(Received for publication, December 26, 1995; and in revised form, January 18, 1996)
From the
The 6 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
6
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
6A cDNAs (
6
,
6
and
6
) were transfected into K562 cells. The mutant
6A integrin subunits were expressed in association with endogenous
1, at levels comparable to that of the wild-type
6A
1. A
single
6A polypeptide chain (150 kDa) was precipitated from
surface-labeled
6
,
6
, and
6
transfectants, while the separate heavy (120 kDa)
and light chains (31 or 30 kDa) were precipitated from the wild-type
6
transfectant. Thus, a change in the RKKR sequence
prevents cleavage of
6. After activation by the anti-
1
stimulatory mAb TS2/16 both cleaved and uncleaved
6A
1
integrins bound and spread on laminin-1. Remarkably, the phorbol ester
phorbol 12-myristate 13-acetate, which activates wild-type
6A
1 to bind to laminin-1, did not activate uncleaved
6A
1. We conclude that uncleaved
6A
1 is capable of
ligand binding and transducing outside/in signals, like wild-type
6A
1. However, inside/out signaling is affected. It appears
that cleavage of
6 is required to generate the proper conformation
in
6 that enables affinity modulation of the
6A
1
receptor by phorbol 12-myristate 13-acetate.
Integrins are transmembrane glycoproteins, composed of
noncovalently associated and
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
1(9, 10) ,
2(11, 12) , and
3 subunits(13) , divalent
cations(14, 15) , and the phorbol ester
PMA(
)(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 subunits identified until now, the
3,
5,
6,
7,
8,
v, and
IIb 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
4 and
E subunits are also proteolytically cleaved but nearer to the
amino-terminal part of the extracellular
domain(1, 2) . The role of proteolytic cleavage of
subunits is not clear. It may have a function in ligand binding.
However, cleavage of the
4 and
IIb chains does not affect
ligand binding of the
4
1 and
IIb
3
integrins(21, 22) . Since the existence of proteolytic
cleavage sites in
subunits is conserved, not only in different
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 6 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
6(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
6 will be described elsewhere. (
)In the present study the importance of cleavage for the
function of
6 was addressed. Two variants of the
6 subunit
exist,
6A and
6B, which have different cytoplasmic
domains(25, 27) . We used the
6A cDNA to
introduce mutations in the codons of the RKKR sequence. Mutant
6A
cDNAs were transfected into K562 cells. Cleavage of the mutant
6A
subunits and ligand binding of the mutant
6A
1 integrins were
investigated.
Figure 1:
Schematic representation of the
wild-type and mutant 6A constructs. The sequence of
6 from
His-873 through Lys-899 is shown. I-VII, homologous
repeat domains; TM, transmembrane
region.
Next, we examined whether the introduced mutations affect
the cleavage of the 6A subunit. The integrin subunits were
immunoprecipitated from cell lysates from
I-labeled cells
using antibodies to
6 and
1, and subsequently analyzed by 10%
SDS-PAGE under reducing conditions. To study cleavage, reduction is
essential since it destroys the disulfide-linkage between the
6
heavy and light chains. As expected, the anti-
6 mAb GoH3 and the
anti-
6A polyclonal antibody p
6A precipitated the
6 heavy
chain (120 kDa) and the
6 light chains (31 and 30 kDa) and
co-precipitated the
1 subunit (120 kDa) from the cells expressing
the wild-type
6A (
6
) (Fig. 2A). The anti-
1 mAb K20 precipitated the
1 subunit and co-precipitated the heavy and light chains of
6
as well as those of
5 (130 and 17 kDa, respectively). No heavy and
light chains of
6 were precipitated from the
6A mutant
expressing transfectants,
6
,
6
,
and
6
(Fig. 2A). Instead, all
antibodies precipitated only one
6 polypeptide chain of 150 kDa (Fig. 2B). K20 precipitated
1 together with
5
and mutant
6 subunits from the
6
,
6
, and
6
transfectants, whereas
GoH3 and p
6A co-precipitated
1. The results show that the
intact RKKR site is essential for cleavage of the
6 subunit, and
that both the cleaved and the uncleaved
6 subunits are expressed
on the cell surface in association with endogenous
1.
Figure 2:
Identification of the cleavage site in the
6 subunit. A,
I-labeled cell lysates of the
6
,
6
,
6
,
and
6
transfectants were immunoprecipitated with
GoH3 (anti-
6; lanes 1), K20 (anti-
1; lanes
2), and p
6A (anti-
6A; 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
6 heavy chain (120 kDa) and the uncleaved
6
polypeptide chain (150 kDa).
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 6
(
6
) 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
6
,
6
, and
6
transfectants did not bind to laminin-1 after PMA treatment.
Thus, the uncleaved
6A
1 integrins appeared unresponsive to
activation by PMA. To test whether the uncleaved
6A
1
integrins possessed a different sensitivity toward PMA, the
transfectants were stimulated with different concentrations of PMA.
Adhesion of the
6
transfectants to laminin-1 was
stimulated in a concentration-dependent manner with optimal binding at
10 ng/ml PMA. In contrast, the
6
,
6
, and
6
transfectants did not
adhere to laminin-1 at any of the concentrations of PMA used (Fig. 3B). The presented data demonstrate that the
uncleaved
6A
1 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
6A
1
integrins is disturbed.
The major finding in the present study is that the uncleaved
6A
1 integrin is not activated by PMA to bind ligand, but its
ligand binding capacity is not different from that of wild-type
6A
1. Furthermore, cell spreading, a post-ligand binding
event, was not affected. We conclude from these findings that: 1)
cleavage of the
6A 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
6A, indicating that these signal transduction pathways
are independent. The results also show that the primary site for
cleavage of the
6 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 6A
1 integrins
was not grossly different from that of the wild-type
6A
1,
since uncleaved
6A associated with endogenous
1, and in the
resting state different
6 mAbs reacted equally well with wild-type
and mutant
6A
1 (not shown). Unfortunately, we could not test
whether PMA activation induces a conformational change in the wild-type
6A
1 integrin, because none of the
6 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 6A
1. 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
6A
1 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 6 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
6A
1(29) . Consistently, PMA induced
phosphorylation of the
6
,
6
,
and
6
cytoplasmic domains (not shown) but failed to
activate the mutant
6A
1 integrins.
As discussed above, it
is not clear how PMA activates the 6A
1 integrin on K562
transfectants. We hypothesize that PMA induces an affinity change in
6A
1. In the mutant
6A
1 integrins, the uncleaved
6 subunit may not be suitable for propagating intracellular
signals elicited by PMA. Additionally, its aberrant structure may
prevent microclustering of
6A
1 in the plane of the membrane.
TS2/16 and Mn induced ligand binding of the
cleaved and uncleaved
6A
1 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
1 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
6A
1 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 4 and
IIb subunits did not alter their
function(21, 22) . Uncleaved
4
1 and
IIb
3 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
6A
1. However,
4
1 on K562 cells and
IIb
3 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
6A
1, if they could be
expressed in a resting state on another cell type.