that can be

considered either cargo or cargo receptors. ArfGAP1 binds

to p24 cargo proteins and to ERD2, a receptor for proteins

with the ER retention signal KDEL that mediates retro-

grade transport of ER-resident proteins from the Golgi to

the ER (56). Peptides from p24 family proteins have been

found to inhibit Arf GAP activity (57). This observation was

the basis of a model explaining GAP control of cargo

sorting (58), which is described in detail in Nie and

Randazzo (26). The effect of ERD2 binding to Arf GAP1

enzymatic activity has not been examined.

ACAP1 interacts with transferrin receptor (TfR), cellubrevin

and integrin b1 (59,60). ACAP1 recognizes two distinct

diphenylalanine-based sequences in the cytoplasmic tail of

TfR. Disruption of the interaction impairs recycling of the

receptor to the plasma membrane. These observations

were the basis for the suggestion that ACAP1 may

function as novel coat or adaptor protein in the recycling

compartment. It has been proposed that ArfGAP1 func-

tions in a similar capacity, directly binding cargo to carry it

into membrane trafficking intermediates (22).

Membrane traffic coat proteins

Three classes of Arf GAPs have been found to bind to

vesicle coat proteins or coat protein adaptors. ArfGAP1 has

been found to bind to coatomer and clathrin AP1 (61,62).

The work addressing the consequences of the interaction

with coatomer is limited. In one study, coatomer was

found to stimulate Arf GAP activity (63); it has also been

reported that coatomer has only a small effect on Arf GAP

activity (64). Whether the coatomer–Arf GAP interaction

has an effect on the formation of vesicles has not been

established. Both SMAP-type GAPs bind to clathrin

(27,28). In addition, SMAP2 interacts with CALM, a clathrin

assembly protein (28). The interaction was found to drive

the formation of transport intermediates from the plasma

membrane and from the TGN. AGAP1 and AGAP2 asso-

ciate with clathrin adaptor proteins, AP3 and AP1, respect-

ively (54,55). In both cases, AGAP was found to affect the

function of the endocytic compartment containing these

clathrin adaptors. Specific molecular consequences of the

SMAP or AGAP – coat proteins interactions – have not

been determined. One possibility that has been proposed

is that the Arf GAPs function as a subunit of a vesicle coat

protein in a manner analogous to the role of Sec23 in ER-

to-Golgi transport mediated by COP-II vesicle coats (65).

Adaptor proteins involved in membrane traffic

ASAP1, ASAP2 and ARAP3 have been reported to bind to

proteins that function as part of the endocytic machinery –

CIN85, POB1 and amphiphysin IIm (42,66,67). CIN85 is an

adaptor protein containing three SH3 domains and a

1468 Traffic 2007; 8: 1465–1475

Inoue and Randazzo

Table 1: Arf GAPs and their interacting proteins

Designation Binding protein Binding site on Arf GAP Function/remarks Reference

ArfGAP1 g-adaptin (AP1) C-terminal Unknown 62

KDEL receptor/ERD2 Unknown ER protein retrieval 56

KDEL protein recruits ArfGAP1 binding

p24a Unknown KDEL protein sorting? 94

ArfGAP3 g-COP (COPI) Unknown Golgi / ER retrograde transport? 61

SMAP1 Clathrin Clathrin box Clathrin-dependent Tfn endocytosis 27

SMAP2 CALM CALM binding domain Unknown; clathrin assembly?

Clathrin Clathrin box EE / TGN retrograde transport 28

GIT1 PIX SHD FA turnover 95

Actin remodeling

Cell spreading, migration

Centrosome maturation

PLCg SHD PLCg activation, IP3 production 69

MEK1 SHD MEK1 activation by AngII or EGF 77

FAK SHD Cell migration 75

Paxillin PBS FA turnover 86

Hic-5 PBS Paxillin-like 86

Huntingtin CC þ PBS Huntington disease pathogenesis

GPCR kinases Unknown b2AR downregulation 78

14-3-3zeta Unknown Actin remodeling 96

GIT2 PIX SHD FA turnover 87

Actin remodeling

Cell spreading, migration

Paxillin PBS FA turnover 86,87

Hic-5 PBS Paxillin-like 86

Leupaxin PBS Paxillin-like; podosome in osteoclast 88

GPCR kinases Unknown b2AR donwnregulation 79

ASAP1 CIN85 Pro (PxxxPR) EGFR recycling 66

Tumor invasion

CD2AP Pro? Unknown 37

Cortactin Pro (PxxP)? Cell migration, tumor invasion 34

Crk Pro (PxxP) Unknown 36

CrkL Pro? FA turnover 35

ASAP1 recruitment to FA

c-Src Pro (PxxP) Unknown 36

FAK SH3 Focal adhesion turnover 83

Cell spreading

Pyk2 SH3 Inhibition of ASAP1 GAP activity 84

POB1 SH3 Cell migration 67

ASAP2 Paxillin Pro? Cell migration 41

Amphiphysin IIm Pro Tac endocytosis 42

Pyk2 SH3? SEAP secretion? 37

ACAP1 NOD1/2 BAR–PH Downregulation of NFkB 90

Integrin b1 pSer around ANK Integrin b1 recycling 60

Cell migration

Tfn receptor Unknown TfR recycling 59

Cellubrevin Unknown Unknown 59

ACAP2 Vaccinia virus K1L protein Unknown Unknown 92

ARAP2 RhoA Rho GAP FA turnover 50

Stress fiber formation

ARAP3 SHIP2 SAM Unknown 74

CIN85 Pro (PxxxPR) Unknown 66

Rap1 RA Stimulation of Rho GAP activity 85

AGAP1 AP3 PH Endosome–lysosome trafficking 54

NO-sensitive soluble guanylyl cyclase PH–GAP–ANK Unknown 92

AGAP2 PLCg N-terminal (Pro) Nucleotide exchange on GLD? 70

Nuclear PI3 kinase N-terminal (Pro) Activation of nuclear PI3K 72

Protein 4.1N N-terminal (Pro) Downregulation of nuclear PI3K 72

Homer N-terminal (Pro) Coupling of mGluR and PI3K 73

Antiapoptosis in neuronal cell

Akt GLD Activation of Akt kinase activity 81

Tumor invasion

AP1 PH AP1-dependent Tfn recycling 55

CC, coiled-coil domain; NFkB, nuclear factor kappa B; SEAP, secreted alkaline phosphatase.

Traffic 2007; 8: 1465–1475 1469


proline-rich domain. CIN85 was first characterized as a

Cbl-interacting protein for epidermal growth factor (EGF)

receptor (EGFR) internalization. The SH3 domains of CIN85

interact with an atypical proline-rich motif (PXPXPR) in

ASAP1 and ARAP3 (66). Overexpression of ASAP1 accel-

erated EGF and EGFR recycling in CHO cells and HeLa

cells (47,66), although the detailed mechanism underlying

this effect is still unknown. CD2AP is a protein highly re-

lated to CIN85 that has also been found to bind to ASAP1

and has been proposed to recruit ASAP1 to the plasma

membrane (39).

The SH3 domain of ASAP1 (PAG2) mediates binding to a

proline-richmotif in the EH-domain-containing protein POB1

(67). Treatment of cells with EGF results in the phosphory-

lation of POB1 and its recruitment to the EGFR (68). POB1

simultaneously binds Ral-binding protein (RalBP), which

contains a Rho GAP domain (67). The complex of ASAP1,

POB1 and RalBP could regulate actin remodeling by control-

ling RhoA�GTP levels, thereby coordinating changes in the

actin cytoskeleton with membrane traffic.

Amphiphysin IIm is a splice variant of amphiphysin II,

which is a protein with BAR and SH3 domains. Amphiphy-

sin II functions in synaptic vesicle endocytosis. The proline-

rich domain of ASAP2 (Papa/PAG3/AMAP2) binds to the

SH3 domain of amphiphysin IIm. Both overexpression and

reduced expression (achieved using siRNA) of ASAP2

inhibit internalization of Tac, the interleukin-2 receptor

a subunit, in HeLa cells (42).

Signaling proteins

Lipid-modifying enzymes

Three types of Arf GAPs – GITs, AGAPs and ARAP3–

interact with enzymes that regulate signaling lipids. Phos-

pholipase Cg (PLCg), an important element of the PIP2 to

IP3 pathway, binds to the SHD of GIT1 (69). This interac-

tion is necessary for PLCg activation induced by angioten-

sin II (AngII) and EGF in vascular smooth muscle cells and

293 cells.

AGAP2 (PIKE/centaurin d1) is another Arf GAP that binds to

lipid metabolizing enzymes. AGAP2 has been reported to

bind to PLC g through its N-terminal proline-rich sequence

(70). In one report, PLCg was found to function as an

exchange factor for nucleotide on the GLD domain of

AGAP2 (70), although recent kinetic analyses indicate that

the affinity for nucleotide of AGAPs is such that basal

exchange rates are extremely rapid, obviating the need for

an exchange factor (71). One form of AGAP (PIKE) binds to

and activates phosphatidylinositol (PI) 3-kinase (PI3K) (72),

preventing neuronal apoptosis. The interaction with PI3K is

thought to be important for the growth and invasion of

glioblastoma cells (73).

Figure 2: Selected Arf GAPs protein complexes involved in receptor trafficking, cell migration and invasion. Arf GAPs that are

related to receptor trafficking, FA turnover, cell migration/spreading or tumor invasion are mapped with the interacting proteins and

expected substrate Arfs. The GAPs that use Arf1 or Arf6 as a substrate are labeled with blue or yellow, respectively. The GAPs whose

substrate specificity is controversial or that may use Arf5 are labeled with green.

1470 Traffic 2007; 8: 1465–1475

Inoue and Randazzo

ARAP3, through its SAM domain, has recently been

reported to bind the PI 5-phosphatase, SHIP2 (74). The

physiological consequences of this interaction have not

been defined; however, given ARAP3 is recruited to the

plasma membrane with PI(3,4,5)P3 and its Arf GAP activity

is regulated by PI(3,4,5)P3, a plausible model is that ARAP3

negatively regulates PI3K signaling by recruiting SHIP2 to

the membrane creating a negative feedback loop.

Protein kinases

Protein kinases have been found to function upstream and

downstream of Arf GAPs. The interaction between GIT

and p21-activated kinase (PAK) (32) is one of the best

studied. It is indirect. PIX, a Rac/Cdc42 exchange factor,

acts as a bridge between GIT and PAK. Interfering with the

association between GIT and PAK has been found to

disrupt cell motility and function of FAs (75,76). The effects

of GIT on actin are thought to be mediated, in part, by the

action of PAK. GITs have also been implicated as regulators

of serine/threonine kinases within signaling pathways.

GIT1, interacts with MEK1, extracellular signal-regulated

kinase kinase. The interaction is constitutive, but GIT1

stimulates MEK activity in response to AngII and EGF

activation of Src-dependent phosphorylation of GIT (77).

GIT is an upstream regulator of PAK andMEK but functions

downstream of another serine/threonine kinase, GPCR

kinase2 (GRK2). Both GIT1 and GIT2, excluding the splice

variant GIT2-short, bind to GRK2 (78,79). GPCR kinase2 is

recruited to GPCR in response to an agonist. GITs are

recruited by GRK2 into this complex and mediate sub-

sequent internalization of the G-protein-coupled receptor.

This function of GIT depends on GIT Arf GAP activity. Point

mutants of GIT that are deficient in GAP activity block

internalization.

AGAP2 (PIKE/centaurin g1/GGAP2) has been reported to

interact with two classes of kinases. Src family proteins

phosphorylate AGAP2 on two tyrosines (80). The phosphory-

lation prevents apoptotic cleavage of AGAP2 during pro-

grammed cell death, which is consistent with the

proposed role of AGAP2 in the anti-apoptotic signaling

pathway. One signal in this pathway that is downstream of

AGAP2 is PI3K. AGAP2 also binds to and activates

a serine/threonine kinase that is critical to this pathway,

Akt (81,82). The AGAP2/Akt complex has been reported to

be dependent on GTP, although given the lack of nucleo-

tide specificity and low affinity recently reported in kinetic

studies (71); the details of the molecular mechanisms

regulating the complex are not obvious and need further

examination.

ASAP1 binds to and functions with two protein tyrosine

kinases, Src and focal adhesion kinase (FAK), including the

FAK homolog Pyk2 (35,83,84). Expression of constitutively

active Src results in the phosphorylation of ASAP1. The

SH3 domain of Src and Src family proteins bind to a PXXP

motif in ASAP1. Focal adhesion kinase binds to the SH3

domain of ASAP1. The first evidence for a role of FAK in

the cellular function of ASAP1 came from studies exam-

ining FAs (83). Disruption of the interaction resulted in

partial dissociation of a FA protein called paxillin from FAs

and changes in cell motility. Deletion of the SH3 domain

of ASAP1 also disrupted the formation of podosomes

consistent with direct binding of FAK to ASAP1 having a role

in this process; however, another protein partner cannot be

excluded on the basis of available data. The phosphorylation

of ASAP1, consequent to expression of activated Src, is

necessary for the formation of podosomes and invadopodia,

which are actin-rich structures on the ventral surface of cells

that mediate adhesion, ECM degradation and tumor inva-

sion. Both Src and Pyk2 have been found to directly

phosphorylate ASAP1, which inhibits the GAP activity for

Arf1 (84). A relationship between the effect of phosphory-

lation on GAP activity and podosome formation has not

been examined to the best of our knowledge.

Small G proteins

Members of other G-protein families, in addition to Arf,

function with Arf GAPs to regulate remodeling of the actin

cytoskeleton. ARAP family Arf GAPs function with Rho and

Rap family proteins in at least two capacities to regulate

actin and actin-associated structures (49,52,85). ARAP1 and

ARAP3 contain active Rho GAP domains that use RhoA as

a substrate in preference to Rac1 and Cdc42. Effects of

ARAP1 and ARAP3 on the actin cytoskeleton are dependent

on the Rho GAP activity, presumably functioning to inacti-

vate RhoA. ARAP2 has an inactive Rho GAP domain,

consequent to lack of the catalytic arginine found in all other

Rho GAPs; however, ARAP2 does bind RhoA�GTP through

its inactive Rho GAP domain. RhoA binding to ARAP2, as

well as ARAP2Arf GAP activity, is required for FA and stress

fiber formation in U118 glioblastoma cells, consistent with

ARAP2 function as a RhoA effector (50).

The ARAPs also contain an RA domain immediately

C-terminal of the Rho GAP domain. Rap1 binds to the RA

domain of ARAP3 and regulates the Rho GAP activity.

Regulation of peripheral actin ruffles that are regulated by

ARAP3 depend on the ability of ARAP3 to bind to Rap1

(52,85).

Actin cytoskeleton

Adhesion and scaffold molecules

The GITs and the ASAPs have been found to regulate FAs

and ASAPs have also been implicated in the regulation of

invadopodia and podosomes. Consistent with this func-

tion, interacting proteins that are components of these

adhesive structures have been identified. Three Arf GAPs,

GIT1, GIT2 and ASAP2 (PAG3/AMAP2), have been re-

ported to interact with paxillin and related proteins, hic-5

and leupaxin (41,79,86–88). Paxillin is an adaptor protein

often used as a marker of FAs that functions in the

transduction of signals mediated through growth factor

Traffic 2007; 8: 1465–1475 1471


receptors and integrins. Paxillin regulates FA dynamics

and, as a consequence, cell migration, spreading and

adhesion. The LD4 motif in paxillin binds to the SHD of

GIT. Interfering with GIT association with paxillin results in

altered FAs and accelerated cell migration.

Crk and CrkL are adaptor proteins that may contribute to

ASAP1 function in FAs and membrane ruffles (35,36). CrkL

is SH2- and SH3-domain-containing protein that binds to

paxillin and Cas at FAs. Two PXXP motifs in ASAP1

mediate binding to Crk and CrkL. The interaction was

found to be necessary for ASAP1 association with FAs (35).

ASAP1 (AMAP1/PAG2) has also been reported to associ-

ate with cortactin, a multidomain protein with N-terminal

acidic, actin-binding, proline-rich and SH3 domains. Cor-

tactin is found in peripheral membrane ruffles and invado-

podia. The SH3 domain of cortactin was found to bind to

a proline-rich motif specific to one splice variant of ASAP1

(34). Subsequent work showed that the SH3 domain of

ASAP1 had a more dominant role in forming the complex

than the splice-variant specific proline-rich motif. Both

ASAP1 and cortactin associate with invadopodia in inva-

sive breast cancer cell lines. Furthermore, disruption of the

ability of ASAP1 to form a complex with cortactin inter-

feres with the formation of invadopodia and structurally

related podosomes (34). ASAP1 expression correlates

with the invasive potential of uveal melanoma cells and

mammary carcinoma cells. Based on these findings, the

ASAP1/cortactin complex has been proposed to function

as a part of an invasive machinery in cancer cells. In

a recently described model, the ASAP1/cortactin complex

links the highly tubulated membranes found in invadopodia

and podosomes to polymerized and branched actin (89).

Miscellaneous proteins

Arf GAPs have been found to bind to a number of proteins

that are not clearly associated with changes in actin or

membranes. ACAP1 (centaurin b1) and ACAP2 (centaurin

b2) have been reported to interact with bacteria-derived

intracellular peptidoglycan sensor proteins, NOD1 and

NOD2, and vaccinia virus protein KILT, respectively (90,91).

AGAP1 associates with nitric oxide (NO)-sensitive soluble

guanylyl cyclase through N-terminal GLD region (92).

Arf GAPs as Regulated Signaling Platforms

Although the roles of Arf GAPs as adaptors or scaffolds

for signaling proteins and as elements of signaling path-

ways have been described for GITs, ARAPs, AGAPs

(PIKE) and ASAPs, the mechanisms by which the distinct

activities of each domain of Arf GAPs may be integrated

have not been elucidated. Recent findings in studies of

ASAP1 (34,47) provide a basis to speculate about mech-

anisms by which integration to control a particular cellular

activity is achieved. We propose a model in which ASAP1

functions as a regulated signaling platform to control the

dynamics of invadopodia and podosomes. These struc-

tures are labyrinths of tubulated membranes associated

with polymerized actin (8). The BAR, PH and Arf GAP

domain of ASAP1 bind to PIP2 and Arf�GTP to induce

membrane tubulation (47). In this way, ASAP1 could be

considered an Arf effector. ASAP1 must be phosphory-

lated on tyrosine, dependent on the nonreceptor tyrosine

kinase Src, to function at podosomes. Thus, ASAP1

integrates three signals – Arf�GTP, PIP2 and Src. ASAP1

associates with the tubulated membranes and may

undergo conformational changes leading to additional

protein–protein interactions (47). ASAP1 binds cortactin,

which induces actin polymerization though interaction

with Arp2/3 and binds to filamentous actin (34). ASAP1

would thus link polymerized actin to the tubulated mem-

branes. ASAP1 binds FAK (83) and Src (36), tyrosine

kinases important to the formation and maintenance of

invadopodia and podosomes. The adaptor protein Crk

bound to ASAP1 has the potential of bringing other signaling

proteins into the complex (35). The association with POB1/

RalBP could control Rho�GTP levels (67), also important for

maintenance of invadopodia. The ASAP1-dependent com-

plex could be rapidly controlled: GAP activity of ASAP1 is

robust and, maintenance of the complex on the tubulated

membranes would depend on continued generation of

Arf�GTP. At other sites, with different signals, ASAP1 could

bind a different group of proteins, resulting in a different

output. For instance, ASAP1 in FAs does not bind cortactin.

Thus, rather than a simple scaffold or adaptor, ASAP1would

function as a multiplexer, providing a unique output for

a particular set of inputs. Thismodel may generalize to other

multidomain Arf GAPs, such as ARAPs or AGAPs. The idea

that Arf GAPs could function as Arf effectors could extend

to simpler Arf GAPs, as suggested for yeast Arf GAPs (93).

Conclusions and Perspective

Arf GAPs are structurally complex proteins. Each has GAP

activity, inducing hydrolysis of GTP bound to Arf. Some of

the functions of Arf GAPs in the regulation of actin and

membranes are attributable to the GAP activity. However,

Arf GAPs affect actin and membranes through additional

mechanisms. In this review, we cataloged GAP-associated

proteins and described potential mechanisms by which

they may contribute to the effects of Arf GAPs on actin or

membranes. The Arf GAPs also directly interact with lipids,

described in Nie and Randazzo (26), to affect the structure

of biological membranes. One challenge at this time is to

understand how protein and lipid association with Arf

GAPs and GAP activity are integrated to coordinate

changes in the actin cytoskeleton and membranes neces-

sary for biological behaviors such as cell migration and

pathological behaviors such as cancer cell invasion. We

speculate in this review about mechanisms of integration

for ASAP1, suggesting that ASAP1 functions as a regulated

1472 Traffic 2007; 8: 1465–1475

Inoue and Randazzo

signaling platform similar to a multiplexer. Ongoing struc-

tural studies focused on the functional relationships

between domains within single Arf GAPs will be important

for understanding the molecular basis of integration of the

distinct activities associated with specific Arf GAPs.

Acknowledgments

This work was supported by the Intramural Research Program of the

National Cancer Institute, Department of Health and Human Services. We

apologize to authors whose work may have been omitted due to restric-

tions in the length of the review, or Paul Randazzo’s or Hiroki Inoue’s

oversight.

References

1. Moss J, Vaughan M. Molecules in the ARF orbit. J Biol Chem 1998;

273: 21431–21434.

2. Randazzo PA, Nie Z, Miura K, Hsu VW.Molecular aspects of the cellular

activities of ADP-ribosylation factors. Sci STKE 2000;1:RE1–RE15.

3. Donaldson JG, Honda A, Weigert R. Multiple activities for Arf1 at the

Golgi complex. Biochim Biophys Acta-Mol Cell Res 2005;1744:

364–373.

4. D’Souza-Schorey C, Chavrier P. ARF proteins: roles in membrane traffic

and beyond. Nat Rev Mol Cell Biol 2006;7:347–358.

5. Donaldson JG. Multiple roles for Arf6: sorting, structuring, and signal-

ing at the plasma membrane. J Biol Chem 2003;278:41573–41576.

6. Randazzo PA, Weiss O, Kahn RA. Preparation of recombinant ADP-

ribosylation factor. Methods Enzymol 1992;219:362–369.

7. CukiermanE,Huber I, RotmanM,Cassel D. TheARF1GTPase-activating

protein: zinc finger motif and Golgi complex localization. Science 1995;

270:1999–2002.

8. Buccione R, Orth JD, McNiven MA. Foot and mouth: podosomes,

invadopodia and circular dorsal ruffles. Nat Rev Mol Cell Biol 2004;5:

647–657.

9. Orth JD, Krueger EW, Weller SG, McNiven MA. A novel endocytic

mechanism of epidermal growth factor receptor sequestration and

internalization. Cancer Res 2006;66:3603–3610.

10. Jaffe AB, Hall A. Rho GTPases: biochemistry and biology. Annu Rev

Cell Dev Biol 2005;21:247–269.

11. Lanzetti L, Palamidessi A, Areces L, Scita G, Di Fiore PP. Rab5 is

a signalling GTPase involved in actin remodelling by receptor tyrosine

kinases. Nature 2004;429:309–314.

12. Randazzo PA, Andrade J, Miura K, Brown MT, Long YQ, Stauffer S,

Roller P, Cooper JA. The Arf GTPase-activating protein ASAP1 regu-

lates the actin cytoskeleton. Proc Natl Acad Sci U S A 2000; 97:

4011–4016.

13. Hashimoto S, Onodera Y, Hashimoto A, Tanaka H, Hamaguchi M,

Yamada A, Sabe H. Requirement for Arf6 in breast cancer invasive

activities. Proc Natl Acad Sci U S A 2004;101:6647–6652.

14. Norman JC, Jones D, Barry ST, Holt MR, Cockcroft S, Critchley DR.

ARF1 mediates paxillin recruitment to focal adhesions and potentiates

Rho-stimulated stress fiber formation in intact and permeabilized Swiss

3T3 fibroblasts. J Cell Biol 1998;143:1981–1995.

15. Randazzo PA, Hirsch DS. Arf GAPs: multifunctional proteins that

regulate membrane traffic and actin remodelling. Cell Signal 2004;16:

401–413.

16. Mesmin B, Drin G, Levi S, Rawet M, Cassel D, Bigay J, Antonny B. Two

lipid-packing sensor motifs contribute to the sensitivity of ArfGAP1 to

membrane curvature. Biochemistry 2007;46:1779–1790.

17. Antonny B, Huber I, Paris S, Chabre M, Cassel D. Activation of

ADP-ribosylation factor 1 GTPase-activating protein by phosphatidyl-

choline-derived diacylglycerols. J Biol Chem 1997;272:30848–30851.

18. Lanoix J, Ouwendijk J, Lin CC, Stark A, Love HD, Ostermann J,

Nilsson T. GTP hydrolysis by Arf-1 mediates sorting and concentration

of Golgi resident enzymes into functional COP I vesicles. EMBO J

1999;18:4935–4948.

19. Tanigawa G, Orci L, Amherdt M, Ravazzola M, Helms JB, Rothman JE.

Hydrolysis of bound GTP by ARF protein triggers uncoating of Golgi-

derived COP-coated vesicles. J Cell Biol 1993;123:1365–1371.

20. Bigay J, Gounon P, Robineau S, Antonny B. Lipid packing sensed by

ArfGAP1 couples COPI coat disassembly to membrane bilayer curva-

ture. Nature 2003;426:563–566.

21. Bigay J, Casella JF, Drin G, Mesmin B, Antonny B. ArfGAP1 responds

to membrane curvature through the folding of a lipid packing sensor

motif. EMBO J 2005;24:2244–2253.

22. Yang JS, Lee SY, Gao M, Bourgoin S, Randazzo PA, Premont RT,

Hsu VW. ARFGAP1 promotes the formation of COPI vesicles, suggest-

ing function as a component of the coat. J Cell Biol 2002;159:69–78.

23. Yang JS, Lee SY, Spano S, Gad H, Zhang L, Nie Z, Bonazzi M, Corda D,

Luini A, Hsu VW. A role for BARS at the fission step of COPI vesicle

formation from Golgi membrane. EMBO J 2005;24:4133–4143.

24. Yang JS, Zhang L, Lee SY, Gad H, Luini A, Hsu VW. Key components of

the fission machinery are interchangeable. Nat Cell Biol 2006;8:

1376–1382.

25. Lee SY, Yang JS, Hong W, Premont RT, Hsu VW. ARFGAP1 plays

a central role in coupling COPI cargo sorting with vesicle formation.

J Cell Biol 2005;168:281–290.

26. Nie Z, Randazzo PA. Arf GAPs and membrane traffic. J Cell Sci 2006;

119:1203–1211.

27. Tanabe K, Torii T, Natsume W, Braesch-Andersen S, Watanabe T,

Satake M. A novel GTPase-activating protein for ARF6 directly interacts

with clathrin and regulates clathrin-dependent endocytosis. Mol Biol

Cell 2005;16:1617–1628.

28. NatsumeW, Tanabe K, Kon S, Yoshida N,Watanabe T, Torii T, SatakeM.

SMAP2, a novel ARF GTPase-activating protein, interacts with clathrin

and clathrin assembly protein and functions on the AP-1-positive early

endosome/trans-Golgi network. Mol Biol Cell 2006;17:2592–2603.

29. Luo R, Ahvazi B, Amariei D, Shroder D, Burrola B, Losert W, Randazzo

PA. Kinetic analysis of GTP hydrolysis catalysed by the Arf1-GTP-

ASAP1 complex. Biochem J 2007;402:439–447.

30. Vitale N, Patton WA, Moss J, Vaughan M, Lefkowitz RJ, Premont RT

GIT proteins, A novel family of phosphatidylinositol 3, 4, 5-trisphosphate-

stimulated GTPase-activating proteins for ARF6. J Biol Chem 2000;275:

13901–13906.

31. Claing A, Chen W, Miller WE, Vitale N, Moss J, Premont RT, Lefkowitz

RJ. b-Arrestin-mediated ADP-ribosylation factor 6 activation and

b 2-adrenergic receptor endocytosis. J Biol Chem 2001;276:

42509–42513.

32. Hoefen RJ, Berk BC. The multifunctional GIT family of proteins. J Cell

Sci 2006;119:1469–1475.

33. Mazaki Y, Hashimoto S, Okawa K, Tsubouchi A, Nakamura K, Yagi R,

Yano H, Kondo A, Iwamatsu A, Mizoguchi A, Sabe H. An ADP-

ribosylation factor GTPase-activating protein Git2-short/KIAA0148 is

involved in subcellular localization of paxillin and actin cytoskeletal

organization. Mol Biol Cell 2001;12:645–662.

34. Onodera Y, Hashimoto S, Hashimoto A, Morishige M, Mazaki Y,

Yamada A, Ogawa E, Adachi M, Sakurai T, manabe T, Wasa H,

Masura N, Sabe H. Expression of AMAP1, an Arf GAP, provides novel

targets to inhibit breast cancer invasive activiteies. EMBO J 2005;

24:963–973.

35. Oda A, Wada I, Miura K, Okawa K, Kadoya T, Kato T, Nishihara H,

Maeda M, Tanaka S, Nagashima K, Nishitani C, Matsuno K, Ishino M,

Traffic 2007; 8: 1465–1475 1473


Machesky LM, Fujita H et al. CrkL directs ASAP1 to peripheral focal

adhesions. J Biol Chem 2003;278:6456–6460.

36. Brown MT, Andrade J, Radhakrishna H, Donaldson JG, Cooper JA,

Randazzo PA. ASAP1, a phospholipid-dependent arf GTPase-activating

protein that associates with and is phosphorylated by Src. Mol Cell Biol

1998;18:7038–7051.

37. Andreev J, Simon JP, Sabatini DD, Kam J, Plowman G, Randazzo PA,

Schlessinger J. Identification of a new Pyk2 target protein with Arf-GAP

activity. Mol Cell Biol 1999;19:2338–2350.

38. Furman C, Short SM, Subramanian RR, Zetter BR, Roberts TM. DEF-1/

ASAP1 is a GTPase-activating protein (GAP) for ARF1 that enhances

cell motility through a GAP-dependent mechanism. J Biol Chem 2002;

277:7962–7969.

39. Liu Y, Yerushalmi GM, Grigera PR, Parsons JT. Mislocalization or

reduced expression of Arf GTPase-activating protein ASAP1 inhibits

cell spreading and migration by influencing Arf1 GTPase cycling. J Biol

Chem 2005;280:8884–8892.

40. Jackson TR, Brown FD, Nie Z, Miura K, Foroni L, Sun J, Hsu VW,

Donaldson JG, Randazzo PA. ACAPs are arf6 GTPase-activating

proteins that function in the cell periphery. J Cell Biol 2000;151:

627–638.

41. Kondo A, Hashimoto S, Yano H, Nagayama K, Mazaki Y, Sabe H. A new

paxillin-binding protein, PAG3/Papa/KIAA0400, bearing an ADP-

ribosylation factor GTPase-activating protein activity, is involved in

paxillin recruitment to focal adhesions and cell migration. Mol Biol Cell

2000;11:1315–1327.

42. Hashimoto S, Hashimoto A, Yamada A, Kojima C, Yamamoto H,

Tsutsumi T, Higashi M, Mizoguchi A, Yagi R, Sabe H. A novel mode

of action of an ArfGAP, AMAP2/PAG3/Pap a, in Arf6 function. J Biol

Chem 2004;279:37677–37684.

43. Kam JL, Miura K, Jackson TR, Gruschus J, Roller P, Stauffer S, Clark J,

Aneja R, Randazzo PA. Phosphoinositide-dependent activation of the

ADP-ribosylation factor GTPase-activating protein ASAP1. Evidence for

the pleckstrin homology domain functioning as an allosteric site. J Biol

Chem 2000;275:9653–9663.

44. CheMM, Boja ES, Yoon HY, Gruschus J, Jaffe H, Stauffer S, Schuck P,

Fales HM, Randazzo PA. Regulation of ASAP1 by phospholipids is

dependent on the interface between the PH and Arf GAP domains. Cell

Signal 2005;17:1276–1288.

45. Mandiyan V, Andreev J, Schlessinger J, Hubbard SR. Crystal structure

of the ARF-GAP domain and ankyrin repeats of PYK2-associated

protein b. EMBO J 1999;18:6890–6898.

46. Peter BJ, Kent HM, Mills IG, Vallis Y, Butler PJ, Evans PR, McMahon

HT. BAR domains as sensors of membrane curvature: the amphiphysin

BAR structure. Science 2004;303:495–499.

47. Nie Z, Hirsch DS, Luo R, Jian X, Stauffer S, Cremesti A, Andrade J,

Lebowitz J, Marino M, Ahvazi B, Hinshaw JE, Randazzo PA. A BAR

domain in the N terminus of the Arf GAP ASAP1 affects membrane

structure and trafficking of epidermal growth factor receptor. Curr Biol

2006;16:130–139.

48. Miura K, Jacques KM, Stauffer S, Kubosaki A, Zhu K, Hirsch DS, Resau

J, Zheng Y, Randazzo PA. ARAP1: a point of convergence for Arf and

Rho signaling. Mol Cell 2002;9:109–119.

49. Krugmann S, Anderson KE, Ridley SH, Risso N, McGregor A,

Coadwell J, Davidson K, Eguinoa A, Ellson CD, Lipp P, Manifava M,

Ktistakis N, Painter G, Thuring JW, Cooper MA et al. Identification of

ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases,

by selective capture on phosphoinositide affinity matrices. Mol Cell

2002;9:95–108.

50. Yoon HY, Miura K, Cuthbert EJ, Davis KK, Ahvazi B, Casanova JE,

Randazzo PA. ARAP2 effects on the actin cytoskeleton are dependent

on Arf6-specific GTPase-activating-protein activity and binding to

RhoA-GTP. J Cell Sci 2006;119:4650–4666.

51. I ST, Nie Z, Stewart A, Najdovska M, Hall NE, He H, Randazzo PA,

Lock P. ARAP3 is transiently tyrosine phosphorylated in cells attaching

to fibronectin and inhibits cell spreading in a RhoGAP-dependent

manner. J Cell Sci 2004;117:6071–6084.

52. Krugmann S, Andrews S, Stephens L, Hawkins PT. ARAP3 is essential

for formation of lamellipodia after growth factor stimulation. J Cell Sci

2006;119:425–432.

53. Nie Z, Stanley KT, Stauffer S, Jacques KM, Hirsch DS, Takei J,

Randazzo PA. AGAP1, an endosome-associated, phosphoinositide-

dependent ADP-ribosylation factor GTPase-activating protein that af-

fects actin cytoskeleton. J Biol Chem 2002;277:48965–48975.

54. Nie Z, Boehm M, Boja ES, Vass WC, Bonifacino JS, Fales HM,

Randazzo PA. Specific regulation of the adaptor protein complex

AP-3 by the Arf GAP AGAP1. Dev Cell 2003;5:513–521.

55. Nie Z, Fei J, Premont RT, Randazzo PA. The Arf GAPs AGAP1 and

AGAP2 distinguish between the adaptor protein complexes AP-1 and

AP-3. J Cell Sci 2005;118:3555–3566.

56. Aoe T, Cukierman E, Lee A, Cassel D, Peters PJ, Hsu VW The KDEL

receptor, ERD2, regulates intracellular traffic by recruiting a GTPase-

activating protein for ARF1. EMBO J 1997;16:7305–7316.

57. Goldberg J. Decoding of sorting signals by coatomer through a GTPase

switch in the COPI coat complex. Cell 2000;100:671–679.

58. Lanoix J, Ouwendijk J, Stark A, Szafer E, Cassel D, Dejgaard K, Weiss

M, Nilsson T. Sorting of Golgi resident proteins into different sub-

populations of COPI vesicles: a role for ArfGAP1. J Cell Biol 2001;155:

1199–1212.

59. Dai J, Li J, Bos E, Porcionatto M, Premont RT, Bourgoin S, Peters PJ,

Hsu VW. ACAP1 promotes endocytic recycling by recognizing recycling

sorting signals. Dev Cell 2004;7:771–776.

60. Li J, Ballif BA, Powelka AM, Dai J, Gygi SP, Hsu VW. Phosphorylation

of ACAP1 by Akt regulates the stimulation-dependent recycling of

integrin b1 to control cell migration. Dev Cell 2005;9:663–673.

61. Watson PJ, Frigerio G, Collins BM, Duden R, Owen DJ. g-COP

appendage domain – structure and function. Traffic 2004;5:79–88.

62. Hirst J, Motley A, Harasaki K, Peak Chew SY, Robinson MS. EpsinR: an

ENTH domain-containing protein that interacts with AP-1. Mol Biol Cell

2003;14:625–641.

63. Goldberg J. Structural and functional analysis of the ARF1-ARFGAP

complex reveals a role for coatomer in GTP hydrolysis. Cell 1999;96:

893–902.

64. Szafer E, Rotman M, Cassel D. Regulation of GTP hydrolysis on

ADP-ribosylation factor-1 at the Golgi membrane. J Biol Chem 2001;

276:47834–47839.

65. Bickford LC, Mossessova E, Goldberg J. A structural view of the COPII

vesicle coat. Curr Opin Struct Biol 2004;14:147–153.

66. Kowanetz K, Husnjak K, Holler D, Kowanetz M, Soubeyran P, Hirsch D,

Schmidt MH, Pavelic K, De Camilli P, Randazzo PA, Dikic I. CIN85

associates with multiple effectors controlling intracellular trafficking

of epidermal growth factor receptors. Mol Biol Cell 2004;15:

3155–3166.

67. Oshiro T, Koyama S, Sugiyama S, Kondo A, Onodera Y, Asahara T,

Sabe H, Kikuchi A. Interaction of POB1, a downstream molecule of

small G protein Ral, with PAG2, a paxillin-binding protein, is involved in

cell migration. J Biol Chem 2002;277:38618–38626.

68. Ikeda M, Ishida O, Hinoi T, Kishida S, Kikuchi A. Identification and

characterization of a novel protein interacting with Ral-binding protein

1, a putative effector protein of Ral. J Biol Chem 1998;273:814–821.

69. Haendeler J, Yin G, Hojo Y, Saito Y, Melaragno M, Yan C, Sharma VK,

Heller M, Aebersold R, Berk BC. GIT1 mediates Src-dependent

activation of phospholipase Cg by angiotensin II and epidermal growth

factor. J Biol Chem 2003;278:49936–49944.

70. Ye K, Aghdasi B, Luo HR, Moriarity JL, Wu FY, Hong JJ, Hurt KJ,

Bae SS, Suh PG, Snyder SH. Phospholipase C g 1 is a physiological

1474 Traffic 2007; 8: 1465–1475

Inoue and Randazzo

guanine nucleotide exchange factor for the nuclear GTPase PIKE.

Nature 2002;415:541–544.

71. SoundararajanM, Yang X, Elkins JM, Sobott F, Doyle DA. The centaurin

g-1 GTPase-like domain functions as an NTPase. Biochem J

2007;401:679–688.

72. Ye K, Hurt KJ, Wu FY, Fang M, Luo HR, Hong JJ, Blackshaw S, Ferris

CD, Snyder SH. Pike. A nuclear GTPase that enhances PI3kinase

activity and is regulated by protein 4.1N. Cell 2000;103:919–930.

73. Rong R, Ahn JY, Huang H, Nagata E, Kalman D, Kapp JA, Tu J, Worley

PF, Snyder SH, Ye K. PI3 kinase enhancer-Homer complex couples

mGluRI to PI3 kinase, preventing neuronal apoptosis. Nat Neurosci

2003;6:1153–1161.

74. Raaijmakers JH, Deneubourg L, Rehmann H, de Koning J, Zhang Z,

Krugmann S, Erneux C, Bos JL. The PI3K effector Arap3 interacts with

the PI(3, 4,5)P(3) phosphatase SHIP2 in a SAM domain-dependent

manner. Cell Signal 2007;19:1249–1257.

75. Zhao ZS, Manser E, Loo TH, Lim L. Coupling of PAK-interacting

exchange factor PIX to GIT1 promotes focal complex disassembly.

Mol Cell Biol 2000;20:6354–6363.

76. Manabe R, Kovalenko M, Webb DJ, Horwitz AR. GIT1 functions in

a motile, multi-molecular signaling complex that regulates protrusive

activity and cell migration. J Cell Sci 2002;115:1497–1510.

77. Yin G, Haendeler J, Yan C, Berk BC. GIT1 functions as a scaffold for

MEK1-extracellular signal-regulated kinase 1 and 2 activation by

angiotensin II and epidermal growth factor. Mol Cell Biol 2004;24:

875–885.

78. Premont RT, Claing A, Vitale N, Freeman JL, Pitcher JA, Patton WA,

Moss J, Vaughan M, Lefkowitz RJ. b2-Adrenergic receptor regulation

by GIT1, a G protein-coupled receptor kinase-associated ADP ribo-

sylation factor GTPase-activating protein. Proc Natl Acad Sci U S A

1998;95:14082–14087.

79. Premont RT, Claing A, Vitale N, Perry SJ, Lefkowitz RJ. The GIT Family

of ADP-ribosylation Factor GTPase-activating Proteins. Functional

diversity of git2 through alternative splicing. J Biol Chem 2000;275:

22373–22380.

80. Tang X, Feng Y, Ye K. Src-family tyrosine kinase fyn phosphorylates

phosphatidylinositol 3-kinase enhancer-activating Akt, preventing its

apoptotic cleavage and promoting cell survival. Cell Death Differ

2007;14:368–377.

81. Ahn JY, Rong R, Kroll TG, Van Meir EG, Snyder SH, Ye K. PIKE

(phosphatidylinositol 3-kinase enhancer)-A GTPase stimulates Akt activity

and mediates cellular invasion. J Biol Chem 2004;279:16441–16451.

82. Hu Y, Liu Z, Ye K. Phosphoinositol lipids bind to phosphatidylinositol 3

(PI3)-kinase enhancer GTPase and mediate its stimulatory effect on

PI3-kinase and Akt signalings. Proc Natl Acad Sci U S A 2005;102:

16853–16858.

83. Liu Y, Loijens JC, Martin KH, Karginov AV, Parsons JT. The association

of ASAP1, an ADP ribosylation factor-GTPase activating protein, with

focal adhesion kinase contributes to the process of focal adhesion

assembly. Mol Biol Cell 2002;13:2147–2156.

84. Kruljac-Letunic A, Moelleken J, Kallin A, Wieland F, Blaukat A. The

tyrosine kinase Pyk2 regulates Arf1 activity by phosphorylation and

inhibition of the Arf-GTPase-activating protein ASAP1. J Biol Chem

2003;278:29560–29570.

85. Krugmann S, Williams R, Stephens L, Hawkins PT. ARAP3 is a PI3K-

and rap-regulated GAP for RhoA. Curr Biol 2004;14:1380–1384.

86. Nishiya N, Shirai T, Suzuki W, Nose K. Hic-5 interacts with GIT1 with

a different binding mode from paxillin. J Biochem (Tokyo) 2002;132:

279–289.

87. Turner CE, Brown MC, Perrotta JA, Riedy MC, Nikolopoulos SN,

McDonald AR, Bagrodia S, Thomas S, Leventhal PS. Paxillin LD4 motif

binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP

protein: a role in cytoskeletal remodeling. J Cell Biol 1999;145:

851–863.

88. Gupta A, Lee BS, Khadeer MA, Tang Z, Chellaiah M, Abu-Amer Y,

Goldknopf J, Hruska KA. Leupaxin is a critical adaptor protein in

the adhesion zone of the osteoclast. J Bone Miner Res 2003;18:

669–685.

89. Randazzo PA, Inoue H, Bharti S. Arf GAPs as regulators of the actin

cytoskeleton. Biol Cell 2007; (In press).

90. Yamamoto-Furusho JK, Barnich N, Xavier R, Hisamatsu T, Podolsky DK.

Centaurin b1 down-regulates nucleotide-binding oligomerization do-

mains 1- and 2-dependent NF-kappaB activation. J Biol Chem 2006;281:

36060–36070.

91. Bradley RR, Terajima M. Vaccinia virus K1L protein mediates host-

range function in RK-13 cells via ankyrin repeat and may interact with

a cellular GTPase-activating protein. Virus Res 2005;114:104–112.

92. Meurer S, Pioch S, Wagner K, Muller-Esterl W, Gross S. AGAP1,

a novel binding partner of nitric oxide-sensitive guanylyl cyclase. J Biol

Chem 2004;279:49346–49354.

93. Zhang CJ, Cavenaugh MM, Kahn RA. A family of Arf effectors defined

as suppressors of the loss of Arf function in the yeast Saccaromyces

cerevisiae. J Biol Chem 1998; 273: 19792–19796.

94. Majoul I, Straub M, Hell SW, Duden R, Soling HD. KDEL-cargo regulates

interactions between proteins involved in COPI vesicle traffic: measure-

ments in living cells using FRET. Dev Cell 2001;1:139–153.

95. Bagrodia S, Bailey D, Lenard Z, Hart M, Guan JL, Premont RT,

Taylor SJ, Cerione RA. A tyrosine-phosphorylated protein that binds

to an important regulatory region on the cool family of p21-activated

kinase-binding proteins. J Biol Chem 1999;274:22393–22400.

96. Angrand PO, Segura I, Volkel P, Ghidelli S, Terry R, Brajenovic M,

Vintersten K, Klein R, Superti-Furga G, Drewes G, Kuster B,

Bouwmeester T, Acker-Palmer A. Transgenic mouse proteomics

identifies new 14-3-3-associated proteins involved in cytoskeletal

rearrangements and cell signaling. Mol Cell Proteomics 2006;5:

2211–2227.

Traffic 2007; 8: 1465–1475 1475


arf gaps and their interacting proteins

Documents