Association of TM4SF proteins with integrins: relevance to cancer

BiorlCnica~

et Biophysics Aga

ELSEVIER

Biochimicaet Biophyca Acta I?(17(1996) 67.71

Mini-Review

Association of TM4SF proteins with integkns: relevance tcocancer Martin E. Hemler *, Brian A. IGannion. Fedor Berditcheyski Ranted 7 March 1996:acceded7 M.rch 1996

Recent biochemical evidence has revealed that proteins in the TMlSF (trammembrane 4 superfamily; tetraspan family) form complexes with certain members of the integrin family of adhesion receptors. This convergence of two protein families has considerable relevance for tumor cells, since both growth and metastasis are regulated by adhesion receptors in the integrin family, and are modulated by TM4SF proteins. The aim of this review is to examine integrin/T?vl4SF associations in the contex: of tumor cell behavior. 1. M&IF protein strurtures The TM4SF proteins include at least 16 members, with 20-30s sequence similarity [I .2]. Each contains 4 highly conserved hydrophobic regions. presumed to be tran\membrane domains, flanked by short N- and C-temrir,, 15-14 aa)_ predicted $0 kcl r~,*~~lnr-:-

. Rei-itwby- -~.~~mc.xw~ C!CXiEiii~.

drophobic domains I and 2 lies a small predicted exrracelIuIar loop (20-27 aa), and between domains 3 and 4 is a larger extracellular loop of 75- 130 aa (Fig. I ). Aside from four highly conserved cysteines. not many other amino acids are conserved within the large loop. Monoclonal antibodies binding extraceliularly to TM4SF protern> most likely bind to sites within the large Icops [3,4]. Six members of the family (CD9. CD37, CD53. CD63. CDEI, CD82) are widely represented on leukocytes. and CD9, CD63, and CD81 are also present on the suriaces #if rszny other cell types [I .2,5].

2.l’hUSF

protein functions

At least three different TM4SF proteins may modulate tumor cell metastasis. In a recent study, the gene for CD82

--’ Corresponding author. Fax: + [email protected].

I

(617)

6322661:

030449X/%/415.00 8 1996 E!sevierSaewe B.V. Ail nghtr nerved P/I SO3O-G119X(96)00007-8

(K.41 I ) on chromosome I 1pl 1.2,suppressed experimental metastasrs when introduced intc a rat prostate cancer cell line AIw. CD82 gene expression WIS reduced in cell lines derived from prosrate tumors [6]. It remains to be determined whether the CD82 gene affects the metastasis of any ot’ler mmor cells. Expression in a mouse melanoma cell line of another TM4SF protein. CD9. inhibited experimental metastasis [7]. Furthermore, in human breast cancer, levels of CD9 were often lower in lymph node metastases than in the primary Nrnor [8]; and in non-small cell lung cancer, CD9 gene expression was associated with higher survival rate [9]. Association of CD9 with decreased metastasis is Perhaps consistent with CD9 overproduction, causing reduced motility in CHO cells. human lung adenocarcinoma. and human myeloma cell lines 171. Like CD9, synthesis of CD63 in melanoma ceils caused reduced expcrincntal metas’ursis. Also, CD63 expression caused a !o:ver in:radermaI growth ra?e of human melanoma cells in nude mice [IO]]. !n general. these studies of TM4SF proteins have emphasized their negative effects on tumor cell motility in viva and metastasis in vitro. However. there are e--eplions. as seen by the positive contributions of CD9 to the migration of B celli lints [I l,lt]. From all of these studier, few hmts have emerged regarding the mechanisms whereby TM4SF protens might regulate cell motility or metastasis. TM4SF proteins may also regulate cell proliferation. Anti-CDS2 mAb co-stimulated T cell proliferation [13.14], whde an anti-CD53 nAh was mitogenic for rat splenic T cells [IS]. and when rrosslinhed. anti-CD53 potentiated B cell entry into S phase [16]. Also. CD9 synthesis caused reduced growTh of a human lung adenocarcinoma cell line 171; the CD81 molecule regulates growth in certain lymphoblastoid cell lines [17]: and a ncwIy discovered TM4SF protein, called il-TMP, regulatis density dependent proliferation of intestinal cpithelial cells [l8]. Growth effects may be a consequence of signaling functions modulated by TM4SF proteins. including regulation of inuacellular cal-

teins together with CDl9, teins 1221. Also, CD81

CD2r

and MHC

cl&s

associates with CD19

a complement

associate together with CD4 or CD8 in T cells [24]; associates with

complex

[231: CD81

to

form CD82

and CD9

receptor

II pro-

and CR2

the heparitt-binding

growth

factor. thus potentiating

activity

[25]. In the mammahan

juxtacrine

EGF-like

growth

urothelium

and

factor

two TM4SF

proteins, UPla and UPlb. assemble with other proteins into highly organized

l6-nm

larger crystalline

protein particles.

chat are part of

anays 1261. Thus. the ability to associate

with each other and with other membrane proteins u itbin larger complexes coutd be a general feature of all ‘I?144 proteins. Recently, TM4SF

proteins have also been found

to form comp!exes with imegrins (see below).

3. lntegrin

tinrtares

Structtnal integrin

and fttn&nns

and functiona;

aspects of the 22 differeat

sr9 t~.tcrodimers have been reviewed extensively

elsewhere [27]. Each integtin recogmze ECM gen). and/or

brtegrindependent volving

functions showing

[2].

However,

[.30.31]

gene induction

protci

. and

Toe effects of TM4SF tuis

and growth

TM4SF CD9

could perhaps be explained

modulation

of cell adhesion.

mAb triggered a 2-3

mrtas-

partially

For example.

by anti-

fold increase in 5 lympiroblu\t

and differentiation

..

and suscepttbdtty to apopto

On tumor cells. integrins play a variety of critical roles. For example. the growth and angiogenesis of human breast cancer cells within

proteins on cell mottli’y.

[33,34].

sis [3X36]

there is not yet much evidence

direct association between TM4FF

signaling molecules or pathways.

weight

mediators (28.291. These inte-

adhesion and signaling events help to regulate

motility

[32]. cell proliferation and PKC dependent

av&

/37].

human skin is dependent on integrin

In contrast. expression

of the fx3,

integrin

iuhib;is tumor cell growth, presumably due to a&depettdent growth inhibitory extracellular

matrix

intemctions

[38,39].

the (19,

in the integrin

abolished

bilized

cells [40]. When synthesized in a rhabdomyosarcoma

anti-CD9

[20]

moted adhesion lated adhesion

and anti-CD82

[ 141 antibodies pro

and spreading of Schwann Also. anti-CD81

cells 2nd T

mAb markedly stimu-

of the 5 ccl! tine, Raji.

line. a$

to isterfollicular a’

correlated

a ftmctional aJ

connection

subunit (211, suggesting

between TM4SF

proteins and the

integrin. However. adheston of many B cells and other

celf

types

(Mannion.

is not stimuhttcd B.A..

L.B. and Hemler Although

by anti-TM4SF

Berditchevski.

F.. Kraeft.

antibodies S.-K.

Chen.

M.E. ( 1996) submitted).

TM4SF

and o”

integrins on tumor cells. Expression of a$, with

enhanced

in viva.

mvasive

potential

of

human prostate carcinoma rells 1421, and reduced survival of patients with human breast carcinoma the ix3/31 integrin

has been correlated

f43]. Similarly,

with invasiveness

and tumor progression of human melanoma cells, in vivo [44]. and in vitro 14551.In another study. the a3

proteins are associated with a host of

cell

had no effect on cell growth in vitro or in viro,

Most important for this review ate the functions of a3.

stromal cells a-d ftbroa~ectm. This adhesion was inhibited ai

of breast carcinoma

but did enhance metastasis (411

by an antibodj

to the integrin

the malignant

with tibmnectin

Similarly,

phenoiype

adhesion to bone marrow tibroblasts [19]. whereas immc-

cells. respec!ive!y.

to

colla-

VCAM).

adhesion leads to signaling events in-

and phosphoiipid

grin-speciftc

cium levels, tyrosinc phosphotylation.

ability

laminin, ICAM,

kinases. adapter proteins. small molecular

GTPases, cell

has a specialized

proteins (e.g.. fibronecdn.

cell surface proteins (e.g.

subunit

cellular activities, their specific molecular functions are not

was transiected into a rhabdomyosarcoma cell line. resulting in the inhibition .of both cell growth in vitro and

yet known. We hypo:hesize that TM4SF

tumorigenicity

proteins may be

transmembrane adaptor proteins. that organize the distribu-

Synthesis of

tion and function of other celi surface molecules and their

metastasis of

associated

carcinoma

oligomeric

signaling

proteins.

In

this

complexes may exist containing

regard, TM4SF

large pro-

in vivo [46]. a’&

positively

human

melanoma

cells (481. The a@,

ently allows attachment

correlates,

in viva. with

1471 and human

renal

on the tumor cell appar-

to VCAM-I

to endotilial

cells,

69

thus leading to extravasation. Also. a’& on a human melanoma line mediated experimental metastasis [49]. and transfection of a$, caused increased bone metastasis of multiple cell lines. probably due to VCAM expression on bone matrow stromal cells [SO]. However. when a spontaneous rather than experimental model was used, oJ synthesis inhibited rather than promoted melanoma cell metastasis [S I].

4. Integrin/TM4SF

asssxiations

Evidence is now emerging that subsets of integrins can specitically associate with comp!rxes of TM4SF proteins. which vary widely depending on cell type. Because integrins. like TM4SF proteins. can regulate cell adhesion and modulate tumor cell growth and me’%tasis. these associations a- IiLly to be htghiy relevant. The first published evidence for TM4SF/integrin complexes showed anti-CD9 antibody induction of CD9/cr”q?, association in platelets [S2]. Subsequently the integtins a$, [I I]. a@, [5,53], and o”p, IS] were found to associate constitutively with the CD9 molecule. as well as with other TM4SF proteins. including CD53, CD63, CDSI. and CD82 (]5.54] and Mannion, B.A., Berditchcvski. F., Kraeft, S.-K., Chen. L.B. and Hemler, M.E. (1996) submitted]. A summary of integtin/TM4SF protein associations is shown in Table I. As indicated, all members of the TM4SF protein family so far tested interact convmcingly and selectiveiy. with the integrins a$,. a’s,. and u”p,. At least one TM4SF protein (,Dftl) also interacts with o$,. Although an interaction between a-$, and CD9 was noted in one report [I I], a-& did not associate with CD9 or any other TM4SF protein in other studies ([5,53.54] and manuscript submitted’t. Deletion or exchange of integrin Q chain cytoplasmic and transmembrane domains did not cause loss of interaction with TM4SF proteins. suggesting that specificity was determined extracellularly. The finding that TM4SF proteins associate with a%;, but not with a$$ indicates that fl chains must also play a role in determining interactions. A major concern has been that the highly hydrophobic TMISF proteins might non-specifically trap other protems into detergent micelles. However, TM4SF interactions ‘ze highly specific for certain integrins, and TM4SF proteins generally do not associaie strongly with the six dtfferent integrins listed at the bottom of Table I. Also, many of the btteractions listed in Table I have now been confirmed in reciprocal co-precipitation experiments (i5.53.541 and manuscript submitted’), and integrin/TM4SF connections have been stabilized by cell surface crosslinking [5.53]. Ft%emtore, interactions seen in detergent lysates have been corroborated by immunofluorescence microscopy showing colocalization of integrins with TM4SF proteins in cell footprints 1541, in the peripheral regions of cells [54], and in cell surface clusters [5]. Aside from

TableI Inte~n/T:.I4SF a*ux,a,~“+ lntegnn

TM4SFprotein> CD9

Rd.

CD53 CD63 CD81 CD82

a3BI ++ a3pI (X3TC5)* ND a3Pl (xx01 ++ a4/3 n4Pl (X4C.O) :: ~4.13 I (X4C2) ND (146I (X4C5) ND ++ atql ND a+3 7 a IIbP3 +’ al/31 NE -_ 02pl

ND ND ND

++ ++ ++

++ ++ ++

++ ND ND

;; ND ND ND ND ND ND ._-

&+ ND ND ++ PD ND ND __

++ ++ i + +T + r ND ND - -

ND ND ND ++ ND ND - ND

~201 (X2C3) a5p I

ND __c

ND _-

- __

- --

ND - ..

06flJ ULj3S fiV’_

-ND - -

ND ND ND

-ND ND

-- ND

ND ND ND

I

++ ++

(5.53.541 unpubl. ’ 1541. unpubl. [I I]. *ilbmittcd ’ rubmiwd submitted cubmind f5.541 cubmilted 1521 submitted 1553.541 submitted [541. unpubl. [s 9154I. wbmated i5.541 submitted 1531

’ SXCS. n3 extracellular domam.cz5san~membrane andcyloplasmic domansX3CO.X4COcytnpksmic domains dcle~ed: X4C5.04 eamcellular.a5 cymplasmic: X2C3 02 extracellular. 03 cytopla\mic ’ Unpubli+bed rewbsof F. Berditcbevckl et al. ’ Mannion.B.A.. Berdacluvrki. F.. Krzefr.S.-K..Chen.L.B. andHemler,M.E. (1996)submitted. ’ Aswciauon WI\ only obervedafterplatelet stxmulation by an&CD9

integrin/TM4SF protein complexes discussed here. perhaps the only other well-documented integrin association with a transmembrane protein is the interaction of & integrins with the CD47/IkP-50 mole-nle [55].

5. Functions of integrin/TM4!3F

complexes

There is some suggestion that association with TM4SF proteins regulates integrin adbesive functions. For example, the nJ&/CDSl association could e:;plain the effects of arm-CD81 antibody on a$,-dependent cell adhesion [2f]. h i\ also notable that two independent a’ mutants (D346E. D408E) that were deticient ht their ability to form celf surface clusters (Pujades, C., Km& S.K., Alon, R., Masumoto, A., Burkly, L.. Springer, T.A., Chen. L.B.. Lobb. R.R. and Hemler, M.E. (1996) submitted) utd mediate cell adhesion [56]. were also deficient in their ability to associate with CD81 (Pujades. C.. Kraeft. S.K.. Alon. R.. Masumoto. A., Burkly. L.. Springer. T.A., Chen. L.B., Lobb. R.R. and Hemler, M.E. (1996) submitted). However. in most cases, effects on adhesion are difficult is demonstrate, stnce synthesis or TM4SF proteins cr addition of anti-TM4SF antibodies has no effect on cell adhesion.

m

ME

Hemkr

et al. / Eiochimicu

et Biophwica

It appears more likely that integrin/TM4SF complexes will have relevance for more complex events, such as those associated with cell motility. Indeed. transfection of a B cell line with CD9 was shown to alter a%, and a$+dependent motiltty [!2], and bofh of these integtins can associate with TM4SF proteins (Table Il. Integrin/TM4SF complexes have been localized to the leading edge of spmading cells, and also to l%podia. as well as within intracellular vesicles concentrated at the traiiing edge (F. Berditchevski et al.. uopublishedl. Notably. such complexes have not been found in focal adhesions (F. Berditchevski et al.. ttnpttblishedl. These results are highly consistent with TMSSF mcdttlation of cell motility. and lead to the hypothesis that TM4SF proteins may regulate the movement and signaling of itttegrins towards and/or away from locations where they are needed for migration. Furtbermore we have recently found evidence for TM4SF proteins and integrins associating with specific enzymes in the phosphatidyl inositol synthesis pathway, leading to production of 4.5PIP, (F. Berditchevski et al., unpublished). Because 45PIP? is an established regulator of cytoskeletal architecture [57], this has obvious relevance for cell spreading and motility. Because integrin-mediated adhesion activates the smal! GTP-binding protein, rho, and because rho may be involved in PIP2 production [58). we hypothesize that htegrin/TM4SF protein complexes could play a major ro!e in regulating rho-dependent functions. A remaining challenge will be to determine in more detail the specific biochemical signaling consequences of integrin/TM4SF protein associations. In addition. it will be important to ascertain the extent to which integrin/TM4SF associations may modulate tumor cell metastasis. For example, it will be useful to learn whether the negative effects of CP82 and o”p, on prostate cancer are influenced by the abi!q of CD82 and a%, to physically associate. In conclusion. the discovery of integrin/TM4SF protein complexes otens up a new area of research, and provides an opporturtky for norei insights into the functions of both types of proteins. Undoubtedly. these novel insights will be highly relevant for urden*zGing of cell motility in vim, and tumor cell mrslstasis in viva.

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I