©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Conserved Membrane-proximal Region of an Integrin Cytoplasmic Domain Specifies Ligand Binding Affinity (*)

(Received for publication, January 23, 1995; and in revised form, March 24, 1995 )

Paul E. Hughes (§) Timothy E. O'Toole (¶) Jari Ylänne (1) Sanford J. Shattil (2) Mark H. Ginsberg (**)

From the  (1) Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, the Department of Biochemistry, University of Helsinki, Helsinki 00170, Finland, and the (2) Department of Cell and Developmental Biology and Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Integrin affinities for ligands can change markedly via a process termed inside-out signaling. We expressed several truncations of the beta(3) cytoplasmic domain in conjunction with an ``activating'' alpha subunit chimera, alphaalpha. Deletion of the 4 C-terminal residues of the beta(3) tail blocked inside-out signaling as assessed by the binding of an activation-specific antibody, PAC1. Several additional truncations remained in the low affinity state, but complete truncation (beta(3)Delta717) caused PAC1 binding. Activation by this truncation mutant did not depend on the alpha subunit cytoplasmic domain and was resistant to inhibitors of cellular metabolism and the over-expression of an isolated beta(3) cytoplasmic domain. Since deletion of beta(3)(Leu-Asp) results in a constitutively activated integrin, this membrane-proximal seven amino acids of the beta(3) cytoplasmic domain is required to maintain alphabeta(3) in a default low affinity state. The amino acid sequence of this region is conserved among integrins. Moreover, the conserved membrane-proximal sequence in alpha subunit tails seems to serve a similar function. Consequently, the conserved membrane-proximal regions of both integrin cytoplasmic domains control the ligand binding affinity of the extracellular domain.


INTRODUCTION

Changes in cell adhesion are often mediated via integrins, a heterodimeric family of cell adhesion receptors composed of two type I transmembrane subunits, alpha and beta (1) . An important feature of integrins is their ability to modulate their affinity for extracellular ligands in response to developmental and environmental cues, a process termed inside-out signaling (2) . Affinity modulation seems to be a general property of integrins, being found in beta(3)(3) , beta(2)(4) , beta(7)(5) , and beta(1) integrins (6) . For example, in order for platelets to aggregate, the integrin alphabeta(3) must bind fibrinogen (7) , which requires prior platelet ``activation.'' Conformational changes in the extracellular domain of alphabeta(3) seem to be responsible for regulation of its affinity (8, 9) . Platelet agonists increase the affinity of alphabeta(3) (activation) via cytoplasmic signaling pathways. These pathways include heterotrimeric GTP-binding proteins, phospholipid metabolism, and serine-threonine kinases and may also involve calcium fluxes, tyrosine kinases, and low molecular weight GTP-binding proteins (2, 9, 10, 11, 12, 13) . How cytoplasmic signals result in changes in the conformation and ligand binding affinity of the extracellular domain (``inside-out signal transduction'') of the integrin remains obscure.

Integrin alpha and beta cytoplasmic domains play key roles in inside-out signaling (2, 14, 15, 16, 17, 18) . As summarized in Fig. 1, chimeras composed of the cytoplasmic domains of various alpha subunits fused to the transmembrane and extracellular domains of alphabeta(3) established that the alpha subunit cytoplasmic domains mediate cell type-specific inside-out signaling (19). Inside-out signaling is an active cellular process as high affinity ligand binding to these chimeras and to alpha(5)beta(1) was reduced by inhibitors of oxidative phosphorylation (NaN(3)) and anaerobic glycolysis (2-deoxyglucose) (19) . The beta cytoplasmic domain is important in this process because partial deletion of the beta(3) cytoplasmic domain disrupted the high affinity state of the chimeric receptors (19) (Fig. 1) and a naturally occurring point mutation, beta(3) S752P, disrupts activation of alphabeta(3)(20, 21) . Furthermore, overexpression of isolated beta(3) or beta(1) cytoplasmic domains can inhibit inside-out signaling (22) (Fig. 1). Thus, physiological inside-out integrin signaling is an energy-dependent process, with cell type-specific signaling machinery operating through both alpha and beta integrin cytoplasmic domains to induce changes in the ligand binding affinity of the extracellular domain.


Figure 1: Control of integrin affinity via the cytoplasmic domains. This schematic summarizes previous studies (19, 22, 26) analyzing the role of integrin cytoplasmic domains in inside-out signaling in CHO cells. The nature of the alpha cytoplasmic domain joined to the transmembrane and extracellular domain of alpha is shown schematically with a shaded box indicating the conserved GFFKR motif. Wild-type alphabeta(3) is in the low affinity state (dash). In contrast, a chimera with the cytoplasmic domain of alpha is activated as indicated by the star. The chimera's active state requires the beta(3) cytoplasmic domain because the chimera is not active when co-expressed with beta(3)Delta724 (see sequence in Fig. 2). Cellular signaling events are involved in the activation of this chimera because it is in the low affinity state in the presence of a combination of inhibitors of oxidative phosphorylation and anaerobic glycolysis (ATP) or over-expressed isolated beta(3) or beta(1) cytoplasmic domains (Free beta). Deletion of the GFFKR motif in alphaDelta991 or alpha(L)Delta also induces the high affinity state. In contrast to the chimeras, high affinity ligand binding to these constructs is not blocked by beta truncation, reduction of ATP, or over-expressed free beta cytoplasmic domains.



The membrane-proximal regions of both alpha and beta cytoplasmic domains are highly conserved across integrin families (23) . The putative membrane-cytoplasm interface of both the integrin alpha and beta subunits is defined by a conserved Lys residue. In both the alpha and beta subunits this Lys is immediately followed by a short (4-6 residue) apolar segment and then a charged region (23) . The corresponding conserved sequences for the alpha and beta subunits are XGFFKR and LLviXhDR (less conserved amino acids are in the lower case, X indicates a non-conserved amino acid). As summarized in Fig. 1, mutations that eliminate the highly conserved membrane-proximal GFFKR motif in the alpha subunit cytoplasmic domain increase ligand binding affinity, i.e. ``activate'' alphabeta(3)(19) . In contrast to the aforementioned chimeras, the high affinity state of these deletion mutants is independent of the cell type, cellular metabolism, and the majority of the beta subunit cytoplasmic domain (19) (Fig. 1). In addition, ligand binding affinity of these deletion mutants is not reduced by over-expressed isolated cytoplasmic domains (22) . Consequently, the activation of these mutants appears to be independent of cytoplasmic signaling pathways, and the GFFKR motif is required to maintain a low affinity state in the absence of physiological activation.

To examine the role of the beta(3) cytoplasmic domain in affinity modulation, we have expressed a series of truncation mutants in Chinese hamster ovary (CHO)^1(^1) cells in conjunction with alpha or an ``activating'' alpha subunit chimera, alphaalpha. We found that the deletion of a conserved membrane-proximal region of the beta(3) cytoplasmic domain results in constitutive high affinity ligand binding that is independent of the usual cellular signaling pathways and the sequence of alpha subunit cytoplasmic domain. Thus, the conserved membrane-proximal portion of the beta subunit, like that of the alpha subunit, maintains the default low affinity state of alphabeta(3).


MATERIALS AND METHODS

Antibodies and Proteins

The anti-alpha beta(3) antibodies D57 (19) , anti-LIBS6 (24) , and PAC1 (25) have been described previously. The antibody to the C terminus of the beta(3) cytoplasmic domain has also been described (26) . The D57 antibody was biotinylated with biotin-N-hydroxy-succinimide (Sigma) according to manufacturer's directions. Fibrinogen was purified as described (8) and fluoresceinated to an F/P ratio of 1.01 with fluorescein isothiocyanate-cellite (Calbiochem) according to the manufacturer's instructions.

cDNA Constructs

The CDM8 expression constructs encoding alpha, alphaDelta991, alphaDelta996, alphaalpha(L)Delta, alphaalpha, alphaalpha(5), beta(3), and beta(3) Delta724 were constructed as described (19) . The beta(3) truncation, Delta717, was made by splice overlap PCR mutagenesis (27) . A 0.9-kb MluI and PstI cut PCR fragment encompassing the mutations was first digested with MluI and PstI and then cloned into MluI and PstI cut pCDM8. This construct was then cut with AflII and DraIII and the 3-kb fragment ligated with the 3.5-kb AflII-DraIII fragment of CD 3a (28) containing the beta(3) extracellular domains. The beta(3) truncations Delta747, Delta752, Delta755, and Delta759 were constructed by PCR using a common 5` primer and a 3` primer containing a stop codon and a XhoI restriction site. The PCR product was cut with XhoI and MluI and cloned into MluI-XhoI cut CDM8. This vector was then digested with MluI and ligated to the 2.4-kb MluI fragment of CD3a encoding the beta(3) extracellular domain. All constructs were verified by DNA sequencing and purified by CsCl centrifugation before transfection. Oligonucleotides were synthesized on a model 391 DNA synthesizer (Applied Biosystems Inc, Foster City, CA).

Cell Culture and Transfection

Chinese hamster ovary (CHO-K1) cells were obtained from American Type Culture Collection (Rockville, MD). They were grown in Dulbecco's modified Eagle's medium (DMEM) (BioWhittaker, Walkersville, MD) containing 10% fetal bovine serum, 1% non-essential amino acids, 2 mM glutamine (Sigma), and 100 units/ml penicillin and 100 µg/ml streptomycin. One day prior to transfection, 10^6 cells were plated in 100-mm culture dishes. Two µg of each integrin alpha and beta cDNA were mixed with 20 µl of Lipofectamine (Life Technologies Inc.) and made up to a final volume of 200 µl with DMEM. After 10 min at room temperature, the mixture was added to the cells in the 100-mm tissue culture dish followed by addition of 3.8 ml of DMEM. The cells were washed once with complete medium. The cells were then grown in complete medium that was changed after 24 h. In the experiments involving the co-expression of Tac-beta(3), the transfection protocol described in Ref. 22 was used. Cells were routinely analyzed 48 h after transfection. In indicated experiments, clonal stable cell lines were produced as described (8) .

Flow Cytometry

Surface expression of integrins was analyzed by flow cytometry with specific antibodies as described (8) . Briefly, 5 times 10^5 cells were incubated on ice for 30 min with primary antibody, washed, and then incubated on ice for 30 min with an fluorescein isothiocyanate-conjugated goat anti-mouse (Tago, Burlingame, CA) secondary antibody. After washing, fluorescent staining was analyzed on a FACScan (Becton Dickinson, Mountain View, Ca) flow cytometer. PAC1 binding was analyzed by two color flow cytometry. Cell staining was carried out in Tyrode's (8) buffer containing 2 mM MgCl(2) and CaCl(2) and 1 mg/ml bovine serum albumin (Sigma) and dextrose. Single cell suspensions were obtained by harvesting with 3.5 mM EDTA, incubating for 5 min in 1 mg/ml L-1-tosyl-amido-2-phenylethyl chloromethyl ketone trypsin (Worthington) and diluting with an equal volume of Tyrode's containing 10% fetal calf serum and 0.1% soybean trypsin inhibitor (Sigma). After washing, 5 times 10^5 cells were incubated in a final volume of 50 µl containing 0.1% PAC1 ascites in the presence or absence of competitive inhibitors of PAC1 binding (either 2 mM peptide GRGDSP or 1 µM peptide mimetic Ro43-5054 (29) , a generous gift of Beat Steiner, F. Hoffmann, La Roche, Basel). After a 30-min incubation at room temperature, cells were washed with cold Tyrode's solution and then incubated on ice with Tyrode's containing a biotinylated anti-alphabeta(3)-specific antibody D57. After 30 min on ice, the cells were washed and incubated with 10% fluorescein isothiocyanate-conjugated goat anti-mouse IgM (TAGO) and 4% phycoerythrin-streptavidin (Molecular Probes Inc, Junction City, OR). Thirty min later cells were diluted to 0.5 ml with Tyrode's solution and analyzed on a FACScan as described (19) . PAC1 binding (fluoescein isothiocyanate staining) was analyzed only on a gated subset of cells positive for alphabeta(3) expression (phycoerythrin staining). To define affinity state, histograms depicting PAC1 staining in the absence or presence of competitive inhibitor were superimposed. A rightward shift in the histogram in the absence of inhibitor is indicative of the presence of high affinity alpha beta(3). To obtain numerical estimates of integrin activation, we calculated an activation index (AI) defined as 100 times (F - F )/(F LIBS6 - F LIBS6), where F is the median fluorescence intensity of PAC1 binding; F median fluorescence intensity of PAC1 binding in the presence of competitive inhibitor; F LIBS6 is the median fluorescence intensity of PAC1 binding in the presence of 2 µM anti-LIBS 6; and F LIBS6 is the median fluorescence intensity of PAC1 binding in the presence of 2 µM anti-LIBS 6 and competitive inhibitor.

In experiments using metabolic inhibitors, the cells were first incubated with glucose-free Tyrode's containing 2 mg/ml 2-deoxyglucose and 0.1% sodium azide for 30 min at room temperature prior to the addition of PAC1. After washing, 5 times 10^5 cells were incubated in a final volume of 50 µl containing 0.1% PAC1 ascites in the presence or absence of 1 µM Ro43-5054 in glucose-free Tyrode's containing 2 mg/ml 2-deoxyglucose and 0.1% sodium azide.

Immunoprecipitation and Western Blotting

Transfectants were surfaced labeled with 1 mM Sulfo-Biotin (Pierce) at room temperature according to the manufacturer's instructions. The reaction was then stopped by the addition of 50 mM Tris-HCl, pH 7.4. The biotin-labeled cells were solubilized in lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 10 mM benzamidine HCl, 2 mM phenylmethylsulfonyl fluoride, 0.1% soybean trypsin inhibitor, 1% Triton X-100, 0.5% Tween 20, 0.02% sodium azide, 5 units/ml aprotinin (Sigma)). Lysates were immunoprecipitated as described (8) , and immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (non-reducing, 7.5% acrylamide gels). The protein was transferred onto Immobilon membranes (Millipore Inc.) and stained with primary antibodies followed by Vectastain peroxidase reagents (Vector Laboratories) and developed with the ECL system (Amersham Corp.) according to the manufacturer's instructions.


RESULTS

Deletion of a Conserved Membrane-proximal Seven Amino Acids of the beta(3) Subunit Activates alphabeta(3)

In order to examine the beta(3) cytoplasmic domain's role in inside-out signal transduction, we transiently expressed a series of truncation mutants in CHO cells with alpha constructs. When expressed in CHO cells, wild-type alphabeta(3) is in the low affinity state (26) . In contrast, chimeras of the extracellular and transmembrane domains of alpha joined to the cytoplasmic domains of certain other alpha subunits (e.g. alpha(5), alpha, alpha, or alpha(2)) bind soluble ligands with high affinity. For an ``activated'' integrin, we arbitrarily chose a chimera comprised of the extracellular and transmembrane domains of alpha fused to the cytoplasmic domain of alpha (alpha alpha). When this chimera is expressed with wild-type beta(3), energy-dependent high affinity ligand binding results (19) .

We generated truncation mutants by placing a series of stop codons at residues Tyr, Thr, Tyr, Arg, and Leu in beta(3). To assess ligand binding affinity, we examined the capacity of the transfectants to bind PAC1, a murine IgMkappa antibody specific for the high affinity conformation of alphabeta(3)(25) . PAC1 mimics the ligand binding characteristics of the natural ligand, fibrinogen (25, 30, 31) and fails to bind to ligand binding defective mutants of alphabeta(3)(32, 33) . Consequently, the binding of this monoclonal antibody faithfully reflects the binding of the physiological ligand.

When coexpressed with alpha alpha (Fig. 2), a beta(3) cytoplasmic domain mutant (beta(3) Delta759) with the 4 C-terminal residues deleted, failed to bind PAC. The lack of PAC1 binding was manifest in all truncations through beta(3) Delta724 (Fig. 2), indicating that an almost complete beta(3) tail is required for inside-out signaling. Surprisingly, high affinity PAC1 binding to alphaalpha beta(3) was restored following the complete truncation (beta(3)Delta717) of the beta(3) cytoplasmic domain. Moreover, this same truncation, when co-expressed with wild-type alpha also bound PAC1 (Fig. 2). Thus, its activating effect did not require the alpha cytoplasmic domain.


Figure 2: The deletion of membrane-proximal residues activates alphabeta(3). CHO cells were co-transfected with 2 µg of cDNA encoding each of the beta(3) constructs and 2 µg of cDNA encoding wild-type alpha () or the chimera alpha alpha (bullet). After 48 h, the cells were harvested and analyzed for PAC1 binding as described under ``Materials and Methods.'' To obtain a quantitative estimate of PAC1 binding, we calculated an activation index, as described under ``Materials and Methods.'' Depicted are the mean ± S.E. of three independent experiments for each beta(3) construct plotted versus beta(3) tail length starting at Leu = 0. The beta(3) cytoplasmic domain sequence is indicated above with the positions of the stop codons marked as inverted triangles. Thus, the first stop codon is at Leu, producing a beta-tail length of 0 in beta(3)Delta717. Depicted are the mean ± S.E. of three independent determinations.



To characterize further the activating effect of the beta(3)Delta717 truncation, we established a stable cell line expressing alphabeta(3)Delta717. This receptor was expressed as a heterodimer that could be immunoprecipitated with an anti-alphabeta(3), but not an antibody directed against the beta(3) cytoplasmic domain (Fig. 3A). Moreover, alphabeta(3)Delta717 bound both PAC1 (not shown) and fibrinogen (Fig. 3B) without prior activation. Furthermore, alphabeta(3)Delta717 was similar to native alphabeta(3) in that it bound a number of conformation-dependent monoclonal antibodies including A2A9 (34), 4F10 (35) , 2G12 (35) , D57 (19) , and mAb15 (36) (data not shown). Since beta(3) Delta724 retains the membrane-proximal region and is not activating, removal of the LLITIHD sequence is responsible for this effect. To test this idea, we internally deleted residues Lys to Ile. When this beta(3) construct was transiently expressed with alpha, it also bound PAC1 spontaneously (not shown). Consequently, this membrane-proximal seven amino acids of beta Lys to Asp, influences the ligand binding affinity of the extracellular domain of alphabeta(3).


Figure 3: A, characterization of alphabeta(3) Delta717. Upper panel, CHO cells stably transfected with alphabeta(3) or alphabeta (3) Delta717 were biotinylated, lysed, and immunoprecipitated with an antibody to the beta(3) cytoplasmic domain (beta(3)tail), a monoclonal anti-alphabeta(3), or a control monoclonal antibody (MOPC21). Following SDS-polyacrylamide gel electrophoresis and electrophoretic transfer, blots were developed with Avidin-Peroxidase (Vectastain ABC). The luminograms were purposely overexposed to establish the absence of precipitated alphabeta(3) Delta717 when the anti-beta(3) tail antibody was used. Lower panel, Western blots of anti-alphabeta(3) or MOPC21 precipitates described in the upper panel were probed with the anti-beta(3) tail antibody, and the blots were developed with biotinylated anti-Immunoglobulin and Avidin-Peroxidase. Note the absence of reactivity in the immunoprecipitate from cells bearing alphabeta(3) Delta717. B, fibrinogen binding to alphabeta(3) Delta717. Fluoresceinated fibrinogen (160 nM) was added to 50 µl of modified Tyrode's solution containing CHO cells stably transfected with the indicated integrin in the presence or absence of 2 µM Ro43-5054. After 30 min at 37° C, 450 µl of Tyrode's solution was added, and fibrinogen binding was measured by flow cytometry as described under ``Materials and Methods.'' Depicted are specific fluorescence (mean fluorescence intensity in the presence of Ro43-5054) for each cell line in the presence or absence of 2 µM Anti-LIBS6, an activating monoclonal antibody. Depicted are mean ± S.E. of three independent determinations.



alpha Chain Cytoplasmic Domain Sequences Do Not Influence the Ligand Binding Affinity of alphabeta(3)Delta717

Sequences C-terminal of the alpha subunit membrane-proximal GFFKR motif are involved in inside-out signal transduction (14, 19, 37, 38, 39, 40, 41, 42, 43) . To examine the requirement of the alpha chain cytoplasmic domain in the activation of alphabeta(3)Delta717, we coexpressed beta(3) Delta717 with an alpha mutant in which the alpha cytoplasmic domain had been truncated following the GFFKR sequence (alphaDelta996). In contrast to wild-type beta(3), coexpression of beta(3) Delta717 with alphaDelta996 in CHO cells resulted in high affinity PAC1 binding (Fig. 4). Thus, the activation of alphabeta(3)Delta717 does not require those amino acids C-terminal of the GFFKR motif in the alpha cytoplasmic domain.


Figure 4: High affinity ligand binding induced by the beta(3) Delta717 truncation is independent of alpha chain cytoplasmic domain sequences. PAC1 binding was measured in CHO cells expressing beta(3) Delta717 (shaded box) with alphaDelta996, alpha alpha(L) Delta, and wild-type alpha as a control. In parallel, we co-expressed these alpha subunit variants with wild-type beta(3) (open box). The data depicted are the mean activation indices ± S.E. of three independent experiments.



The conserved membrane-proximal GFFKR motif of the alpha subunit could interact with beta(3) (Lys-Asp) (23). To determine whether a GFFKR deletion could reverse the activating effect of the beta(3) (Lys-Asp) deletion, we co-transfected beta(3) Delta717 and the alphaalpha (L)Delta chimera, in which the GFFKR motif of the alpha (L) cytoplasmic domain is disrupted (19) . The transient expression of alpha alpha (L)Delta leads to high affinity PAC1 binding with both beta(3) Delta717 and wild-type beta(3) (Fig. 4). Similar data were obtained with the GFFKR truncation mutant alphaDelta991 (26) (not shown). Thus, no portion of the alpha subunit cytoplasmic domain is required for the activation of alphabeta(3)Delta717.

High Affinity Ligand Binding to alphabeta(3)Delta717 Is Independent of Physiological Signaling Mechanisms

Inside-out signaling requires active cellular processes since it can be inhibited by a combination of NaN(3) and 2-deoxyglucose, inhibitors of anaerobic glycolysis and oxidative phosphorylation. Furthermore, integrin activation can also be inhibited by isolated beta(3) or beta(1) cytoplasmic domains. However, mutations in the alpha subunit that delete the GFFKR motif activate alpha beta(3) even in the presence of metabolic inhibitors or excess isolated beta(3) cytoplasmic domain (19, 22) . Consequently, these mutants do not seem to require the usual cellular signaling machinery for activation. To gain insight into the mechanism of activation conferred by beta(3) Delta717, we examined the effect of metabolic inhibitors and the co-expression of isolated beta(3) cytoplasmic domains on PAC1 binding to alphabeta(3)Delta717.

First, we examined the effect of treatment with the metabolic inhibitors NaN(3) and 2-deoxyglucose on activation of alphabeta(3)Delta717. There was no change in PAC1 binding to CHO cells stably transfected with alphabeta(3)Delta717 (Fig. 5). In contrast, the metabolic inhibitors caused an 8-fold reduction of PAC1 binding to CHO cells expressing a constitutively active alpha chimera. Therefore, high affinity PAC1 binding to alphabeta(3)Delta717 does not require intact cellular ATP metabolism.


Figure 5: beta(3) Delta717 does not require cellular metabolism to activate alphabeta(3). Stable CHO cell lines expressing alphabeta(3)Delta717 and the constitutively active chimera alphaalpha(5)beta(3) were assayed for PAC1 binding in the presence and absence of the metabolic inhibitors NaN(3) and deoxyglucose. Depicted are the mean activation indices ± S.E of three independent experiments in the presence (open box) and absence (shaded box) of the inhibitors.



Next, we examined the effect of coexpression of an isolated beta(3) tail on the affinity state of alphabeta(3)Delta717. The free beta(3) tail was transiently expressed in the form of a chimera of the cytoplasmic domain of integrin beta(3) subunit fused to the extracellular and transmembrane domains of the Tac subunit of the interleukin-2 receptor (44) . The high affinity state of alphabeta(3)Delta717 was resistant to Tac-beta(3) coexpression (Fig. 6). However, high affinity PAC1 binding was inhibited in the constitutively active chimera, alphaalpha(5)beta(3), demonstrating the potency of Tac-beta(3) as an inhibitor of inside-out signal transduction. This result, together with the observation that the high affinity state of alphabeta(3)Delta717 does not require cellular metabolism suggests that the activation of this receptor is independent of the usual cellular signaling machinery.


Figure 6: Overexpression of an isolated beta(3) cytoplasmic domain fails to inhibit PAC1 binding to alphabeta(3)Delta717. Depicted are flow cytometry histograms in which fluorescence intensity is plotted on the abscissa and cell number on the ordinate. PAC1 binding in the presence (open histogram) and absence (filled histogram) of competitive inhibitor, 1 µM Ro43-5054, is depicted in panels A-D. alphabeta(3)Delta717 specifically binds PAC1 (panel A); this binding is not effected by co-expression of Tac-beta(3) (panel B). The integrin chimera alpha alpha(5)beta(3) binds PAC1 with high affinity (panel C); coexpression of Tac-beta(3) blocks this high affinity PAC1 binding (panel D). High affinity PAC1 binding was restored following the addition of the activating antibody anti-LIBS6. Tac-beta(3) expression levels were similar in both transfections (data not shown).




DISCUSSION

The most important findings presented here are: 1) the removal of the 7-residue highly conserved inner membrane-proximal region of the beta(3) subunit (beta(3)Delta717) increases the ligand binding affinity, i.e. activates the integrin alphabeta(3). 2) The high affinity state of alphabeta(3)Delta717 is independent of the cellular signaling machinery as: (a) high affinity ligand binding is preserved in the absence of the distal portion of the beta(3) cytoplasmic domain, a region required for ``physiological'' inside-out signal transduction. (b) High affinity ligand binding was not reduced by the addition of inhibitors of oxidative phosphorylation and anaerobic glycolysis. (c) The overexpression of an isolated beta(3) cytoplasmic domain, a potent inhibitor of inside-out signaling, failed to inhibit the activation of alphabeta(3)Delta717. 3) The high affinity ligand binding conferred by the beta(3) Delta717 truncation is independent of the alpha chain cytoplasmic domain sequences. Thus, the highly conserved inner membrane proximal portion of the integrin beta subunit influences the affinity state of the extracellular domain of alphabeta(3).

A complete truncation of the beta(3) cytoplasmic domain, beta(3)Delta717, promotes high affinity ligand binding. In contrast, beta(3) Delta724, a truncation that spares beta(3)(Leu-Asp), is not activating when co-expressed with either alpha or alphaalpha. Lys is proposed to be the first cytoplasmic domain residue in beta(3)(23) , consequently the Leu-Asp would be the most membrane proximal portion of the beta(3) cytoplasmic domain. Disruption of this sequence seems to be the cause of the increased ligand binding affinity because a similar result was observed with a 6-residue ``loop-out'' mutation of this sequence that spared the distal cytoplasmic domain. Moreover, alphabeta(3) Delta717 was expressed as a heterodimer and bound several conformation-sensitive monoclonal antibodies. Since integrin surface expression is sensitive to mutations and deletions (45, 46, 47, 48, 49, 50) , these results suggest that alphabeta(3) Delta717 is not grossly misfolded. Moreover, this integrin also bound the physiological ligand, fibrinogen, suggesting that its high affinity ligand binding state is similar to that of the physiologically activated receptor. beta(3)(Leu-Asp) is a conserved sequence in other integrin beta cytoplasmic domains (Fig. 7). Moreover, mutations that disrupt this sequence in beta(2) stimulate the binding of iC3b-coated particles (51) and in beta(7)(5) stimulate VCAM-1 binding. Thus, this region appears to be important in controlling the affinity state of multiple integrin families.


Figure 7: Alignment of beta subunit cytoplasmic domains. The cytoplasmic domains of human beta subunits, with the exception of beta(4), were aligned using PILEUP and displayed using PRETTY programs of the UWGCG package (61). Consensus residues were defined by their presence in at least four of the sequences and are displayed in capital letters. Note the conservation of the sequence in beta(3)(Leu-Asp) (dark gray box) in all beta subunits. The alpha actinin-binding decapeptide (56) of beta(1) is surrounded by the hatched box. A light gray box surrounds the conserved NPXY that has been implicated in particle phagocytosis (51) in beta(2). Ser is circled; substitution of a Pro at this position disrupts bidirectional integrin signaling (21). The TTT sequence involved in the adhesive function of beta(2) is boxed (62).



The high affinity state of alphabeta(3)Delta717 is independent of the usual cellular signaling machinery. Previous studies established that C-terminal sequences of the beta cytoplasmic domain are required for the active high affinity state of several integrins (5, 19, 21) as well as for normal adhesive function (52, 53) . We found that the high affinity state of the chimera, alphaalphabeta(3), was abolished by removal of as little as 4 C-terminal residues. Yet, the beta(3)Delta717 constructs were ``active'' in the complete absence of these distal sequences. Conversely, the distal portions of the alpha cytoplasmic domain are also required for both the active high affinity state (19) and for normal cell adhesion (14, 37, 38, 39, 40, 41, 42, 43) . Nevertheless, these sequences were not required for high affinity ligand binding to alphabeta(3)Delta717 because alphaDelta996 beta(3)Delta717, a mutant in which the alpha cytoplasmic domain has been truncated following the GFFKR sequence, bound PAC1. In addition, the physiological integrin high affinity state is sensitive to depletion of cellular ATP (19, 54) or overexpression of isolated beta(3) cytoplasmic domains (22). The binding of PAC1 to alphabeta(3)Delta717 was unaffected by either of these treatments. Consequently, the high affinity state of alphabeta(3)Delta717 does not appear to require the signaling mechanisms involved in integrin activation.

The capacity of the membrane-proximal portion of the beta cytoplasmic domain to regulate integrin affinity may be due to its interaction with an intracellular partner. The cytoskeletal proteins alpha actinin (55, 56) and talin (57) bind to beta subunit cytoplasmic domain peptides, but the relevant binding sites do not include beta(3)(Leu-Asp). pp125, a tyrosine kinase, was recently reported to bind to a beta(3) peptide that contains this region (58) . Consequently, the role of pp125 in integrin affinity modulation may bear further study. Finally, the alpha subunit cytoplasmic domain may contain a binding site for beta(3)(Leu-Asp). As already noted, the membrane proximal alpha cytoplasmic domain is highly conserved, and deletions here change ligand binding affinity (19) . The proximal portions of the alpha and beta cytoplasmic tails must be parallel to each other and contiguous with presumptively helical transmembrane regions (23) . Further, there appear to be interactions between the alpha and beta tails in synthetic neo-protein mimics of the integrin cytoplasmic face (59) . Thus, the conserved membrane-proximal region of the integrin cytoplasmic domains may serve to constrain these receptors into a default low affinity conformation. In addition, it could act as a conduit for the proposed long range allosteric rearrangements (60) involved in inside-out integrin signaling.


FOOTNOTES

*
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. This work was supported by National Institutes of Health Grants HL48728 and AR 27214 (to M. H. G.) and HL 40387 (to S. J. S.). This is manuscript number 9116-VB from the Scripps Research Institute.

§
Post-doctoral fellow of the California Heart Association.

Supported by TRDP 3RT0320 from the California Tobacco Diseases Research Program and an Established Investigator of the American Heart Association.

**
To whom correspondence should be addressed. Tel.: 619-554-7124; Fax: 619-554-6403.

(^1)
The abbreviations used are: CHO, Chinese hamster ovary; LIBS, ligand-induced binding site; DMEM, Dulbecco's modified Eagle's medium; PCR, polyacrylamide chain reaction; kb, kilobase(s).


ACKNOWLEDGEMENTS

We gratefully acknowledge the superb technical assistance of Tracy Shipley and Jane Forsyth.


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