©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Cleavage of the 6A Subunit Is Essential for Activation of the 6A1 Integrin by Phorbol 12-Myristate 13-Acetate (*)

(Received for publication, December 26, 1995; and in revised form, January 18, 1996)

Gepke O. Delwel Frans Hogervorst Arnoud Sonnenberg (§)

From the Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The alpha6 integrin subunit is proteolytically cleaved during biosynthesis in a covalently associated heavy and light chain. To examine the importance of cleavage for the function of the alpha6 subunit, we introduced mutations in the cDNA encoding the RKKR (876-879) sequence, the presumed cleavage site, in which either one or two basic residues were replaced by glycine. Wild-type and mutant alpha6A cDNAs (alpha6, alpha6 and alpha6) were transfected into K562 cells. The mutant alpha6A integrin subunits were expressed in association with endogenous beta1, at levels comparable to that of the wild-type alpha6Abeta1. A single alpha6A polypeptide chain (150 kDa) was precipitated from surface-labeled alpha6, alpha6, and alpha6 transfectants, while the separate heavy (120 kDa) and light chains (31 or 30 kDa) were precipitated from the wild-type alpha6 transfectant. Thus, a change in the RKKR sequence prevents cleavage of alpha6. After activation by the anti-beta1 stimulatory mAb TS2/16 both cleaved and uncleaved alpha6Abeta1 integrins bound and spread on laminin-1. Remarkably, the phorbol ester phorbol 12-myristate 13-acetate, which activates wild-type alpha6Abeta1 to bind to laminin-1, did not activate uncleaved alpha6Abeta1. We conclude that uncleaved alpha6Abeta1 is capable of ligand binding and transducing outside/in signals, like wild-type alpha6Abeta1. However, inside/out signaling is affected. It appears that cleavage of alpha6 is required to generate the proper conformation in alpha6 that enables affinity modulation of the alpha6Abeta1 receptor by phorbol 12-myristate 13-acetate.


INTRODUCTION

Integrins are transmembrane glycoproteins, composed of noncovalently associated alpha and beta subunits, that are involved in cell-extracellular matrix and cell-cell interactions(1, 2) . Besides mediating cell adhesion, they participate in both inside/out and outside/in cell signaling processes(1, 3) .

Integrins can exist in multiple functional states(4, 5, 6) , and for most integrins the conversion of an inactive state to an active state is required for high affinity ligand binding. Such activation or inside/out signaling involves intracellular signals that act through integrin cytoplasmic domains and which cause conformational changes in the extracellular domains, by which their ligand binding properties are regulated(1, 7, 8) . Agents that can activate integrins are, among others: monoclonal antibodies (mAbs) to the beta1(9, 10) , beta2(11, 12) , and beta3 subunits(13) , divalent cations(14, 15) , and the phorbol ester PMA(^1)(16, 17) . Multimerization or redistribution of integrins may also contribute to increased ligand binding(18, 19) . Ligand binding results in various biological responses such as, cell spreading, migration, differentiation, formation of focal adhesion sites, and gene induction (outside/in signaling)(1, 20) .

From the 16 different alpha subunits identified until now, the alpha3, alpha5, alpha6, alpha7, alpha8, alphav, and alphaIIb subunits are proteolytically cleaved near the carboxyl-terminal part of the extracellular domain, resulting in a heavy chain that is disulfide-linked to a membrane spanning light chain. The alpha4 and alphaE subunits are also proteolytically cleaved but nearer to the amino-terminal part of the extracellular domain(1, 2) . The role of proteolytic cleavage of alpha subunits is not clear. It may have a function in ligand binding. However, cleavage of the alpha4 and alphaIIb chains does not affect ligand binding of the alpha4beta1 and alphaIIbbeta3 integrins(21, 22) . Since the existence of proteolytic cleavage sites in alpha subunits is conserved, not only in different alpha chains but also across species, cleavage is likely to be of functional importance. Furthermore, it has been well documented that many proteins, e.g. peptide hormones, cell adhesion proteins, and growth factor receptors, are synthesized as inactive precursors that acquire their fully active form by endoproteolytic cleavage at specific basic residues(23) .

The alpha6 subunit is proteolytically cleaved during biosynthesis into a 120-kDa heavy chain that remains covalently associated with a light chain (31 or 30 kDa)(24) . Several potential proteolytic cleavage sites are present in the primary structure of alpha6(25, 26) . One of them, as we have previously suggested, may be the RKKR sequence (25) (Fig. 1). A detailed structural analysis of the different cleavage sites used in alpha6 will be described elsewhere. (^2)In the present study the importance of cleavage for the function of alpha6 was addressed. Two variants of the alpha6 subunit exist, alpha6A and alpha6B, which have different cytoplasmic domains(25, 27) . We used the alpha6A cDNA to introduce mutations in the codons of the RKKR sequence. Mutant alpha6A cDNAs were transfected into K562 cells. Cleavage of the mutant alpha6A subunits and ligand binding of the mutant alpha6Abeta1 integrins were investigated.


Figure 1: Schematic representation of the wild-type and mutant alpha6A constructs. The sequence of alpha6 from His-873 through Lys-899 is shown. I-VII, homologous repeat domains; TM, transmembrane region.




MATERIALS AND METHODS

Cell Culture and Antibodies

K562 transfectants were grown in RPMI 1640 supplemented with 7.5% fetal calf serum, penicillin, streptomycin and 1 mg/ml Geneticin (G-418 sulfate, Life Technologies Inc.). The following monoclonal antibodies (mAbs) to integrin subunits were used: the rat mAb GoH3 (28) and the mouse mAb J8H (29) to alpha6; the rabbit polyclonal antibody palpha6A directed against the carboxyl-terminal sequence of alpha6A(30) ; the mouse mAb Sam-1 to alpha5 (11) , a gift from Dr. C. G. Figdor (University of Nijmegen, The Netherlands). The mouse mAbs K20 (31) and TS2/16 (32) directed against the beta1 subunit were obtained from the 4th International Workshop on Leukocytes and the American Type Culture Collection (Rockville, MD), respectively.

Construction of Mutant alpha6A cDNAs

The overlap extension method (33) was used for site-directed mutagenesis of the RKKR site. Briefly, for each mutant two overlapping primers were synthesized encoding glycine at the appropriate positions. These primers were: for alpha6 primer A (sense), positions 2668-2688 (5`-CACAACTCAGGAAAGAAACGG-3`) and primer B (antisense) the same positions; for alpha6 primer C (sense) positions 2677-2699 (5`-AGAAAGAAAGGGGAAATTACTGA-3`) and primer D (antisense) the same positions; for alpha6 primer E (sense) positions 2668-2691 (5`-CACAACTCAAGAGGCGGCCGGGAA-3`) and primer F (antisense) the same positions. Two flanking primers were: primer X (sense) positions 2425-2449 (5`-GTTGGCGAGCAAGCTATGAAATCTG-3`) and primer Y (antisense) positions 2896-2917 (5`-TGAAGGCTCGCATGAGAATGTC-3`). Primer positions for alpha6 correspond to the sequence published by Hogervorst et al.(26) . First-round PCRs were performed with primers A, C, or E and primer Y, and with primers B, D, or F and primer X using Pfu polymerase (Stratagene, La Jolla, CA) and pUC18-alpha6A cDNA as template. PCR products were isolated from agarose gel, and a mixture of the two products of each mutant was used in a second-round PCR with primers X and Y. The resulting PCR products were digested with BstXI and XbaI and subcloned into pUC18-alpha6A cDNA. Mutant clones were identified by sequence analysis. Correct alpha6A cDNA fragments were isolated after HindIII digestion and cloned into the HindIII site of pRc/CMV (Invitrogen, San Diego, CA). Construction of wild-type alpha6A cDNA in pRc/CMV has been described(30) .

Transfection and Flow Cytometry

alpha6A cDNA constructs were transfected into K562 cells by electroporation using a Bio-Rad Gene Pulser, and cells expressing high levels of the transfected alpha6A subunit were sorted on the FACScan (Becton Dickinson, Mountain View, CA) three times using mAb J8H, as described previously(30) .

Immunoprecipitation of I-Labeled Cells

Transfectants were surface labeled with I by the lactoperoxidase/hydrogen peroxide method(28) , washed, and solubilized in lysis buffer containing 1% Nonidet P-40 and used for immunoprecipitation as described previously(30) .

Cell Adhesion and Spreading Assays

Microtiter plates (96-well, Greiner GmbH, Frickenhausen, Germany) were coated with 20 µg/ml laminin-1 (Collaborative Biomedical Products, Bedford, MA) for 16 h at 4 °C. Plates were washed with phosphate-buffered saline, and incubated with phosphate-buffered saline containing 1% bovine serum albumin for 60 min at room temperature to block nonspecific adhesion. Cells were labeled with Cr (400 µCi), washed in Iscove's modified Dulbecco's medium containing 0.35% bovine serum albumin, and resuspended at 10^6 cells/ml. Cells were stimulated either by PMA (dissolved at various concentrations in Me(2)SO, Sigma) or TS2/16 (1:5 hybridoma supernatant). Cell adhesion was performed as described previously(30) . Cell spreading analysis has been described(34) .


RESULTS

The RKKR Site in the alpha6 Subunit Is Required for Cleavage

To investigate the role of cleavage of the alpha6A subunit, mutations were introduced in the cDNA encoding the RKKR site producing the following alpha6A mutants: alpha6, alpha6, and alpha6 (Fig. 1). The different alpha6A cDNA constructs were transfected into K562 cells, which only express the alpha5beta1 integrin. Fluorescence-activated cell sorter analysis of the different alpha6A transfectants showed that the alpha6 subunit was expressed on all transfectants. The alpha6 expression levels were approximately similar for all transfectants, as were the levels of the endogenous alpha5 and beta1 subunits (not shown).

Next, we examined whether the introduced mutations affect the cleavage of the alpha6A subunit. The integrin subunits were immunoprecipitated from cell lysates from I-labeled cells using antibodies to alpha6 and beta1, and subsequently analyzed by 10% SDS-PAGE under reducing conditions. To study cleavage, reduction is essential since it destroys the disulfide-linkage between the alpha6 heavy and light chains. As expected, the anti-alpha6 mAb GoH3 and the anti-alpha6A polyclonal antibody palpha6A precipitated the alpha6 heavy chain (120 kDa) and the alpha6 light chains (31 and 30 kDa) and co-precipitated the beta1 subunit (120 kDa) from the cells expressing the wild-type alpha6A (alpha6) (Fig. 2A). The anti-beta1 mAb K20 precipitated the beta1 subunit and co-precipitated the heavy and light chains of alpha6 as well as those of alpha5 (130 and 17 kDa, respectively). No heavy and light chains of alpha6 were precipitated from the alpha6A mutant expressing transfectants, alpha6, alpha6, and alpha6 (Fig. 2A). Instead, all antibodies precipitated only one alpha6 polypeptide chain of 150 kDa (Fig. 2B). K20 precipitated beta1 together with alpha5 and mutant alpha6 subunits from the alpha6, alpha6, and alpha6 transfectants, whereas GoH3 and palpha6A co-precipitated beta1. The results show that the intact RKKR site is essential for cleavage of the alpha6 subunit, and that both the cleaved and the uncleaved alpha6 subunits are expressed on the cell surface in association with endogenous beta1.


Figure 2: Identification of the cleavage site in the alpha6 subunit. A, I-labeled cell lysates of the alpha6, alpha6, alpha6, and alpha6 transfectants were immunoprecipitated with GoH3 (anti-alpha6; lanes 1), K20 (anti-beta1; lanes 2), and palpha6A (anti-alpha6A; lanes 3). Precipitates were analyzed by 10% SDS-PAGE under reducing conditions. B, shorter exposure of part of the autoradiogram shown in A, revealing the alpha6 heavy chain (120 kDa) and the uncleaved alpha6 polypeptide chain (150 kDa).



Uncleaved alpha6Abeta1 Integrins Are Not Activated by PMA

The alpha6Abeta1 integrin is a receptor for laminin-1 and various other laminin isoforms(14, 30, 34) . To examine the functional activity of the mutant alpha6Abeta1 integrins, adhesion of cells to laminin-1 was tested. The unstimulated wild-type and mutant alpha6A transfectants did not bind to laminin-1 (Fig. 3A). After treatment of the cells with the stimulatory anti-beta1 mAb TS2/16 (9, 10) all four transfectants adhered to laminin-1, and the percentages of bound cells were approximately similar. Binding of all transfectants could be completely blocked by the anti-alpha6 mAb GoH3, demonstrating that the observed adhesions were mediated by the wild-type and mutant alpha6Abeta1 integrins (not shown).


Figure 3: Effect of TS2/16 and PMA stimulation of the transfectants on adhesion to laminin-1. A, adhesion of unstimulated, TS2/16 (1:5 hybridoma supernatant) and PMA (10 ng/ml) stimulated transfectants to laminin-1. B, adhesion to laminin-1 at different concentrations of PMA. Binding percentages are expressed as percentage of the total input/well (100%). Adhesion was measured in triplicate; error bars represent standard deviations of the averaged results. One representative experiment out of five is shown. C, cell spreading of the TS2/16-stimulated transfectants on laminin-1.



The phorbol ester PMA was subsequently used to stimulate the transfectants. Fig. 3A shows that the PMA-stimulated wild-type alpha6 (alpha6) transfectants adhered to laminin-1, and that the percentage of bound cells was lower than after stimulation with TS2/16, as previously reported(30) . Surprisingly, the alpha6, alpha6, and alpha6 transfectants did not bind to laminin-1 after PMA treatment. Thus, the uncleaved alpha6Abeta1 integrins appeared unresponsive to activation by PMA. To test whether the uncleaved alpha6Abeta1 integrins possessed a different sensitivity toward PMA, the transfectants were stimulated with different concentrations of PMA. Adhesion of the alpha6 transfectants to laminin-1 was stimulated in a concentration-dependent manner with optimal binding at 10 ng/ml PMA. In contrast, the alpha6, alpha6, and alpha6 transfectants did not adhere to laminin-1 at any of the concentrations of PMA used (Fig. 3B). The presented data demonstrate that the uncleaved alpha6Abeta1 integrin functions as a laminin-1 receptor, but only when activated by conformational changes induced by an antibody. The PMA-induced inside/out signaling by the uncleaved alpha6Abeta1 integrins is disturbed.

All Transfectants Spread on Laminin-1

We also investigated whether cleavage of the alpha6A subunit influences cell spreading. All four transfectants spread on laminin-1 when stimulated with TS2/16, and the number of spread cells was comparable (Fig. 3C). Only a few cells of the wild-type alpha6 transfectant spread after stimulation with PMA (not shown).


DISCUSSION

The major finding in the present study is that the uncleaved alpha6Abeta1 integrin is not activated by PMA to bind ligand, but its ligand binding capacity is not different from that of wild-type alpha6Abeta1. Furthermore, cell spreading, a post-ligand binding event, was not affected. We conclude from these findings that: 1) cleavage of the alpha6A integrin subunit is required for PMA induced inside/out signaling; 2) activation by PMA meets different requirements than activation by Mn (not shown) or TS2/16; 3) inside/out but not outside/in signal transduction is dependent on cleavage of alpha6A, indicating that these signal transduction pathways are independent. The results also show that the primary site for cleavage of the alpha6 subunit is the RKKR site, which is a consensus sequence for the endoprotease furin(23) .

PMA stimulates inside/out signaling processes that ultimately result in activation of the integrin(1, 7) . Activation by PMA may cause conformational changes in the integrin that induce the high affinity binding state. The structure of the uncleaved alpha6Abeta1 integrins was not grossly different from that of the wild-type alpha6Abeta1, since uncleaved alpha6A associated with endogenous beta1, and in the resting state different alpha6 mAbs reacted equally well with wild-type and mutant alpha6Abeta1 (not shown). Unfortunately, we could not test whether PMA activation induces a conformational change in the wild-type alpha6Abeta1 integrin, because none of the alpha6 mAbs detected a difference between the activated and nonactivated form of the integrin.

It has been suggested that not changes in affinity but rather post-ligand binding events such as cell spreading are involved in the PMA-induced adhesion(16, 35) . This seems unlikely for our cells, since PMA induced spreading of only a few K562 transfectants expressing wild-type alpha6Abeta1. PMA has also been shown to induce clustering of integrins that correlated with increased ligand binding(18) . We did not observe ligand-independent clustering of wild-type or mutant alpha6Abeta1 upon PMA treatment (not shown) by confocal laser scanning microscopy, but the presence of microclusters cannot be ruled out.

Since PMA is an activator of protein kinase C, the phosphorylation of intracellular proteins, in particular integrin cytoplasmic domains, has been suggested to play a role in integrin activation. Phosphorylation of the alpha6 subunit in response to PMA has been reported(29, 36, 37) , although we have recently demonstrated that such phosphorylation is not required for activation of alpha6Abeta1(29) . Consistently, PMA induced phosphorylation of the alpha6, alpha6, and alpha6 cytoplasmic domains (not shown) but failed to activate the mutant alpha6Abeta1 integrins.

As discussed above, it is not clear how PMA activates the alpha6Abeta1 integrin on K562 transfectants. We hypothesize that PMA induces an affinity change in alpha6Abeta1. In the mutant alpha6Abeta1 integrins, the uncleaved alpha6 subunit may not be suitable for propagating intracellular signals elicited by PMA. Additionally, its aberrant structure may prevent microclustering of alpha6Abeta1 in the plane of the membrane.

TS2/16 and Mn induced ligand binding of the cleaved and uncleaved alpha6Abeta1 integrins. In contrast to PMA, activation by TS2/16 or Mn does not involve inside/out signaling pathways, since these agents bind directly to the integrin. TS2/16 binds to the beta1 subunit and induces a conformational change in the integrin(38) , whereas Mn may increase the affinity for ligand (5) or stabilize the high affinity state of the integrin(15) . Although TS2/16 and Mn induce conformational changes in alpha6Abeta1 to activate the integrin, these may be different from those induced by PMA. Alternatively, the conformational changes induced by TS2/16 or Mn may be sufficient for integrin activation, while activation by PMA may require integrin clustering, as well.

It has been previously reported that cleavage of the alpha4 and alphaIIb subunits did not alter their function(21, 22) . Uncleaved alpha4beta1 and alphaIIbbeta3 were expressed on the cell surface and adhered to their ligands VCAM-1 and fibronectin or fibrinogen, respectively. Our results do not contradict these studies, since we also observed cell surface expression and ligand binding of uncleaved alpha6Abeta1. However, alpha4beta1 on K562 cells and alphaIIbbeta3 on COS-1 cells are constitutively active and thus do not require activation to mediate ligand binding. Possibly, PMA treatment of these integrins will reveal similar results as described here for alpha6Abeta1, if they could be expressed in a resting state on another cell type.


FOOTNOTES

*
This work was supported by Grant NKI-91-260 from the Dutch Cancer Society. 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.

§
To whom correspondence should be addressed: Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel.: 31-20-5121942; Fax: 31-20-5121944; asonn{at}nki.nl.

(^1)
The abbreviations used are: PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody; PCR, polymerase chain reaction.

(^2)
G. O. Delwel, I. Kuikman, A. A. de Melker, and A. Sonnenberg, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank E. Noteboom for assistance with FACScan analysis and Drs. E. Roos and C. P. Engelfriet for critical reading of the manuscript.


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