©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutation of the Cytoplasmic Domain of the Integrin Subunit
DIFFERENTIAL EFFECTS ON CELL SPREADING, RECRUITMENT TO ADHESION PLAQUES, ENDOCYTOSIS, AND PHAGOCYTOSIS (*)

Jari Ylänne (1)(§), Jarkko Huuskonen (1), Timothy E. O'Toole (3), Mark H. Ginsberg (3), Ismo Virtanen (2), Carl G. Gahmberg (1)

From the (1) Departments of Biochemistry and (2) Anatomy, University of Helsinki, Helsinki, Finland and the (3) Department of Vascular Biology, The Scripps Research Institute, La Jolla, California

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cytoplasmic domain of the subunit of the integrin is required for cell spreading on fibrinogen. Here we report that deletion of six amino acids from the COOH terminus of the (ITYRGT) totally abolished cell spreading and formation of adhesion plaques, whereas retaining Ilepartially preserved these functions. We further found that substitution of Tyrwith Ala also abolished -mediated cell spreading.

The effects of these and other mutations on additional functions of were also studied. Progressive truncations of , in which stop codons were inserted at amino acid positions 759-756, caused partial defects in the recruitment of to preestablished adhesion plaques and a gradual decrease in the ability of to mediate internalization of fibrinogen-coated particles. The Tyr Ala substitution mutant was almost totally inactive in both of these assays. Point mutations at Tyr, and at a conserved area close to the transmembrane domain of , decreased integrin recruitment to preestablished adhesion plaques but allowed -mediated formation of these structures and partial cell spreading. Deletion of the cytoplasmic domain of did not affect the constitutive endocytosis of .


INTRODUCTION

Integrins are transmembrane glycoproteins that mediate cell adhesion and migration by functionally linking extracellular structures to the cytoskeleton (see Refs. 1-5). Integrins can induce intracellular signals, which are needed for cytoskeletal rearrangements (see Ref. 6), for maintenance of anchorage-dependent cell growth (see Refs. 1 and 7) and for prevention of apoptosis (8, 9) . The signals require integrin clustering, which may generate signaling complexes of cytoskeletal proteins and regulatory proteins such as tyrosine kinases (see Refs. 10 and 11). In several adherent cells, adhesion plaques or focal contacts may represent integrin-induced signaling complexes (11) .

The cytoplasmic domains of integrin -subunits are highly conserved in all integrins that are linked to adhesion plaques (see Ref. 12). The cytoplasmic domains of and mediate interactions with the cytoskeletal proteins talin and -actinin, which are concentrated in adhesion plaques (13, 14, 15) . Two kinds of observations point out the critical role of the cytoplasmic domains of integrin -subunits in integrin function: their removal inhibits integrin-mediated cell adhesion (16) and cell spreading (17) , and their autonomous overexpression causes cell rounding by uncoupling integrin-induced signals from cell adhesion (18, 19) .

In the present work, we have used recombinant integrins expressed in CHO() cells to study which areas of the cytoplasmic domain of the subunit are needed for cell spreading and other functions of . This system allows us to study mutants with minimal interference by endogenously expressed integrins. It has been previously shown that no prior activation events are needed for -mediated cell adhesion to fibrinogen-coated surfaces (20) , although the binding of to soluble fibrinogen requires activation (21) . We show that an area of containing Tyrand Ileis essential for -mediated cell spreading. On the other hand, mutations at amino acid positions 727-733 or 759 caused defects in the recruitment of to preestablished adhesion plaques and in -mediated internalization of fibrinogen-coated particles but still allowed the formation of adhesion plaques and cell spreading. We also show that neither the cytoplasmic domain of the integrin subunit nor the NPLYsequence motif are needed for constitutive endocytosis of .


MATERIALS AND METHODS

Plasmid Constructs and Mutations

The pCDM8 (Invitrogen, San Diego, CA) derived plasmids containing human or integrin cDNAs or their mutants (996), (728), and (S752P) have been described previously (17, 22, 23, 24, 25, 26) . In some experiments, a pCDM8 construct, CN2b, was used containing and neomycin resistance genes (22) . Mutations were introduced into the plasmid by amplifying by PCR a segment of the insert with a common 5` primer (5`GGCTGGCTGGGATCCCAGTGTGAG3`) and a 3` primer containing the appropriate mutation. The mutant primers were as follows: 747, 5`-ATATCTCGAGTCATGGGTTGTTGGCTGTGTCCC-3`; 752, 5`-ATATCTCGAGTCACGTGGCCTCTTTATACAGTGG-3`; 755, 5`-ATATCTCGAGTCAG-AAGGTAGACGTGGCCTC-3`; 756, 5`-ATATCTCGAGTCAGGTGAAGGTAGACGTGGCC-3`; 757, 5`-ATATCTCGAGTCAATTGGTGAAGGTAGACG3`; 758, 5`-ATATCTCGAGTCAGATATTGGTGAAGGTAGACG-3`; 759, 5`-ATATCTCGAGTCACGTGATATTGGTGAAGGT-AG-3`; S752A, 5`-ATATCTCGAGTTAAGTGCCCCGGTACGTGATATTGGTGAAGGTAGCCGTGGCCTC-3`; and Y759A, 5`-ATATCTCGAGTTAAGTGCCCCGGGCCGTGATATTGG-3`. All of these primers contained a XhoI restriction site at their 3` end. Internal point mutations were generated by a two-step PCR reaction, where the common 5` primer and a mutant 3` primer were first used (mutant primers were (F727A/F730L/E733A): 5`-GCTCTGGCGCGTACTTCCTCAAGTTTAGCGGCTTCTT-3` and Y747A, 5`-GGCCTCTTTAGCCAGTGGGTTGTTGGC-3`). The PCR product was then purified and used as a 5` primer together with a 3` primer containing a XhoI site (5`-ATATCTCGAGGACATTCTCCCAACCTACCC-3`). The oligonucleotides were made with a PCR-mate 391 DNA synthesizer (Applied Biosystems, Foster City, CA). All PCR reactions were done by using the Dynazymethermostable DNA polymerase (Finnzymes, Espoo, Finland) and a Perkin-Elmer/Cetus DNA thermal cycler (Perkin-Elmer).

From the final PCR products, a MluI- XhoI fragment or an AflII- XhoI fragment (restriction enzymes were from New England Biolabs, Beverly, MA) was purified from an agarose gel by using the Geneclean II kit (Bio 101, La Jolla, CA) and cloned into the original vector. The PCR-derived parts of the mutant clones were verified by sequencing (T7 sequencing kit, Pharmacia Biotech). Supercoiled plasmid DNA was purified using CsCl (Pharmacia) gradient centrifugation (27) .

Cell Culture and Transfections

Chinese hamster ovary cells (CHO-K1, ATCC CCL 61) were from the American Type Culture Collection (Rockville, MD). They were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beth Haemek, Israel), 100 units/ml penicillin, 0.1 mg/ml streptomycin (Biological Industries), and nonessential amino acids (Life Technologies, Inc.). The expression vectors were transiently introduced into the cells by using the LipofectAMINE reagent (Life Technologies, Inc.). Briefly, 2 10cells were seeded on a 100-mm diameter cell culture dish (Falcon, Becton Dickinson UK, Plymouth, United Kingdom) 24 h before transfection. Two µg of and plasmids were incubated in 200 µl of nonsupplemented medium with 20 µl of LipofectAMINE for 10 min; the cells were washed twice with nonsupplemented medium, and the DNA-LipofectAMINE mixture was added to the cells in 3.8 ml of nonsupplemented medium. After incubation for 6 h, the medium was changed to 10 ml of normal cell culture medium, which was further changed 24 h after transfection. The cells were analyzed 48 h after the start of transfection. For stable transfections, either the LipofectAMINE method or a calcium-phosphate precipitation method was used (27) . Stable cell lines expressing the transfected integrins were selected in 0.75 mg/ml Geneticin(G418)-disulfate (Sigma), and further cloned until cell lines of similar expression levels were established. The stable cell lines expressing (728) and (S752P) have been characterized earlier (25, 26) .

For cell adhesion experiments, fibrinogen and fibronectin were purified from human plasma (28, 29) . Contaminating fibronectin was removed from the fibrinogen preparations by gelatin-Sepharose affinity chromatography. Round glass coverslips (13 mm in diameter, Menzel-Gläzer, Germany) or cell culture plastics (Falcon) were treated with 30 µg/ml of fibrinogen or 10 µg/ml of fibronectin in phosphate-buffered saline (PBS: 0.14 M NaCl 10 m M sodium phosphate, pH 7.4) overnight at +4 °C, followed by treatment with 1% bovine serum albumin (Sigma) in PBS for 1 h at 22 °C, and washed twice with PBS before the cells were seeded in serum-free medium. For phase contrast photographs or counting of spread cells (30) , the specimens were fixed with 2% paraformaldehyde in PBS for 10 min on ice. The proportions of spread cells to adherent cells were determined from triplicate samples by counting more than 150 cells/sample in a double-blind manner. For immunostaining, cells were fixed with 2% paraformaldehyde, containing 0.5% Triton X-100 (BDH Ltd., Poole, UK) in PBS for 10 min on ice.

Antibodies, Flow Cytometry, and Microscopy

The antibodies used in the study were as follows: monoclonal anti-PL98DF6 (31) , monoclonal anti-PL90BB10 (31) , monoclonal anti-fibrinogen (clone D1G10VL2, Immunotech, Marseille, France), polyclonal anti-fibronectin receptor (Telios Pharmaceuticals, La Jolla, CA), polyclonal anti-talin (32) , and polyclonal anti-vinculin (rabbit 901, 33). For flow cytometry and immunofluorescence microscopy, diluted polyclonal antisera or 10 µg/ml purified IgG of the monoclonal antibodies were used, followed by appropriate secondary antibodies (fluorescein isothiocyanate-coupled rabbit anti-mouse immunoglobulin (Dakopatts, Glostrup, Denmark), tetramethylrhodamine isothiocyanate-coupled goat anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA), or fluorescein isothiocyanate-coupled goat anti-rabbit IgG (Cappel, Organon Teknika, West Chester, PA)). For the immunofluorescence stainings, controls were routinely made that showed negligible nonspecific staining with the primary and secondary antibodies. Flow cytometry was done with a FACScaninstrument (Becton Dickinson Immunocytometry Systems, San Jose, CA). A Leiz Aristoplan fluorescence microscope (Leica Mikroskopie und Systeme, Wetzlar, Germany) was used with a plan Apochromat 63 oil immersion objective, and photographs were taken using Kodak Tmax 400 film (Eastman Kodak Co.).

The ability of the mutations to mediate recruitment of (996)to adhesion plaques (17) was scored, after transient transfections, by counting the proportion of (996)-expressing spread cells in which adhesion plaque-like immunolocalization could be revealed with the anti-monoclonal antibody. For this purpose, the cells were allowed to adhere on fibronectin for 2 h. Triplicate coded samples were counted, and the results of one representative experiment, out of at least three, are shown.

Endocytosis

Endocytosis of integrins was measured by a method modified from Bretscher (34) . Briefly, 2 10cells were cultured overnight on 35-mm diameter tissue culture dishes, washed twice with Dulbecco's modified PBS (Life Technologies, Inc.), and labeled for 1 h on ice with 1 ml of 0.2 mg/ml NHS-SS-biotin (Pierce) in Dulbecco's modified PBS. Labeled cells were washed twice and incubated in serum-free culture medium containing 10 m M HEPES (Sigma), either at 37 °C or on ice, for 15 min followed by washing with Dulbecco's modified PBS and three reductions of 5 min on ice with a solution containing 50 m M 2-mercaptoethanesulfonic acid (Sigma), 10 m M NaCl, 1 m M EDTA, 50 m M Tris, 0.2% bovine serum albumin, pH 8.6. The cells were lysed with 200 µl of 200 m M n-octyl-- D-glycopyranoside (Sigma), 1 m M phenylmethylsulfonyl fluoride (Sigma) in Dulbecco's modified PBS at +4 °C and centrifuged at 12,000 g, for 5 min. After this, an equal volume of 100 m M Tris, 150 m M NaCl, 1 m M CaCl, 1% Triton X-100, 0.1% SDS (BDH Ltd), 0.1% Nonidet P-40 (BDH Ltd), pH 7.4, was added. Preabsorption was done for 30 min at 4 °C by the addition of 25 µl of a 50% suspension of protein-G-Sepharose 4 fast flow (Pharmacia) and 1 µl of normal mouse serum to the lysate. The Sepharose was discarded, and 25 µl of protein-G-Sepharose, equilibrated with 2 µg of PL98DF6 IgG, was added to the lysate. After continuous rotation for 2 h at +4 °C, the supernatant was discarded, the Sepharose was washed 4 times, and bound proteins were eluted with 70 µl of electrophoresis sample buffer (35) without reducing agents. A 6.5% acrylamide slab gel was run and the proteins were transferred to Immobilon membrane (Millipore, Bedford, MA) using 25 m M Tris, 192 m M glycine, 20% methanol, pH 8.3 (36) . Biotin-labeled proteins were visualized using 2 µg/ml peroxidase-coupled streptavidin (Pierce) and a chemiluminescence Western blotting kit (Boehringer Mannheim).

Internalization of Fibrinogen-coated Beads

Polystyrene beads (5 10(4.5 µm), Polysciences, Inc, Warrington, PA) were coated for 20 h at 22 °C with 100 µg/ml of fibrinogen in 250 µl of 0.25 M NaBO, pH 9.5, followed by incubation for 20 h at 4 °C with 0.1% bovine serum albumin in PBS. 3 10cells were seeded in 400 µl of serum-free media on fibronectin-coated coverslips on 24-well cell culture plates for 2 h, and 5 10fibrinogen-coated beads were added to the wells. After further incubation for 2 h, the cells were washed with PBS, fixed with 4% paraformaldehyde in PBS, and stained simultaneously with PL98DF6 and monoclonal anti-fibrinogen followed by the fluorescein isothiocyanate-labeled secondary antibody. The proportion of cell bound nonfluorescent beads was determined using a fluorescence microscope. The samples were coded so that the examiners could not identify them. The results are shown as mean values from six different fields containing about 100 beads.


RESULTS

Cell Spreading

To study which residues of the cytoplasmic domain of are needed for -induced cell spreading, we constructed a series of point mutations and COOH-terminal deletions. The mutations used in this study are listed in . All of the mutants were expressed in CHO cells together with the subunit, which is not expressed at the CHO cell surface without transfected (17, 23) . Cells transiently transfected with and mutants were allowed to adhere to fibrinogen. All of the constructs were expressed at similar levels, but only some transfectants were able to promote cell spreading on fibrinogen (Fig. 1). Deletions at which the stop codon was inserted at or before Ileas well as the point mutations Y747A and S752P were not able to mediate cell spreading. Few partially spread cells were detected among these cells. The deletions 758 and 759 as well as the point mutations F727A/F730L/E733V, S752A, and Y759A were able to mediate cell spreading on fibrinogen. Double immunofluorescence studies by using anti-talin (Fig. 2, a, c, e, and g) or anti-vinkulin (not shown) and anti-(Fig. 2, b, d, f, and h) revealed that the wild-type (Fig. 2, a and b) and expressed together with the deletion mutations up to the 758 (Fig. 2, g and h) were able to form and localize to adhesion plaques in cells cultured on fibrinogen. However, in cells expressing the 757 (Fig. 2, e and f) or further deletion (747; Fig. 2, c and d), no such structures were found. Similarly, cells transiently expressing the point mutations Y747A (Fig. 3, b and c) or S752P (Fig. 3 d) did not reveal any adhesion-plaque like structures when cultured on fibrinogen. Even the few partially spread cells expressing these mutations (Y747A; Fig. 3 c) lacked adhesion plaques. However, those point mutations that were able to mediate clear cell spreading, were also found in adhesion plaques (Fig. 3, a, e, and f).


Figure 1: Ability of cells transiently transfected with and or its mutants to spread on fibrinogen. In each case, the percentage of -expressing cells, determined by flow cytometry analysis using anti-, is indicated. Phase contrast photomicrographs are shown from samples allowed to adhere on fibrinogen for 2 h. Note that the microscope was focused on the level of the substratum so that mostly only the adherent cells are seen. There were no or very few spread cells transfected with and the 757 deletions of or with the mutations Y747A or S752P.




Figure 2: Double immunofluorescence micrograph of CHO cells transiently transfected with and some COOH-terminal deletion mutants of . Cells were allowed to adhere on fibrinogen for 2 h and stained with anti-talin ( a, c, e, and g) and anti-( b, d, f, and h). Cells were transfected with wild-type ( a and b), or with the mutants 747 ( c and d), 757 ( e and f), or 758 ( g and h). Bar, 10 µm.




Figure 3: Immunofluorescence micrographs with transiently transfected cells expressing with the mutants F727A/F730L/E733V ( a), Y747A ( b and c), S752P ( d), S752A ( e), or Y759A ( f). All of the samples were stained with anti-. Note clear reactivity at adhesion plaques in a, e, and f, whereas in b, c, and d no adhesion plaques were detected either in round or partially spread cells. Bar, 10 µm.



For more detailed studies of cell spreading, cell lines stably expressing the various mutants with were established. When analyzed by flow cytometry, the expression levels of the different mutants were similar, only the S752P- and 728-bearing cells had somewhat higher expression levels of than the others (Fig. 4). By using the stable cell lines, we could verify the results obtained with the transiently transfected cells, indicating that deletion mutants up to the 757 and the point mutant Y747A were not able to mediate cell spreading. The mutants 759, 758, F727A/F729L/E733V, S752P, and Y759A were not as effective in mediating cell spreading as wild-type (Fig. 5). The stable cell line expressing the S752P mutant behaved slightly differently than transient transfectants with the same mutation. This was apparently due to the relatively high expression level of the transfected integrin. The S752P cell line was able to form small but well organized adhesion plaques on fibrinogen (26) . In control experiments, all of the cell lines spread equally well on fibronectin (not shown). Together, the experiments using transiently transfected cells and stably transfected cell lines indicated that a difference of one amino acid (Ile) in COOH-terminal deletion mutants or the single Y747A substitution of determined whether adhesion plaques were formed and the cells spread or not. On the other hand, the point mutation F727A/F729L/E733V and point and deletion mutations around Tyrcaused small defects in cell spreading, but still allowed the formation of adhesion plaques.


Figure 4: Flow cytometry analysis of stable cell lines expressing with wild-type or the mutants 728, 747, 756, 757, 758, 759, F727A/F730L/E733V, Y747A, S752A, S752P, or Y759A. In each case, fluorescence histograms with negative control antibody ( dashed line) or anti-( solid line) with fluorescein isothiocyanate-labeled secondary antibody are shown.



Recruitment to Adhesion Plaques

As there may be differences in the ability of the mutants to mediate de novo formation of adhesion plaques, and to be recruited to preestablished adhesion plaques, we studied the recruitment by utilizing the ability of truncated (996) to allow ligand-independent movement of to adhesion plaques (17) . In this assay, wild-type or the mutants are expressed transiently in CHO cells together with the (996) cells are cultured on fibronectin-coated surfaces, fixed, and stained for immunofluorescence with an anti-monoclonal antibody. The percentage of spread positive cells having an adhesion plaque-like localization of (996)was calculated. Fig. 6 shows that all of the mutants except S752A and Y759A were significantly defective in recruitment to adhesion plaques. A repeatable, but less significant defect was also detected in the case of the S752A and Y759A mutants. No correlation was found between the level of transient expression of the mutants and their ability to be recruited to adhesion plaques. This suggests that we had managed to mutate residues that are involved in integrin-cytoskeleton interactions at adhesion plaques.

Constitutive Endocytosis

The tyrosine residues at positions 747 and 759 of are located in putative internalization signal sequences (37, 38) . Because some integrins are known to be constitutively endocytosed and recycled (34, 39) , mutations deleting or altering these signals could also alter the endocytotic cycle of integrins. To study this, cell lines expressing wild-type or with the extensive COOH-terminal deletion of (728) were surface-labeled with NHS-SS-biotin at 0 °C, incubated either at 0 or 37 °C for 15 min, and exposed to a membrane-impermeable reducing agent. Immunoprecipitation followed by blotting with streptavidin-coupled peroxidase showed that in both cell lines 10-20% of became protected from reduction at 37 °C (Fig. 7 A), indicating that constitutive endocytosis of took place in CHO cells, regardless of the presence or absence of the cytoplasmic domain. The Y747A (Fig. 7 B) and Y759A mutants (not shown) were also found to be endocytosed similar to wild-type . Thus, neither the cytoplasmic domain of nor the sequences NPKYor NITYare needed for the constitutive endocytosis of in CHO cells.


Figure 7: Measurement of integrin endocytosis. Panel A, comparison of wild-type and (728); panel B, comparison of wild-type and (Y747A). Sample 1, surface labeling with NHS-SS-biotin without reduction. Indicated amounts, given as percentage of duplicate samples, were pipetted on the gel to facilitate quantitation; sample 2, surface labeling, 15-min incubation at 0 °C, and reduction with 2-mercaptoethanesulfonic acid; sample 3, surface labeling, 15-min incubation at 37 °C, and reduction with 2-mercaptoethanesulfonic acid. Under each lane, corresponding results of densitometric scanning of the subunit band is shown ( OD, optical density, arbitrary units). In A, comparison of the sample 3 bands to the standards of sample 1 indicated that 17% of the wild-type and 13% of the 728 mutant were detected (reduction controls in samples 2 subtracted). In B, samples 3 constituted 12% of the wild-type and 18% of the Y747A mutant.



Phagocytosis of Ligand-coated Particles

Certain integrins participate in phagocytosis (40, 41) or in the internalization of particles coated with their ligands (42, 43) . To study the effects of the cytoplasmic domain mutations in -mediated particle internalization, fibrinogen-coated polystyrene beads were allowed to settle on the cells, and internalized beads were counted by using immunolabeling techniques. CHO cells expressing wild-type , or the mutants S752A and Y759A internalized 60-80% of the beads in 2 h, whereas a significantly smaller intake was detected in cells expressing other mutations (Fig. 8). Control experiments showed that the beads bound poorly to parental CHO cells, and that less than 1% of the bound beads were taken into untransfected CHO cells under these conditions. The results show that the subunit cytoplasmic domain is necessary for -mediated internalization of fibrinogen-coated particles, and that the same mutations that cause defects in recruitment to adhesion plaques also cause defects in particle internalization.


DISCUSSION

The major findings of this study are as follows.

1) By using COOH-terminal deletions we could determine that the sequence up to Ileof was necessary for integrin-induced cell spreading and formation of adhesion plaques.

2) The Y747A substitution abolished -mediated cell spreading.

3) Residues Phe-Gluand Tyrdefine areas involved in integrin-adhesion plaque interactions so that mutations at these positions slow down cell spreading, but still allow formation of adhesion plaques.

4) The cytoplasmic domain of is not needed for the constitutive endocytosis of in CHO cells.

5) The cytoplasmic domain of is needed for -mediated internalization of ligand-coated particles, and the same mutations that affect recruitment to adhesion plaques also affect internalization.

The results are summarized in . Fig. 9 summarizes our current thinking about the functional roles of the different parts of the cytoplasmic domain of the integrin subunit.

The ability of our mutations to be recruited to preestablished adhesion plaques largely correlated to what has been previously found by using corresponding mutation in the integrin subunit (44) . However, functional differences of COOH-terminal deletion mutations of showed that the cytoplasmic domain up to Ileis specifically needed for cell spreading. The mutant, in which Ilewas the first of the deleted amino acids, was unable to mediate -dependent formation of adhesion plaques and cell spreading, but retained partial activity in recruiting the integrin to preestablished adhesion plaques and in mediating internalization of ligand-coated particles. Although Ileof is not conserved in other integrins, the sequences around this position are homologous (VTTVVNPKYin , TTTVMNPKFin , and TSTFTNITYin ). Which residues of this stretch are critical for cell spreading has not been studied in the case of other integrins. In point mutation studies of and , this conserved stretch has, however, been implicated for cytoskeletal interactions (44) and integrin activation (45) . Comparison of the different studies is difficult, due to different experimental conditions. It is, however, possible that the area around Ileof could be part of a binding site for some signal-transducing proteins, which are necessary for the formation of adhesion plaques. Such signal-transducing proteins (7) may be scarce at adhesion plaques, and, thus, the addition or removal of the binding site would not increase integrin recruitment to preestablished structures. Actually, COOH-terminal deletions ending at amino acid positions 755-758 were all similarly active in the recruitment. Mutations at Proin , which corresponds to Ileof , have no effect of recruitment either (44) . In addition to amino acid homology, interchangeable functions of the cytoplasmic domains of some integrin -subunits (46) favor the hypothesis of a common signaling function for this area.

We also mutated Ser, which is located at the conserved stretch discussed above. A serine to proline substitution at this position has been found in a patient with Glanzmann thrombasthenia (47) . This mutation makes incapable to be activated by intracellular signals (26) and decreases its capacity to mediate cell spreading (this study and Ref. 26). Here we showed that a serine to alanine mutation at this position had only minimal effects on the recruitment of to adhesion plaques and had no detectable effects on -mediated cell spreading. Previously, substitution of the homologous sequence in , TTT, with AAA, has been shown to cause a major decrease in leukocyte adhesion to ICAM-1, whereas Thr Ala mutation in had only a a minor effect (45) . It is possible that this area is involved in both integrin activation and in integrin-cytoskeletal interactions in and integrins.

The mutation Y747A disrupted all of the functions dependent on the COOH-terminal part of the cytoplasmic domain. The same defects were observed in the COOH-terminal deletion mutants where a stop-codon was inserted before or at position 755. Thus, the simplest explanation for the effects of the Y747A mutation is that it disrupts the folding of the COOH terminus of . Sequences such as NPIYof make a -turn in several proteins (37) , and a tyrosine to alanine substitution in this motif may disrupt this turn. A similar explanation has been suggested in the case of the corresponding mutation in (12) .

As far as no physical data are available on the three-dimensional structure of the cytoplasmic domains of integrin subunits, we must also consider other than conformational hypotheses for the functional effects of the Y747A mutation. There is evidence that talin-integrin interaction is inhibited by phosphorylation of the corresponding tyrosine in the subunit (13, 48) . Tyrosine to phenylalanine substitution has, however, no effect to the recruitment of the subunit to adhesion plaques (44) . A synthetic peptide from this area inhibits integrin-talin interaction (13) . If this is the case also in , our results imply that the cytoskeletal interactions mediated by the region around Tyrwould be absolutely required for integrin-mediated formation of adhesion plaques. This is further supported by findings that cytochalasin D inhibits integrin-mediated signals (49) .

We found that point mutations at Phe-Gluand Tyrand COOH-terminal deletions ending at Ileor Tslowed down -mediated cell spreading on fibrinogen but were able to support formation of adhesion plaques. Furthermore, we found that these mutants were defective in integrin recruitment to preestablished adhesion plaques. Thus, these mutations abrogated some, but not all, -cytoskeletal interactions. They could still perform functions that need sequences around Tyror before and at Ile(that is, the organization of adhesion plaques). This suggests that the mutations abrogate binding sites for some proteins at adhesion plaques. The area defined by Phe-Glumay be an interaction site of -actinin, because peptides from the corresponding region of have been found to bind to this protein (15) .

Integrins are known to participate in phagocytosis or in internalization of particles coated with ligands. The major complement iC3b receptor in leukocytes is (CD11b/CD18) (see Refs. 2 and 40). This integrin binds to complement-coated particles and cells, and mediates their phagocytosis. Some pathogens also directly use integrins for entering the cells (43, 50) ( e.g. enteropathogenic Yersinia strains, which bind to integrins through their surface protein invasin (42) ). It has been proposed that for effective invasion of bacteria, the bacterial protein-integrin interaction must be of sufficiently high affinity and that the host cells must be able to undertake extensive reorganization of the cytoskeleton induced by ligand binding to integrins (51) . Our results support this by showing that similar determinants of the cytoplasmic domain of the integrins are needed for internalization of ligand-coated particles and for integrin recruitment to adhesion plaques.

In contrast to integrin-mediated internalization of ligand-coated particles, the constitutive intake of was not found to be dependent on the cytoplasmic domain of . Several integrins are internalized via the endocytotic pathway and are rapidly recycled back to the cell surface (34, 39) . Different integrins seem to have different abilities to take part in the endocytotic cycle. For example, , , and (CD11b/CD18) were found to circulate in all cells studied, whereas , , and (CD11a/CD18) did not circulate in any cells (39) . Typically, circulating and noncirculating integrins have circulating rates of 1-2% and less than 0.1% of surface protein/min, respectively (39) . The size of the intracellular pool is 8-20% for circulating integrins but less than 1.3% for noncirculating integrins (39) . We showed here that 10-20% of cell surface-labeled was internalized in 15 min in spite of the large COOH-terminal deletion of or the mutations Y747A and Y759A. These values clearly fall in the range expected for circulating integrins.

The present identification of important residues required for integrin-mediated cell spreading should be helpful in resolving the detailed mechanisms of integrin function and transmission of integrin-induced signals. Our results imply that factors that are absolutely required for integrin-mediated cell spreading bind to the 758 deletion mutant of , but do not bind either to the 757 deletion mutant or the Y747A substitution mutant. It is possible that binding of these putative factors to integrins represent early, still unidentified steps in the post-ligand binding events of integrin function.

  
Table: Mutations used in this study


  
Table: Summary of the results

The data in the table have been presented in Figs. 2-8. ND, not determined.



FOOTNOTES

*
This study was supported in part by grants from the Academy of Finland (to J. Y., C. G. G., and I. V.), the Finnish Cancer Society (to C. G. G.), the Sigrid Jusélius Foundation (to C. G. G.), the Finnish Society of Science and Letters (to J. Y.), the Ella and Georg Ehrnrooth Foundation (to J. Y.), the Walter and Lisi Wahl Foundation (to J. Y.), and by National Institutes of Health Grants HL48728 and HL28235 (to M. H. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Postdoctoral fellow of the Arthritis Foundation during these studies. To whom correspondence should be addressed: Div. of Biochemistry, Dept. of Biosciences, P. O. Box 5 (Unioninkatu 35), 00014 University of Helsinki, Finland. Tel.: 358-191-7793; Fax: 358-191-7769; E-mail: Jari.Ylanne@csc.fi.

The abbreviations used are: CHO, Chinese hamster ovary; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Aili Grundström and Pipsa Kaipainen for technical help, Dr. Matti Korhonen for helpful discussions, and Dr. Keith Burridge (Chappel Hill, NC) for providing anti-talin antiserum.


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