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Phosphoinositide specificity determines whichcytohesins regulate b1 integrin recycling

Seung Ja Oh* and Lorraine C. Santy`

Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA

*Present address: Department of Dermatology, University of Pennsylvania Medical School, 415 Curie Boulevard, Philadelphia, PA 19104, USA`Author for correspondence (lcsanty@psu.edu)

Accepted 27 February 2012Journal of Cell Science 125, 3195–3201� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.101683

SummaryRecycling of internalized integrins is a crucial step in adhesion remodeling and cell movement. Recently, we determined that the ADP-ribosylation factor–guanine nucleotide exchange factors (ARF-GEFs) cytohesin 2/ARNO and cytohesin 3/GRP1 have opposing effects

on adhesion and stimulated b1 integrin recycling even though they are very closely related proteins (80% sequence identity). We havenow determined the sequence differences underlying the differential actions of cytohesin 2/ARNO and cytohesin 3/GRP1. We found thatthe ability of cytohesins to promote b1 integrin recycling and adhesion depends upon the presence or absence of a key glycine residue intheir pleckstrin homology (PH) domains. This glycine residue determines the phosphoinositide specificity and affinity of cytohesin PH

domains. Switching the number of glycines in the PH domains of cytohesin 2 and cytohesin 3 is sufficient to reverse their effects onadhesion and spreading and to reverse their subcellular locations. Importantly, we also find that a mutant form of cytohesin 3/GRP1 thathas three rather than two glycines in its PH domain rescues b1 integrin recycling in cytohesin 2/ARNO knockdown cells. Conversely, amutant form of cytohesin 2/ARNO with two glycines in its PH domain fails to rescue b1 integrin recycling. Therefore, we conclude thatphosphoinositide specificity is the sole functional difference that determines which cytohesin can promote integrin recycling.

Key words: Cytohesin, ARF, Integrin, Endocytosis, Recycling

IntroductionIntegrin-based cell to ECM adhesion formation, turnover and

remodeling are required to convert cytoskeletal contractile forces

into productive movement (Caswell and Norman, 2006). During

migration of epithelial cells, the balance between E-cadherin

based cell–cell adhesion and integrin based cell–ECM adhesion

influences both the mode and extent of epithelial movement

(Hay, 2005; Perl et al., 1998). Increased integrin based cell–ECM

adhesion strength and contractility promotes epithelial migration

and scattering (de Rooij et al., 2005; Sander et al., 1998).

Conversely, enhanced E-cadherin based adhesion reduces

lamellipodia formation, scattering and invasive movements

(Borghi et al., 2010; Hordijk et al., 1997). These data suggest

that upregulation of integrin exocytosis and cell surface integrin

levels can promote epithelial migration.

Integrins can be internalized from the plasma membrane via a

number of pathways (Altankov and Grinnell, 1995; Ylänne et al.,

1995). Some internalized integrin is immediately recycled via the

‘short-loop’ pathway. The remainder of the internalized integrin

recycles through the ‘long-loop’ pathway via the recycling

endosome (Caswell and Norman, 2008). This pathway is subject

to regulation by serum, growth factors and PKC activity (Gao

et al., 2000; Ng et al., 1999; Powelka et al., 2004). These factors

can promote both integrin recycling and migration.

The formation of trafficking carriers is initiated by the actions

of small GTPases of the ADP-ribosylation factor (ARF) family.

ARF6 is the GTPase that regulates the stimulated recycling of b1integrin (Powelka et al., 2004). Activation of ARFs requires the

action of guanine nucleotide exchange factors (GEFs). We have

recently shown that the ARF-GEF cytohesin 2/ARNO and its

closely related homologue, cytohesin 3/GRP1, have distinct

functions in cell adhesion, spreading and migration. Furthermore,

only cytohesin 2/ARNO regulates b1 integrin recycling. Finally,these proteins have distinct subcellular locations (Oh and Santy,

2010).

We have now identified the key differences underlying

differential actions of cytohesin 2/ARNO and cytohesin 3/

GRP1 in b1 integrin recycling. We find that the identity of thepleckstrin homology (PH) domain determines the effect of

cytohesins on cell adhesion and spreading. The PH domain is

highly conserved in cytohesin 2 and cytohesin 3. However, the

most abundant splice variants of these proteins differ

significantly in the phosphoinositide binding specificity of their

PH domains (Klarlund et al., 2000; Ogasawara et al., 2000).

Alternative splicing of a single three-nucleotide exon produces

variants of cytohesin 1, 2 and 3 PH domains that contain either

two or three glycine residues (Klarlund et al., 2000; Ogasawara

et al., 2000). The presence or absence of this single glycine has a

profound effect on their phosphoinositide-binding specificity

(Klarlund et al., 2000; Ogasawara et al., 2000). The diglycine

form has a strong selectivity for phosphatidylinositol 3,4,5-

trisphosphate [PtdIns(3,4,5)P3 (PIP3)], while the triglycine form

has an equal affinity for PtdIns(3,4,5)P3 and PtdIns(4,5)P2 (PIP2)

(Klarlund et al., 2000). Cytohesin 2/ARNO primarily exists in the

triglycine form and cytohesin 3/GRP1 is predominately in the

diglycine form (Ogasawara et al., 2000). We have found that this

difference in glycine number is the sole sequence difference that

underlies their different effects on adhesion and b1 integrin

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recycling. These results implicate the phosphoinositide

composition of the b1 integrin recycling compartment as a keyplayer in stimulated integrin recycling.

ResultsThe PH domains of cytohesin 2/ARNO and cytohesin

3/GRP1 are responsible for the differential effects of

these proteins on cell adhesion to fibronectin

We constructed hybrid constructs where a single domain of

cytohesin 2 was replaced with the equivalent domain of cytohesin

3. The coiled-coil (cc) domain, the polybasic (pb) domain or the

PH domain of cytohesin 2 was replaced with the equivalent

cytohesin 3 domain (Fig. 1A). Cytohesin 2/ARNO expression

enhances cell adhesion to fibronectin, while cytohesin 3/GRP1expression reduces cell adhesion to fibronectin (Fig. 1B) (Oh and

Santy, 2010). If the relevant domain of cytohesin 3 has beenswapped into cytohesin 2, then the cells expressing that constructshould show impaired rather than enhanced binding to fibronectin.Cytohesin 2 with the cytohesin 3 cc domain (2 w/3cc) or cytohesin

2 with the cytohesin 3 pb domain (2 w/3pb) expression stillsignificantly enhanced cell adhesion suggesting that these domainsdo not underlie the differences between cytohesin 2 and 3

(Fig. 1A,B). On the other hand, cells expressing cytohesin 2 withthe cytohesin 3 PH domain (2 w/3PH) had significantly reducedcell adhesion when compared to the either control cells or to cells

expressing wild-type cytohesin 2/ARNO (Fig. 1A,B). Importantly,cells expressing 2 w/3PH adhered to fibronectin similarly to cellsexpressing wild-type cytohesin 3/GRP1 (Fig. 1B). Therefore, weconclude that the key differences that determine if a cytohesin

regulates integrin recycling lie in the PH domain.

The number of glycine residues in the PH domain ofcytohesin 2/ARNO (3G) and cytohesin 3/GRP1 (2G)determines their effects on cell adhesion and spreading

Since we found that the identity of the PH domain determines the

effect of cytohesin expression on cell adhesion to fibronectin, wetested the hypothesis that the phosphoinositide affinity of the PHdomain is the critical difference. We made cytohesin constructswith reversed phosphoinositide binding affinity by altering the

number of glycine residues in the PH domains. Specifically, weconstructed cytohesin 2 with a diglycine PH (GG cytohesin 2)and cytohesin 3 with a triglycine PH (GGG cytohesin 3), which

only differ in glycine numbers when compared to wild-typecytohesin 2/ARNO (3G) and wild-type cytohesin 3/GRP1 (2G).We then tested whether these mutant constructs alter cell

adhesion to fibronectin. As shown in Fig. 2A, HeLa cellsexpressing GGG cytohesin 3 had significantly enhanced celladhesion to fibronectin to a level similar to the cells expressing

wild-type cytohesin 2/ARNO (3G). Conversely, GG cytohesin 2expression reduced cell adhesion. This reduced adhesion wassignificantly different from the adhesion of control cells or cellsexpressing wild-type cytohesin 2/ARNO (3G), but was similar to

the adhesion of cells expressing wild-type cytohesin 3/GRP1(2G; Fig. 2A). We have previously shown that expression ofcytohesin 2 increases cell surface b1 integrin levels whileexpression of cytohesin 3 reduces surface b1 integrin (Oh andSanty, 2010). We measured the levels of surface integrin in HeLacells expressing wild-type cytohesin 2 or 3 or the cytohesins with

altered glycine numbers. Cells expressing cytohesins with threeglycines had enhanced levels of cell surface b1 integrin whilethose expressing cytohesins with two glycines had reduced

surface levels of b1 integrin (supplementary material Table S1).Therefore, we concluded that the number of glycine residues ofthe PH domain is the critical determinant of the effect ofcytohesin expression of cell adhesion.

As cells bind more tightly to fibronectin, they also spread morerapidly on the matrix. Consistent with the adhesion data, GGcytohesin 2 expressing HeLa or MCF-7 cells spread less than

control cells and similar to cells expressing wild-type cytohesin3/GRP1 (2G; Fig. 2B–D). Furthermore, GGG cytohesin 3expressing HeLa or MCF-7 cells spread more than control cells

and similar to cells expressing wild-type cytohesin 2/ARNO (3G;Fig. 2B–D). Spreading of MCF-7 cells were more effected bycytohesin expression than the spreading of HeLa cells, suggesting

Fig. 1. The identity of the PH domain determines the effect of cytohesin

expression on cell adhesion. (A) Cartoon depicting the hybrid cytohesin

2/cytohesin 3 constructs utilized and summary of their effects on adhesion.

(B) HeLa cells were transfected with the indicated constructs and their

adhesion to various concentrations of fibronectin tested as described in the

Materials and Methods. Adhesion levels were normalized to the adhesion of

control cells to 10 mg/ml fibronectin. Adhesion of cytohesin-expressing cellswas significantly different from adhesion of control cells at the indicated

points *P,0.01, t-test. Data are means 6 s.e.m. Control (blue) n524,

cytohesin 2 (red) n513, cytohesin 3 (green) n510, 2 w/3cc (magenta) n56, 2

w/3pb (cyan) n58, 2 w/3PH (orange) n53. Adhesion of cells expressing 2

w/3cc or 2 w/3pb was not statistically different from adhesion of cells

expressing wild-type cytohesin 2 at any fibronectin concentration. Adhesion

of cells expressing 2 w/3PH was statistically different from the adhesion of

cells expressing cytohesin 2 at 3, 5 and 10 mg/ml fibronectin (P,0.01).Adhesion of cells expressing 2 w/3PH was not statistically different from the

adhesion of cells expressing cytohesin 3 at any fibronectin concentration.

(C) Saved samples of the cells from one experiment shown in B were lysed

and western blotted to show the expression level of the myc-tagged cytohesin

constructs and glyceraldehyde 3-phosphate dehydrogenase (GapDH).

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that HeLa cells may have alternative non-cytohesin dependent

mechanisms for recycling integrins. We conclude that the

different phophoinositide affinity of cytohesin 2 and 3 is

responsible for their opposing effects on cell adhesion and

spreading.

Differential localization of GG cytohesin 2 and GGGcytohesin 3 in spreading cells

We previously demonstrated that in spreading cells wild-type

cytohesin 2/ARNO (3G) is localized at both the cell periphery and

interior regions, while wild-type cytohesin 3/GRP1 (2G) is

exclusively located at the cell periphery (Oh and Santy, 2010).

Therefore, we determined where the GG cytohesin 2 and GGG

cytohesin 3 mutants localize in spreading cells. GG cytohesin 2

was exclusively located at the cell periphery region and is

restricted to the most basal level of the cell, which is the same

localization as wild-type cytohesin 3/GRP1 (Fig. 3A). Conversely,

GGG cytohesin 3 was localized at both peripheral and interior

regions of spreading cells (Fig. 3B). GGG cytohesin 3 was

extensively co-localized with wild-type cytohesin 2/ARNO (3G)

and was less well co-localized with cytohesin 3 (Fig. 3B,C,E).

Similarly GG cytohesin 2 was better co-localized with cytohesin 3

than with cytohesin 2 (Fig. 3A,D,E). These data all suggest that the

number of glycine residues (3Gs vs. 2Gs) in the PH domain is also

the important determinant of the differential localization of

cytohesin 2 and cytohesin 3.

GGG cytohesin 3 expression rescues impaired b1 integrin

recycling caused by knockdown of cytohesin 2/ARNO

Cytohesin 2/ARNO knockdown inhibits b1 integrin recycling(Oh and Santy, 2010). If phosphoinositide affinity is the only

functional difference between cytohesin 2 and 3, then GGG

cytohesin 3 should rescue b1 integrin recycling in cells wherecytohesin 2/ARNO has been knocked-down while GG cytohesin

2 should not rescue b1 integrin recycling. MCF-7 cells weretransfected with siRNA targeting cytohesin 2/ARNO or control

Fig. 2. The number of glycine residues in the PH domain is the critical determinant of cytohesin effects on cell adhesion and spreading. (A) Reversing the

phosphoinositide specificity of the cytohesin PH domains, reverses their effect on adhesion. HeLa cells were transfected with the indicated DNA constructs, and

the adhesion assay was performed as described in the Materials and Methods. Adhesion of all cells expressing cytohesin constructs was significantly different

from the adhesion of control cells, as indicated. The adhesion of cells expressing cytohesin 3 GGG (cyto 3 GGG) was not significantly different from the adhesion

of cells expressing cytohesin 2 at any fibronectin concentration. Similarly, the adhesion of cells expressing cytohesin 2 GG (cyto 2 GG) was not significantly

different from the adhesion of cells expressing cytohesin 3. Data are means 6 s.e.m. (B,C) MCF-7 (B) or HeLa (C) cells were transfected with constructs

expressing the indicated proteins and allowed to spread on fibronectin as described in the Materials and Methods. The surface area of the spreading cells was

measured using Slidebook 5.0 and normalized to the surface area of spreading cells transfected with empty vector. The spreading of all cytohesin-expressing cells

was significantly different from the spreading of control cells (t-test, indicated by asterisks over each bar). The spreading of pairs of cytohesin-expressing cells was

compared using a t-test. Data are means 6 s.e.m. of more than 30 cells. *P,0.01; n.s., not significantly different. (D) Examples of the spreading MCF-7 cells

measured in B. Scale bar: 10 mm. (E) Expression levels of cytohesin constructs transfected into HeLa cells as in A and C.

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siRNA. A day later the cells were transfected again with either

empty plasmid or plasmid encoding myc–GGG-cytohesin-3 or

myc-GG-cytohesin-2. After 24 hours of recovery, the recycling

of b1 integrin was assayed. As shown in Fig. 4, the integrin isrecycled back to the plasma membrane in 5 minutes in control

cells. However, the cells treated with siRNA targeting cytohesin

2/ARNO have significantly impaired b1 integrin recycling(Fig. 4A,B) (Oh and Santy, 2010). Importantly, expression of

GGG cytohesin 3 completely rescued b1 integrin recycling in thecells treated with siRNA against wild-type cytohesin 2/ARNO

(Fig. 4A,B). Furthermore expression of GG cytohesin 2 failed

to rescue b1 integrin recycling in these cells (Fig. 4A,B). Thisdata strongly support the conclusion that the different

phosphoinositide specificity of the cytohesin PH domains

determines their differential localization and promotes distinct

functions of the cytohesins in cell adhesion and migration.

DiscussionAlthough cytohesin 2/ARNO and cytohesin 3/GRP1 share 80%

sequence identity, cytohesin 2 and cytohesin 3 have distinct

functions (Casanova, 2007; Oh and Santy, 2010). Cytohesin 2

and 3 have opposing effects on cell adhesion, spreading and

migration (Oh and Santy, 2010). Additionally, only cytohesin 2 is

required in b1 integrin recycling (Oh and Santy, 2010). In thisstudy, we demonstrated that the number of glycine residues in the

PH domain is the key difference underlying the differential

actions of cytohesin 2/ARNO and cytohesin 3/GRP1 in cell

adhesion, spreading and b1 integrin recycling. Previous studieshave demonstrated that the number of glycine residues in

cytohesin PH domains is the key determinant of the

phosphoinositide binding affinity of these PH domains

(Klarlund et al., 2000). Cytohesin PH domains with 2 glycines

bind PtdIns(3,4,5)P3 with high affinity and specificity. The

Fig. 3. The number of glycine residues (2Gs versus 3Gs) in

the PH domains determines cytohesin localization. MCF-7

cells were transfected with FLAG-tagged cytohesin 3 and myc-

tagged GG cytohesin 2 (A), FLAG-tagged cytohesin 3 and myc-

tagged GGG cytohesin 3 (B), FLAG-tagged GGG cytohesin 3 and

myc-tagged cytohesin 2 (C) or myc-tagged GG cytohesin 2 and

mCherry-tagged cytohesin 2 (D), and the transfected cells were

spread on fibronectin for 30 minutes, fixed, stained and analyzed

by deconvolution microscopy as described in the Materials and

Methods. Left panels, a maximum projection along the Z-axis is

shown. Right panels, a maximum projection along the X-axis is

shown. In the merged images the proteins shown individually in

the left most panels are shown in green and the proteins shown

individually in the middle panels are shown in red. Scale bars:

10 mm. (E) The extent of co-localization of GG cytohesin 2 andGGG cytohesin 3 with the wild-type cytohesins was analyzed

using Slidebook in 7–15 spreading cells. Data are means 6

standard error of the Pearson’s correlation coefficient.

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addition of a third glycine residue reduces the affinity for

PtdIns(3,4,5)P3 and greatly increases the affinity of the PH

domain for PtdIns(4,5)P2 (Klarlund et al., 2000). The structures

of PH domains of cytohesin 3/Grp1 (2G) bound to the headgroup

of PtdIns(3,4,5)P3 and cytohesin 2/ARNO (3G) bound to the

headgroups of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 have been

solved and demonstrate the structural basis of the differential

binding affinities (Cronin et al., 2004; Lietzke et al., 2000). The

addition of a third glycine disrupts interactions of PtdIns(3,4,5)P3with the peptide backbone thereby reducing the PtdIns(3,4,5)P3binding affinity (Cronin et al., 2004). Furthermore the addition of

this glycine causes PtdIns(4,5)P2 to bind in a different orientation

into a high affinity pocket (Cronin et al., 2004). Our data support

the conclusion that phosphoinositide specificity is the key

functional difference determining which cytohesin functions in

cell adhesion, spreading and integrin recycling.

We also found that localization of cytohesin 2 and cytohesin 3

depends on number of glycines in the PH domain. This

observation is somewhat surprising since these proteins are

recruited to membranes both by their PH domains and by

binding to scaffold proteins via their coiled-coil domains, which

is the least conserved region (Donaldson and Jackson, 2000;

Lim et al., 2010; White et al., 2010). However, we found that a

change in the number of glycine residues in the PH domain is

sufficient to alter localization of cytohesin 2 and cytohesin 3.

Protein–protein interactions mediated by the coiled-coil domain

may therefore serve to stabilize cytohesins at membranes once

they have been recruited there by PH domain–phosphoinositide

interactions. Alternatively, we have shown that coiled-coiled

domain mediated interactions are required for assembly of

protein complexes that have signal transduction roles (White

et al., 2010). The coiled-coil domain may therefore promote

Fig. 4. Expression of GGG cytohesin 3 but not GG cytohesin 2 rescues b1 integrin recycling in cells treated with cytohesin 2 siRNA. MCF-7 cells were

transfected with either control siRNA or siRNA targeting cytohesin 2 as described in the Materials and Methods. 24 hours later the cells were transfected again

with the indicated plasmids. After another 24 hours b1 integrin recycling was tested as described in the Materials and Methods. (A) Quantification of b1

integrin recycling. Mean b1 integrin surface fluorescence in cell-containing areas was measured and the background fluorescence of cell-free areas was subtracted

using Slidebook 5.0: control 0 min n517; control 5 min n517; cytohesin 2 knockdown 0 minutes n518; cytohesin 2 knockdown 5 minutes n517;

GGG rescue 0 minutes n518; GGG rescue 5 minutes n517; GG rescue 0 minutes n518; GG rescue 5 minutes n518. Data are means 6 s.e.m. The surface

fluorescence levels were compared using a t-test; *P,0.01, n.s., not significant. (B) Representative fields showing surface b1 integrin after 0 or 5 minutes of

serum stimulation. Scale bar: 10 mm. (C) Expression of cytohesin rescue constructs in cytohesin 2 knockdown cells. MCF-7 cells were transfected as in A and B.The cells were then lysed and western blotted with 9E10 anti-myc and mouse anti-actin antibodies. (D) Knockdown of cytohesin 2 in the rescue experiments.

MCF-7 cells were transfected with the indicated siRNAs and control vector as in A and B. Total RNA was isolated and used as template for RT-PCR to amplify

cytohesin 2 or GapDH as described in the Materials and Methods.

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cytohesin-dependent signal transduction rather than cytohesinlocalization.

Our data implicate binding to PtdIns(4,5)P2 as the key functionthat allows a cytohesin to promote stimulated b1 integrin recycling.Other studies have reported the presence of PtdIns(4,5)P2 in

early endosomal compartments but not in the later recyclingcompartments (Brown et al., 2001; Donaldson et al., 2009;Jovanovic et al., 2006). Therefore, it is not clear how

PtdIns(4,5)P2 would be acting to recruit cytohesin 2 and induceintegrin recycling. It is worth noting that our studieshave investigated stimulated, not basal, integrin recycling.

PtdIns(4,5)P2 might be produced transiently at recyclingcompartments in response to serum or other signals, but play norole in unstimulated recycling of proteins to the plasma membrane.Alternatively cytohesin 2 could be recruited onto early endosomal

membranes by binding to PtdIns(4,5)P2 and then be maintained onthese membranes as they traffic through the endosomal system byinteractions with other proteins. In this scenario cytohesin 2 would

be present on the endosomal membranes in an inactive state until it isturned on by additional signals. One final possibility is that as ARF6is activated during recycling, it can activate phosphatidylinositol-4-

phosphate 5 kinase to produce PtdIns(4,5)P2 (Brown et al., 2001;Donaldson et al., 2009; Grant and Donaldson, 2009; Jovanovic et al.,2006). Thus, PtdIns(4,5)P2 and cytohesin 2 may set up a positivefeedback loop that promotes the recycling of endocytic cargo.

Clearly further work will be necessary to determine wherePtdIns(4,5)P2 and cytohesin 2 intersect with internalized b1 integrin.

The inhibitory action of cytohesin 3/GRP1

The increased adhesion and spreading in cells expressing cytohesin

2 is explained by the ability of this protein to promote b1 integrinrecycling and thereby elevate surface integrin levels. Cytohesin 3,on the other hand, inhibits adhesion and migration. In contrast to

cytohesin 2/ARNO, cytohesin 3/GRP1 is not involved in b1integrin recycling due to its exclusive localization at the plasmamembrane (Oh and Santy, 2010). However, cytohesin 2 andcytohesin 3 share many up- and downstream signaling molecules.

Therefore, cytohesin 3/GRP1 expression may reduce cell adhesionand spreading by sequestering these common up- or downstreamcomponents at the plasma membrane, thereby perturbing

formation of a functional complex at the recycling endosome.Scaffolding proteins such as GRASP, Cybr, CNK1 and CNK3/IPCEF regulate ARF6 activity by binding to cytohesins (Attar

et al., 2012; Lim et al., 2010; White et al., 2010). A numberof molecules including Rac, phosphatidylinositol-4-phosphate 5kinase and PLD act downstream of ARF6 in signaling pathways

(Logan and Mandato, 2006). Cytohesin 3/GRP1 expression in cellsmay affect all of these signaling pathways. Further investigation ofthe inhibitory action of cytohesin 3/GRP1 on b1 integrin recyclingis required to determine which of these pathways is impaired by

cytohesin 3 overexpression. Identifying these pathways willidentify additional pathways that are limiting for b1 integrinrecycling.

Cytohesin specificity

Cytohesin 2 localizes at both cell edges and interior regions,while cytohesin 3 exclusively locates at the plasma membrane(Oh and Santy, 2010). Therefore, we hypothesize that only

cytohesin 2 functions at internal compartments, while bothcytohesin 2 and cytohesin 3 should be involved with events at theplasma membrane (Oh and Santy, 2010). Consistent with this

idea, recent studies have shown that both cytohesin 2 and

cytohesin 3 are involved in insulin signal transduction.

Knockdown of either cytohesin 2 or cytohesin 3 produces

insulin resistance in mice (Hafner et al., 2006). Additionally,

cytohesin function is required to generate PtdIns(4,5)P2-rich

environment at the plasma membrane for recruitment of insulin

receptor substrate 1 (IRS1) (Lim et al., 2010). Although both

cytohesins should act at the plasma membrane, cytohesin 3 might

still have unique functions in PtdIns(3,4,5)P3 dependent events

due to its high affinity for this lipid. Even though both insulin and

PDGF increase PtdIns(3,4,5)P3 levels at the plasma membrane, a

rise is less and slower in response to PDGF (Oatey et al., 1999).

Therefore, total cytohesin activity at the plasma membrane,

which will be a sum of cytohesin 2 binding to either lipid and

cytohesin 3 binding to PtdIns(3,4,5)P3, may be different in

response to these different signals. Alternatively, cytohesin 3

recruitment to the plasma membrane in response to

PtdIns(3,4,5)P3 may act as a coincidence detector to increase

cytohesin activity when PtdIns(3,4,5)P3 production occurs in

conjunction with other signals.

In this study, we demonstrated that the presence of a single

additional glycine in the PH domain of cytohesin 2/ARNO

differentiates its functions and localization from the functions and

localization of cytohesin 3/GRP1. This result implicates the

phophoinositide binding specificity of cytohesin 2/ARNO and

cytohesin 3/GRP1 as a key determinant of their localization and

functions. Furthermore these results implicate phosphoinositide

production as a key signal in the stimulated recycling of b1 integrin,and therefore as a key player in the initiation of cell migration.

Materials and MethodsCells and reagents

HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)

with antibiotics (Penicillin, streptomycin, Fungizone), 10% FBS and glutamine.MCF-7 cells were cultured in DMEM/F-12 with antibiotics (Penicillin,streptomycin, Fungizone), 10% FBS and nonessential amino acids. The TS2/16anti-b1 integrin antibody was purchased from Santa Cruz Biotechnology (SantaCruz, CA, USA). M2 anti-flag antibodies (labeled and unlabeled) were purchased

from Sigma-Aldrich (St. Louis, MO, USA). 9E10 anti-myc antibodies (labeled andunlabeled), anti-actin and anti-GapDH antibodies were purchased from Covance(Princeton, NJ, USA). Alexa-Fluor-647–TS2/16 was obtained from BioLegend(San Diego, CA, USA).

siRNAs and expression constructs

siRNA duplexes against the sequence 59-GCAAUGGGCAGGAAGAAGU-39targeting human cytohesin 2/ARNO and control siRNA against firefly luciferasewere purchased from Dharmacon (Layfayette, CO, USA). The siRNAs weretransfected into MCF-7 cells by using Neon Transfection System (Invitrogen,Carlsbad, CA, USA) according to the manufacturer’s recommended protocol.

Plasmids encoding myc-tagged cytohesin 2/ARNO or Myc-tagged cytohesin 3/GRP1 in pcDNA3 were constructed by PCR from myc-tagged cytohesin 2/ARNOor FLAG-tagged cytohesin 3/GRP1 in pCB7 provided by James Casanova(University of Virginia). Mutations were introduced by using QuikChange site-directed mutagenesis (Stratagene, Wilmington, DE, USA). Hybrid constructs were

constructed by creating unique restriction sites in equivalent locations of cytohesin2 and 3. The construct for expressing siRNA-resistant GG cytohesin 2 was createdby introducing two silent point mutations into the siRNA site using site-directedmutagenesis. Transient transfections were performed according to themanufacturer’s instructions using Lipofectamine LTX (Forward transfection

(Invitrogen, Carlsbad, CA, USA) for MCF-7 cells and Lipofectamine 2000(Forward transfection) for HeLa.

Adhesion assay

HeLa cells were transfected with various DNA constructs as described above.After 24 hours of expression, adhesion of the cells to fibronectin was tested aspreviously described (Oh and Santy, 2010). Briefly, the cells were harvested by

incubation in PBS, 4 mM EDTA, 1 mM EGTA. The cells were resuspended inOptimem medium with 0.2% BSA. Equal numbers of cells were plated in wells

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coated with varying concentrations of fibronectin. After adhesion for 1 hour thecells were washed, fixed and stained with crystal violet.

Spreading assaySpreading of cells transfected with cytohesin constructs was performed aspreviously described (Oh and Santy, 2010). Briefly cells were transfected andharvested as described for the adhesion assay. They were plated on coverslipscoated with 10 mg/ml of fibronectin and allowed to spread for 15 minutes (HeLacells) or 30 minutes (MCF-7 cells). The cells were then fixed and stained with9E10 anti-myc followed by Alexa-Fluor-488-conjugated anti-mouse secondaryantibody and Rhodamine-conjugated phalloidin.

Co-localization of cytohesinsMCF-7 cells were transfected with the indicated cytohesin constructs and the nextday allowed to spread on fibronectin as described above for the spreading assay.The cells were fixed with 4% paraformaldehyde and then stained with FITC-conjugated M2 anti-FLAG and biotinylated 9E10 anti-myc followed by Alexa-Flour-546-conjugated streptavidin or with Cy3-conjugated M2 anti-FLAG andAlexa-Fluor-488-conjugated 9E10 anti-myc antibodies. Co-localization of thecytohesins was assayed using deconvolution microscopy as previously described(Oh and Santy, 2010). The extent of co-localization was quantified by calculatingthe Pearson’s correlation coefficient in multiple cells using Slidebook 5.0.

Analyzing rescue of b1 integrin recycling by microscopyTwo days before the assay, siRNA targeting cytohesin 2/ARNO or control siRNAwas transfected into MCF-7 cells. The next day empty plasmid, plasmid encodingmyc–GGG-cytohesin-3 or plasmid encoding siRNA-resistant myc–GG-cytohesin-2 were transfected into these cells using Lipofectamine LTX. After 8 hours ofrecovery, the cells were serum starved overnight. The next day, b1 integrinrecycling was assayed as previously described (Oh and Santy, 2010). Briefly, TS2/16 anti-b1 integrin was bound to the surface of the cells in the cold for 2 hours.The cells were allowed to internalized the bound antibody for 1 hour at 37 C̊, andthen remaining surface antibody was removed with by washing the cells twice with0.5 M NaCl, 0.5% acetic acid, pH 3.0. Recycling of internalized integrin wasinitiated by the addition of warm DMEM with 20% FBS. At designated times thecells were chilled and fixed with 4% paraformaldehyde. The cells were leftunpermeabilized and antibody that had been recycled to the cell surface wasdetected by incubation with Alexa-Fluor-594-conjugated anti-mouse secondaryantibody.

RT-PCRMCF-7 cells were transfected with siRNAs and plasmid as described above for therescue assay. The day after plasmid transfection total RNA was isolated using theRNeasy kit according to the manufacturer’s instructions (Qiagen, Inc., Valencia,CA). RT-PCR to amplify cytohesin 2 and GapDH was performed as previouslydescribed (Oh and Santy, 2010), using 0.2 mg of the RNA as template.

Detection of surface b1 integrin levelsHeLa cells were transfected with the indicated cytohesin constructs and a GFPvector as described above. After 24 hours of expression, the cells were harvestedas described above for the adhesion assay, fixed and stained with Alexa-Fluor-647-conjugated TS2/16 anti-b1 integrin. Levels of GFP and Alexa Fluor 647fluorescence were measured using an FC500 benchtop flow cytometer. AlexaFluor 647 levels in GFP positive cells were analyzed using the FlowJo (Tree Star,Inc., Ashland, OR, USA) PB histogram comparison algorithm.

FundingThis work was supported by grants from the American HeartAssociation [grant number SDG 0730229N to L.C.S.]; and theAmerican Cancer Society [grant number RSG-09-168-01-CSM toL.C.S.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.101683/-/DC1

ReferencesAltankov, G. and Grinnell, F. (1995). Fibronectin receptor internalization and AP-2

complex reorganization in potassium-depleted fibroblasts. Exp. Cell Res. 216, 299-309.

Attar, M. A., Salem, J. C., Pursel, H. S. and Santy, L. C. (2012). CNK3 and IPCEF1produce a single protein that is required for HGF dependent Arf6 activation andmigration. Exp. Cell Res. 318, 228-237.

Borghi, N., Lowndes, M., Maruthamuthu, V., Gardel, M. L. and Nelson, W. J.

(2010). Regulation of cell motile behavior by crosstalk between cadherin- andintegrin-mediated adhesions. Proc. Natl. Acad. Sci. USA 107, 13324-13329.

Brown, F. D., Rozelle, A. L., Yin, H. L., Balla, T. and Donaldson, J. G. (2001).Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic. J. CellBiol. 154, 1007-1018.

Casanova, J. E. (2007). Regulation of Arf activation: the Sec7 family of guaninenucleotide exchange factors. Traffic 8, 1476-1485.

Caswell, P. T. and Norman, J. C. (2006). Integrin trafficking and the control of cellmigration. Traffic 7, 14-21.

Caswell, P. and Norman, J. (2008). Endocytic transport of integrins during cellmigration and invasion. Trends Cell Biol. 18, 257-263.

Cronin, T. C., DiNitto, J. P., Czech, M. P. and Lambright, D. G. (2004). Structuraldeterminants of phosphoinositide selectivity in splice variants of Grp1 family PHdomains. EMBO J. 23, 3711-3720.

de Rooij, J., Kerstens, A., Danuser, G., Schwartz, M. A. and Waterman-Storer,C. M. (2005). Integrin-dependent actomyosin contraction regulates epithelial cellscattering. J. Cell Biol. 171, 153-164.

Donaldson, J. G. and Jackson, C. L. (2000). Regulators and effectors of the ARFGTPases. Curr. Opin. Cell Biol. 12, 475-482.

Donaldson, J. G., Porat-Shliom, N. and Cohen, L. A. (2009). Clathrin-independentendocytosis: a unique platform for cell signaling and PM remodeling. Cell. Signal. 21,1-6.

Gao, B., Curtis, T. M., Blumenstock, F. A., Minnear, F. L. and Saba, T. M. (2000).Increased recycling of (alpha)5(beta)1 integrins by lung endothelial cells in responseto tumor necrosis factor. J. Cell Sci. 113, 247-257.

Grant, B. D. and Donaldson, J. G. (2009). Pathways and mechanisms of endocyticrecycling. Nat. Rev. Mol. Cell Biol. 10, 597-608.

Hafner, M., Schmitz, A., Grüne, I., Srivatsan, S. G., Paul, B., Kolanus, W., Quast,T., Kremmer, E., Bauer, I. and Famulok, M. (2006). Inhibition of cytohesins bySecinH3 leads to hepatic insulin resistance. Nature 444, 941-944.

Hay, E. D. (2005). The mesenchymal cell, its role in the embryo, and the remarkablesignaling mechanisms that create it. Dev. Dyn. 233, 706-720.

Hordijk, P. L., ten Klooster, J. P., van der Kammen, R. A., Michiels, F., Oomen,

L. C. J. M. and Collard, J. G. (1997). Inhibition of invasion of epithelial cells byTiam1-Rac signaling. Science 278, 1464-1466.

Jovanovic, O. A., Brown, F. D. and Donaldson, J. G. (2006). An effector domainmutant of Arf6 implicates phospholipase D in endosomal membrane recycling. Mol.Biol. Cell 17, 327-335.

Klarlund, J. K., Tsiaras, W., Holik, J. J., Chawla, A. and Czech, M. P. (2000).Distinct polyphosphoinositide binding selectivities for pleckstrin homology domainsof GRP1-like proteins based on diglycine versus triglycine motifs. J. Biol. Chem. 275,32816-32821.

Lietzke, S. E., Bose, S., Cronin, T., Klarlund, J., Chawla, A., Czech, M. P. andLambright, D. G. (2000). Structural basis of 3-phosphoinositide recognition bypleckstrin homology domains. Mol. Cell 6, 385-394.

Lim, J., Zhou, M., Veenstra, T. D. and Morrison, D. K. (2010). The CNK1 scaffoldbinds cytohesins and promotes insulin pathway signaling. Genes Dev. 24, 1496-1506.

Logan, M. R. and Mandato, C. A. (2006). Regulation of the actin cytoskeleton by PIP2in cytokinesis. Biol. Cell 98, 377-388.

Ng, T., Shima, D., Squire, A., Bastiaens, P. I., Gschmeissner, S., Humphries, M. J.and Parker, P. J. (1999). PKCalpha regulates beta1 integrin-dependent cell motilitythrough association and control of integrin traffic. EMBO J. 18, 3909-3923.

Oatey, P. B., Venkateswarlu, K., Williams, A. G., Fletcher, L. M., Foulstone, E. J.,Cullen, P. J. and Tavaré, J. M. (1999). Confocal imaging of the subcellulardistribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes. Biochem. J. 344, 511-518.

Ogasawara, M., Kim, S.-C., Adamik, R., Togawa, A., Ferrans, V. J., Takeda, K.,

Kirby, M., Moss, J. and Vaughan, M. (2000). Similarities in function and genestructure of cytohesin-4 and cytohesin-1, guanine nucleotide-exchange proteins forADP-ribosylation factors. J. Biol. Chem. 275, 3221-3230.

Oh, S. J. and Santy, L. C. (2010). Differential effects of cytohesins 2 and 3 on b1integrin recycling. J. Biol. Chem. 285, 14610-14616.

Perl, A. K., Wilgenbus, P., Dahl, U., Semb, H. and Christofori, G. (1998). A causalrole for E-cadherin in the transition from adenoma to carcinoma. Nature 392, 190-193.

Powelka, A. M., Sun, J., Li, J., Gao, M., Shaw, L. M., Sonnenberg, A. and Hsu,

V. W. (2004). Stimulation-dependent recycling of integrin beta1 regulated by ARF6and Rab11. Traffic 5, 20-36.

Sander, E. E., van Delft, S., ten Klooster, J. P., Reid, T., van der Kammen, R. A.,

Michiels, F. and Collard, J. G. (1998). Matrix-dependent Tiam1/Rac signaling inepithelial cells promotes either cell-cell adhesion or cell migration and is regulated byphosphatidylinositol 3-kinase. J. Cell Biol. 143, 1385-1398.

White, D. T., McShea, K. M., Attar, M. A. and Santy, L. C. (2010). GRASP andIPCEF promote ARF-to-Rac signaling and cell migration by coordinating theassociation of ARNO/cytohesin 2 with Dock180. Mol. Biol. Cell 21, 562-571.

Ylänne, J., Huuskonen, J., O’Toole, T. E., Ginsberg, M. H., Virtanen, I. and

Gahmberg, C. G. (1995). Mutation of the cytoplasmic domain of the integrin beta 3subunit. Differential effects on cell spreading, recruitment to adhesion plaques,endocytosis, and phagocytosis. J. Biol. Chem. 270, 9550-9557.

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http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.101683/-/DC1http://dx.doi.org/10.1006%2Fexcr.1995.1038http://dx.doi.org/10.1006%2Fexcr.1995.1038http://dx.doi.org/10.1006%2Fexcr.1995.1038http://dx.doi.org/10.1016%2Fj.yexcr.2011.10.018http://dx.doi.org/10.1016%2Fj.yexcr.2011.10.018http://dx.doi.org/10.1016%2Fj.yexcr.2011.10.018http://dx.doi.org/10.1073%2Fpnas.1002662107http://dx.doi.org/10.1073%2Fpnas.1002662107http://dx.doi.org/10.1073%2Fpnas.1002662107http://dx.doi.org/10.1083%2Fjcb.200103107http://dx.doi.org/10.1083%2Fjcb.200103107http://dx.doi.org/10.1083%2Fjcb.200103107http://dx.doi.org/10.1111%2Fj.1600-0854.2007.00634.xhttp://dx.doi.org/10.1111%2Fj.1600-0854.2007.00634.xhttp://dx.doi.org/10.1111%2Fj.1600-0854.2005.00362.xhttp://dx.doi.org/10.1111%2Fj.1600-0854.2005.00362.xhttp://dx.doi.org/10.1016%2Fj.tcb.2008.03.004http://dx.doi.org/10.1016%2Fj.tcb.2008.03.004http://dx.doi.org/10.1038%2Fsj.emboj.7600388http://dx.doi.org/10.1038%2Fsj.emboj.7600388http://dx.doi.org/10.1038%2Fsj.emboj.7600388http://dx.doi.org/10.1083%2Fjcb.200506152http://dx.doi.org/10.1083%2Fjcb.200506152http://dx.doi.org/10.1083%2Fjcb.200506152http://dx.doi.org/10.1016%2FS0955-0674%2800%2900119-8http://dx.doi.org/10.1016%2FS0955-0674%2800%2900119-8http://dx.doi.org/10.1016%2Fj.cellsig.2008.06.020http://dx.doi.org/10.1016%2Fj.cellsig.2008.06.020http://dx.doi.org/10.1016%2Fj.cellsig.2008.06.020http://www.ncbi.nlm.nih.gov%2Frntre2query.fcgi%3Fcmd%3DRetrieve&db%3DPubMed&list%5Fuids%3D10633076&dopt%3DAbstracthttp://www.ncbi.nlm.nih.gov%2Frntre2query.fcgi%3Fcmd%3DRetrieve&db%3DPubMed&list%5Fuids%3D10633076&dopt%3DAbstracthttp://www.ncbi.nlm.nih.gov%2Frntre2query.fcgi%3Fcmd%3DRetrieve&db%3DPubMed&list%5Fuids%3D10633076&dopt%3DAbstracthttp://dx.doi.org/10.1038%2Fnrm2755http://dx.doi.org/10.1038%2Fnrm2755http://dx.doi.org/10.1038%2Fnature05415http://dx.doi.org/10.1038%2Fnature05415http://dx.doi.org/10.1038%2Fnature05415http://dx.doi.org/10.1002%2Fdvdy.20345http://dx.doi.org/10.1002%2Fdvdy.20345http://dx.doi.org/10.1126%2Fscience.278.5342.1464http://dx.doi.org/10.1126%2Fscience.278.5342.1464http://dx.doi.org/10.1126%2Fscience.278.5342.1464http://dx.doi.org/10.1091%2Fmbc.E05-06-0523http://dx.doi.org/10.1091%2Fmbc.E05-06-0523http://dx.doi.org/10.1091%2Fmbc.E05-06-0523http://dx.doi.org/10.1074%2Fjbc.M002435200http://dx.doi.org/10.1074%2Fjbc.M002435200http://dx.doi.org/10.1074%2Fjbc.M002435200http://dx.doi.org/10.1074%2Fjbc.M002435200http://dx.doi.org/10.1016%2FS1097-2765%2800%2900038-1http://dx.doi.org/10.1016%2FS1097-2765%2800%2900038-1http://dx.doi.org/10.1016%2FS1097-2765%2800%2900038-1http://dx.doi.org/10.1101%2Fgad.1904610http://dx.doi.org/10.1101%2Fgad.1904610http://dx.doi.org/10.1042%2FBC20050081http://dx.doi.org/10.1042%2FBC20050081http://dx.doi.org/10.1093%2Femboj%2F18.14.3909http://dx.doi.org/10.1093%2Femboj%2F18.14.3909http://dx.doi.org/10.1093%2Femboj%2F18.14.3909http://dx.doi.org/10.1042%2F0264-6021%3A3440511http://dx.doi.org/10.1042%2F0264-6021%3A3440511http://dx.doi.org/10.1042%2F0264-6021%3A3440511http://dx.doi.org/10.1042%2F0264-6021%3A3440511http://dx.doi.org/10.1074%2Fjbc.275.5.3221http://dx.doi.org/10.1074%2Fjbc.275.5.3221http://dx.doi.org/10.1074%2Fjbc.275.5.3221http://dx.doi.org/10.1074%2Fjbc.275.5.3221http://dx.doi.org/10.1074%2Fjbc.M109.043935http://dx.doi.org/10.1074%2Fjbc.M109.043935http://dx.doi.org/10.1038%2F32433http://dx.doi.org/10.1038%2F32433http://dx.doi.org/10.1038%2F32433http://dx.doi.org/10.1111%2Fj.1600-0854.2004.00150.xhttp://dx.doi.org/10.1111%2Fj.1600-0854.2004.00150.xhttp://dx.doi.org/10.1111%2Fj.1600-0854.2004.00150.xhttp://dx.doi.org/10.1083%2Fjcb.143.5.1385http://dx.doi.org/10.1083%2Fjcb.143.5.1385http://dx.doi.org/10.1083%2Fjcb.143.5.1385http://dx.doi.org/10.1083%2Fjcb.143.5.1385http://dx.doi.org/10.1091%2Fmbc.E09-03-0217http://dx.doi.org/10.1091%2Fmbc.E09-03-0217http://dx.doi.org/10.1091%2Fmbc.E09-03-0217

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