CD151, CD81 and (Alpha)3(Beta)1 Integrin Function in Neurite Outgrowth
Total Page：16
File Type：pdf, Size：1020Kb
Journal of Cell Science 113, 1871-1882 (2000) 1871 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1409 Transmembrane-4-superfamily proteins CD151 and CD81 associate with α3β1 integrin, and selectively contribute to α3β1-dependent neurite outgrowth Christopher S. Stipp and Martin E. Hemler* Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, MA 02115, USA *Author for correspondence (e-mail: [email protected]) Accepted 22 March; published on WWW 10 May 2000 SUMMARY Proteins in the transmembrane-4-superfamily (TM4SF) number, length, and rate of extension were all affected by form many different complexes with proteins in the anti-TM4SF antibodies. In summary: (1) these substrate- integrin family, but the functional utility of these complexes dependent inhibition results strongly suggest that CD151 has not yet been demonstrated. Here we show that TM4SF and CD81 associations with α3β1 are functionally relevant, proteins CD151, CD81, and CD63 co-distribute with α3β1 (2) TM4SF proteins CD151 and CD81 make a strong integrin on neurites and growth cones of human NT2N positive contribution toward neurite number, length, and cells. Also, stable CD151-α3β1 and CD81-α3β1 complexes rate of outgrowth, and (3) NT2N cells, a well-established were recovered in NT2N detergent lysates. Total NT2N model of immature central nervous system neurons, can be neurite outgrowth on laminin-5 (a ligand for α3β1 integrin) a powerful system for studies of integrin function in neurite was strongly inhibited by anti-CD151 and -CD81 outgrowth and growth cone motility. antibodies either together (~85% inhibition) or alone (~45% inhibition). Notably, these antibodies had no inhibitory effect on NT2N neurites formed on laminin-1 or Key words: Integrin, Transmembrane-4-superfamily, Neuron, fibronectin, when α3β1 integrin was not engaged. Neurite Neurite, Laminin-5 INTRODUCTION example, α3β1-CD151 association is selectively maintained in 1% Triton X-100; additional interactions of α3β1 and α6β1 TM4SF proteins (also called tetraspanins) are present at with TM4SF proteins are selectively seen in 1% Brij-96 reasonably high levels on nearly every mammalian cell detergent; and many other integrin-TM4SF interactions are and tissue type. There are at least 21 core family members seen only in less stringent detergent conditions (e.g. 1% (Hemler et al., 1996; Maecker et al., 1997; Todd et al., 1998; CHAPS). Wright and Tomlinson, 1994), each containing 4 presumed The role of integrins during cell adhesion and motility is well transmembrane domains, short N- and C-terminal cytoplasmic established (Huttenlocher et al., 1995; Sheetz et al., 1998). domains (containing 5-14 amino acids), a small intracellular TM4SF proteins likewise regulate cell motility, although they loop (typically 4 aa), and two extracellular loops (20-27 aa; 75- generally do not participate in cell adhesion (Hemler, 1998; 130 aa). Maecker et al., 1997). Thus it seems likely that integrin- Like TM4SF proteins, proteins in the integrin family of cell TM4SF protein complexes might play a role in cell motility. adhesion receptors are widespread on many cell types (Hynes Indeed, sometimes in the same experiment, antibodies to both and Lander, 1992). Notably, the α2β1, α3β1, α4β1, α4β7, integrins and TM4SF proteins (CD81, CD151, CD9, CD63) α5β1, α6β1, α6β4, αIIbβ3, and αLβ2 integrins have all been inhibited cell motility (Sincock et al., 1999; Yanez-Mo et al., suggested to associate with various TM4SF proteins including 1998; Yauch et al., 1998). However, in those studies the issue CD9, CD63, CD81, CD82, and/or CD151 (Berditchevski et al., of integrin substrate specificity was not addressed, and it was 1995; Fitter et al., 1999; Hadjiargyrou et al., 1996; Hasegawa not clear the extent to which the integrin-TM4SF proteins et al., 1998; Hemler, 1998; Hemler et al., 1996; Jones et al., complexes needed to be actively engaged on integrin ligand. In 1996; Nakamura et al., 1995; Rubinstein et al., 1994; Schmidt fact, another study has specifically demonstrated that α6Aβ1 et al., 1996; Sincock et al., 1999; Skubitz et al., 1996; Yanez- integrin can induce CD81-dependent motility without Mo et al., 1998; Yauch et al., 1998). However, detection of obviously engaging the integrin substrate (Domanico et al., integrin-TM4SF protein interactions varies considerably 1997). depending on detergent conditions (Berditchevski et al., 1996; To address whether specific α3β1-TM4SF complexes might Serru et al., 1999; Sincock et al., 1999; Yauch et al., 1998). For be functionally relevant, we have chosen to study NT2N cells. 1872 C. S. Stipp and M. E. Hemler NT2N cells are neuron-like cells derived from the human NT2 integrin cytoplasmic domain (Takada and Hemler, 1989); an embryonic carcinoma cell line (Pleasure et al., 1992). These antiserum raised against intact, denatured β4 integrin (unpublished), cells express many integrins and TM4SF proteins for which and horseradish peroxidase (HRP)-conjugated goat-anti-mouse and antibodies are available, and thus had a strong potential to goat anti-rabbit antibodies (Sigma). provide insight into the roles of TM4SF proteins in neurite Cell culture outgrowth and growth cone motility. In addition, NT2N α β NT2 precursor cells (Stratagene) were maintained in OPTI-MEM neurites grew on many different substrates, including the 3 1 (Gibco-BRL) with 10% fetal bovine serum, penicillin-streptomycin, ligand laminin-5, thus allowing an evaluation of the substrate- and 2 mM glutamine. NT2N neuron-like cells were obtained as dependent inhibitory effects of anti-TM4SF antibodies. described by Pleasure et al. (1992). Briefly, NT2 precursor cells were The roles of TM4SF proteins themselves, or of integrin- treated with 10 µm retinoic acid for 4-5 weeks then split 1:6 into TM4SF complexes during neurite outgrowth have not been medium with mitotic inhibitors and cultured an additional 10-14 days. studied, except for a few reports involving the CD9 protein. In NT2N cells, growing as clusters over a monolayer of non-neuronal one case, CD9 associated with integrin α6β1 under conditions cells, were isolated by rinsing with phosphate-buffered saline (PBS) of relatively low detergent stringency, and anti-CD9 antibody and treating for 2-3 minutes with trypsin/EDTA diluted 30-fold in stimulated neurite outgrowth of small cerebellar neurons on PBS. Purified neurons were maintained on plates coated with Matrigel (Becton Dickinson) diluted 1:50. laminin-1, but not poly-L-lysine (Schmidt et al., 1996). α β Since the exact number of clustered NT2N cells in a culture is However, the functional role of 6 1 was not elucidated. In difficult to determine, NT2N cell purity was estimated as follows: another study, immobilized anti-CD9 antibody supported morphologically non-neuronal cells in 20 random fields were counted, neurite outgrowth from sympathetic neurons, which was partly and the amount of detergent extractable protein contributed by these inhibited by anti-α3β1 mAb (Banerjee et al., 1997). However, cells was calculated using a value of protein/cell obtained from lysates because anti-α3 mAb also inhibited neurite outgrowth on of known numbers of NT2 precursor cells. The protein derived from immobilized anti-Thy1 mAb and on collagen (Banerjee et al., non-neuronal cells was then expressed as a fraction of the total protein 1997), the specific functional involvement of α3β1-CD9 obtained from the NT2N cell lysates, and found to be less than 1% of complexes was not clear. Thus far, antibodies to TM4SF the total. For neurite outgrowth and staining experiments, NT2N cells were proteins had not been shown to inhibit neurite outgrowth. rinsed with PBS and treated with trypsin/EDTA diluted 10-fold in Here we show for the first time strong inhibition of neurite PBS. Cells were dislodged, and trypsin treatment was stopped by outgrowth by antibodies to TM4SF proteins (in this case, adding DME with 5 mg/ml crystalline bovine serum albumin (BSA) CD151 and CD81). Furthermore, we show inhibition by anti- (ICN) and 0.2 mg/ml soybean trypsin inhibitor (Sigma). Cells were TM4SF antibodies that depends on functional engagement of collected by centrifugation, resuspended in a serum-free medium TM4SF-associated α3β1 integrin with its laminin-5 ligand. consisting of DMEM with B27 additives (Brewer et al., 1993), (Gibco Such integrin-substrate dependence had not been previously BRL) and 1 mM glutamine, and plated to substrate-coated coverslips. demonstrated in studies of integrin-TM4SF complexes on Preparation of coverslips; video-microscopy neurons or other cell types. Together these results strongly Acid-washed glass coverslips were coated with 20 µg/ml mouse suggest that integrin-TM4SF complexes can act as a functional laminin-1 (Gibco BRL), diluted in PBS, with 2 µg/ml rat laminin-5 unit, dependent on integrin engagement with ligand, and of (a gift of Desmos Inc.) diluted in PBS with 0.005% Tween-20, or with critical importance during neurite outgrowth. During the 20 µg/ml fibronectin (Becton-Dickinson). Substrates were coated course of these studies we also show that NT2N cell either for 1 hour at 37°C or overnight at 4°C. Coverslips were rinsed differentiation is accompanied by pronounced changes in 3 times in PBS and blocked with 10 mg/ml heat-inactivated BSA for integrin and TM4SF protein expression, and that NT2/NT2N 1 hour at 37°C. Coverslips were rinsed three times and used for cells are an excellent system for studying human neurite experiments described below. differentiation and outgrowth. Video images were acquired using a Zeiss Axiovert 135 microscope equipped with (1) a VS25 electronic shutter driven by a Uniblitz D122 controller (Vincent Associates) and (2) a black and white video camera (TM-7AS, PULNiX America Inc.) connected via a focusing MATERIALS AND METHODS monitor (PVM-137, Sony Corp.) to a Power Macintosh 6500 computer containing a VG-5 frame grabber (Scion Corp.). Image Antibodies acquisition was performed with the program Scion Image 1.60 (Scion Anti-integrin mAbs used were anti-α1, TS2/7 (Hemler et al., 1984), Corp.).