(Received for publication, November 2, 1994; and in revised form, December 27, 1994)
From the
In this study we have used several complementary techniques to
explore the interaction between the membrane linker molecule, ankyrin,
and the inositol 1,4,5-trisphosphate (IP) receptor in mouse
T-lymphoma cells. Using double immunolabeling and laser confocal
microscopy, we have found that both cytoplasmic IP
receptor
and ankyrin are preferentially accumulated within ligand-induced
lymphocyte receptor-capped structures. The binding between ankyrin and
IP
receptor appears to be very specific. Further analyses
indicate that the amino acid sequence GGVGDVLRKPS in the IP
receptor shares a great deal of structural homology with the
ankyrin-binding domain located in certain well characterized
ankyrin-binding proteins such as the cell adhesion molecule, CD44.
Biochemical studies using competition binding assays and a synthetic
peptide identical to GGVGDVLRKPS (a sequence detected in rat brain
IP
receptor (amino acids 2548-2558) and mouse brain
IP
receptor (amino acids 2546-2556)) indicate that
this 11-amino acid peptide binds specifically to ankyrin (but not
fodrin or spectrin). Furthermore, this peptide competes effectively for
ankyrin binding to IP
receptor-containing vesicles and/or
purified IP
receptor, and it blocks ankyrin-induced
inhibitory effects on IP
binding and
IP
-mediated internal Ca
release in mouse
T-lymphoma cells. These findings suggest that this amino acid sequence,
GGVGDVLRKPS, which is located close to the C terminus of the IP
receptor, resides on the cytoplasmic side (not the luminal side)
of IP
receptor-containing vesicles. This unique region
appears to be an important part of the IP
receptor
ankyrin-binding domain and may play an important role in the regulation
of IP
receptor-mediated internal Ca
release during lymphocyte activation.
Cytoskeletal proteins are known to be important participants in
multiple functions involving cell regulation(1, 2) .
Because the cytoskeletal network (e.g. microfilaments,
microtubules, and intermediate filaments) can potentially form a
physical link between the plasma membrane, organelle membranes, and
nuclear membrane, it has been suggested that they are involved in
transmitting important regulatory signals during cell activation by
agonists(1, 2) . Specifically, Putney and his
co-workers (3) have reported that IP(
)receptor-containing vesicles may be attached to the
plasma membrane through cytoskeletal elements such as actin. In
addition, our laboratory has found that both IP
binding and
IP
-induced Ca
release activities are
significantly inhibited by cytochalasin D (an inhibitor for
microfilament function) and colchicine (an agent known to disassemble
microtubules)(4) . These findings support the notion that the
cytoskeleton is involved in the regulation of IP
receptor-mediated function.
Recently, ankyrin (a cytoskeletal
protein known to link membrane proteins such as erythrocyte band 3 (1) and lymphocyte GP85(CD44) (5, 6, 7, 8, 9, 10) to
spectrin/fodrin-associated microfilaments) has been shown to bind
IP receptor in brain (11) and lymphoma
cells(12) . Furthermore, the binding of ankyrin to its receptor
displays inhibitory effects of IP
binding and
IP
-mediated internal Ca
release(12) . Consequently, certain cytoskeletal proteins (e.g. ankyrin) are strongly implicated in regulating internal
Ca
release(11, 12, 13) .
In this paper we have identified a unique 11-amino acid sequence,
GGVGDVLRKPS, which is located close to the C terminus of the IP receptor and appears to be an important part of the
ankyrin-binding domain of the IP
receptor. The binding of
ankyrin to this sequence is critically important for the regulation of
IP
-mediated internal Ca
release during
lymphocyte activation.
Ankyrin is known to link various transmembrane proteins to
the actin network through its interaction with spectrin or fodrin (a
spectrin-like protein)(1, 2, 16) . For
example, in erythrocytes ankyrin connects the band 3 anion exchange
protein to spectrin(1, 16, 17) . In
non-erythrocytes, ankyrin is associated with a number of
physiologically important membrane proteins including the
Na/K
-ATPase(18, 19) ,
the voltage-dependent (20) , and the amiloride-sensitive
Na+ channels(21) , as well as
CD44(GP85)(5, 6, 7, 8, 9, 10) ,
possibly via its binding to fodrin.
Previously, we have shown that
ankyrin interacts with the IP receptor in mouse T-lymphoma
cells(12) . In this study, using double immunofluorescence
staining and laser confocal microscopic analyses, we have found that
ligands such as anti-CD3 antibody and ConA induce an accumulation of
the IP
receptor (Fig. 1, B and D)
underneath receptor cap structures (Fig. 1, A and C). These results are consistent with previous findings
indicating that lymphocyte IP
receptors are aggregated
underneath certain surface receptor-capped structures(22) .
Importantly, the IP
receptor (Fig. 1, E and G) appears to be co-localized with ankyrin (Fig. 1, F and H) within surface receptor-capped structures
induced by ConA (Fig. 1, E and F) and anti-CD3
antibody (Fig. 1, G and H). Because both
anti-CD3 antibody and ConA have been shown to be involved in T cell
receptor-mediated signal transduction and lymphocyte
activation(23, 24) , these results suggest that the
close interaction between IP
receptor and ankyrin during
ligand-induced receptor capping events may be critically important for
IP
receptor-mediated internal Ca
release
at the onset of lymphocyte activation.
Figure 1:
Double immunofluorescence staining of
IP receptor and ankyrin in ligand-induced lymphocyte-capped
structures. A, surface ConA-capped structures induced by
fluorescein-labeled ConA. B, intracellular IP
receptor staining using rhodamine-labeled mouse monoclonal
anti-IP
receptor antibody in the same ConA-capped cells
shown in A. C, surface CD3-capped structures induced
by hamster anti-CD3 antibody followed by fluorescein-labeled rabbit
anti-hamster IgG. D, intracellular IP
receptor
staining using rhodamine-labeled mouse monoclonal anti-IP
receptor antibody in the same CD3-capped cells shown in C. E, intracellular IP
receptor staining
using rhodamine-labeled mouse monoclonal anti-IP
receptor
antibody in ConA-capped cells induced by unlabeled ConA. F,
intracellular ankyrin staining using fluorescein-labeled mouse
monoclonal anti-ankyrin antibody in the same ConA-capped cells induced
by unlabeled ConA as shown in E. G, intracellular
IP
receptor staining using rhodamine-labeled mouse
monoclonal anti-IP
receptor antibody in CD3-capped cells
(induced by hamster anti-CD3 antibody plus rabbit anti-hamster IgG). F, intracellular ankyrin staining using fluorescein-labeled
mouse monoclonal anti-ankyrin antibody in the same CD3-capped cells
(induced by hamster anti-CD3 antibody plus rabbit anti-hamster IgG as
shown in G). Arrowheads indicate surface
receptor-capped structures and accumulation of intracellular IP
receptor and ankyrin.
We have also reported
recently that mouse T-lymphoma cell IP receptor (
260
kDa) is preferentially located in a light density vesicle fraction
(15-25% sucrose interface with a density of 1.07 g/ml) containing
a unique vesicle population different from Golgi, endoplasmic
reticulum, plasma membranes, and lysosomes (12) . Scatchard
plot analyses have shown the presence of a high affinity
ankyrin-binding site (K
of 0.2 nM) on the
IP
receptor in mouse T-lymphoma cells(12) .
Furthermore, we have shown that the binding of ankyrin to the IP
receptor-containing vesicles (light density vesicles)
significantly modulates IP
binding and
IP
-induced internal Ca
release activities (12) .
Using a panel of monoclonal and polyclonal antibodies
against IP receptor, we have established that the
T-lymphoma cell IP
receptor displays immunological
cross-reactivity with the rat brain IP
receptor(12) . Ankyrin has also been shown to bind to the
IP
receptor in brain(11) . These data suggest that
T-lymphoma cell IP
receptor and brain IP
receptor share a great deal of structural and functional
similarities including ankyrin binding properties. In order to
understand the regulatory role of ankyrin in modulating IP
receptor-mediated function, it is clearly important to first
determine the ankyrin-binding domain of the IP
receptor.
Because the primary structure of T-lymphoma cell IP
receptor is not available, the well established protein sequence
of IP
receptor obtained from rat and mouse brain (25, 26) has been used for sequence comparison
analyses in this study. As shown in Fig. 2, both rat and mouse
brain IP
receptor contains the sequence GGVGDVLRKPS,
located close to the C terminus of the molecule (i.e. at amino
acids 2548-2558 of rat brain IP
receptor and amino
acids 2546-2556 of mouse brain IP
receptor). This
11-amino acid peptide shares a great deal of sequence homology with the
CD44 ankyrin-binding domain (e.g. GGNGTVEDRKPS), which is
involved in the regulation of cell adhesion function(10) . To
test whether the sequence GGVGDVLRKPS of the IP
receptor is
involved in ankyrin binding, we have examined the ability of an
11-amino acid synthetic peptide identical to GGVGDVLRKPS to bind
various cytoskeletal proteins. As shown in Fig. 3A,
this synthetic peptide binds ankyrin specifically in a dose-dependent
manner. The binding of this synthetic peptide to ankyrin is specific,
because it does not bind other cytoskeletal proteins such as fodrin or
spectrin (Fig. 3A). A control peptide containing a
scrambled sequence (GRDVKSPGLVG) with the same amino acid composition
as that of the synthetic peptide does not bind to any cytoskeletal
proteins tested (e.g. ankyrin, fodrin, and spectrin) (Fig. 3B).
Figure 2:
Sequence comparisons between IP receptor and CD44.
Figure 3:
Binding of I-labeled
cytoskeletal proteins (e.g. ankyrin, spectrin, and fodrin) to
synthetic peptides. Various concentrations (20, 40, 60, and 80 ng/ml)
of
I-labeled cytoskeletal proteins, including ankyrin
(
), fodrin (
), and spectrin (
), were incubated
with the nitrocellulose discs coated with either the 11-amino acid
peptide (GGVGDVLRKPS) or a scramble peptide (GRDVKSPGLVG) at 4 °C
for 4 h as described under ``Materials and Methods.''
Nonspecific binding was determined in the presence of a 100-fold excess
of the respective unlabeled cytoskeletal proteins and subtracted from
the total binding. The results represent the average of duplicate
determinations for each of the ligands used. A, binding of
I-labeled cytoskeletal proteins to the 11-amino acid
peptide (GGVGDVLRKPS). B, binding of
I-labeled
cytoskeletal proteins to scramble peptide
(GRDVKSPGLVG).
In this study we have found that the
11-amino acid synthetic peptide (GGVGDVLRKPS) competes effectively for
the ankyrin-binding site on both purified mouse T-lymphoma cell
IP receptor and IP
receptor-containing vesicles
(light density vesicles). We have determined the apparent inhibition
constants (K
) to be
25 and
50 nM,
respectively (Fig. 4A). As a positive control, we have
also found that this synthetic peptide competes effectively with CD44
(a well characterized ankyrin-binding protein) for ankyrin binding (Fig. 4B). These findings strongly suggest that the
amino acid sequence GGVGDVLRKPS is an important part of the
ankyrin-binding domain of T-lymphoma cell IP
receptor.
Figure 4:
Binding of I-labeled
IP
receptor, CD44, and IP
receptor-containing
vesicles to ankyrin.
I-Labeled samples (e.g. IP
receptor, CD44, or IP
receptor-containing vesicles) were incubated with ankyrin in the
presence of various concentrations of unlabeled 11-amino acid peptide
(GGVGDVLRKPS) as described under ``Materials and Methods.''
The specific binding observed in the absence of any of the competing
peptides is designated as 100%. The results represent an average of
duplicate determinations for each concentration of the competing
peptide used. A, binding of
I-labeled IP
receptor (
) and IP
receptor-containing vesicles
(
) to ankyrin. B, binding of
I-labeled
CD44 to ankyrin in the absence (a) and presence (b)
of the competing peptides.
Furthermore, we have confirmed that the binding of ankyrin to the
IP receptor in IP
receptor-containing vesicles
(light density vesicles) causes a remarkable inhibition on IP
binding (Table 1) and IP
-stimulated internal
Ca
release (Table 2) as shown
previously(12) . Most importantly, we have found that this
synthetic peptide (at concentrations ranging from 1 nM to 1
µM, comparable with those used to inhibit ankyrin binding
to IP
receptor (Fig. 4)) is capable of reversing the
inhibitory effects of ankyrin-induced IP
binding (Table 1) and IP
-mediated Ca
release (Table 2) in a concentration-dependent manner.
These findings suggest that the 11-amino acid sequence of GGVGDVLRKPS
(located close to the C terminus of the IP
receptor)
resides on the cytoplasmic side (not the luminal side) of IP
receptor-containing vesicles in mouse T-lymphoma cells. The exact
location of this 11-amino acid ankyrin-binding domain within the
peptide of mouse T-lymphoma cell IP
receptor remains to be
determined. We feel that identification of the ankyrin-binding domain
has allowed us to experimentally define the topology of the IP
receptor with regard to the side (the cytoplasmic side versus luminal side) on which the 11-amino acid sequence GGVGDVLRKPS
resides in the C terminus region. However, computer-generated models
(based on limited rat and mouse brain IP
receptor
sequences) predict that a region such as GGVGDVLRKPS would be located
on the luminal side of the Ca
storage
vesicles(25, 26, 27) . It is possible that
there is a differential arrangement of this sequence between mouse
T-lymphoma cell IP
receptor and brain IP
receptors. This point awaits further investigation. We believe,
however, that the unique 11-amino acid region GGVGDVLRKPS is not only
important for ankyrin binding, but it also plays a pivotal role in the
regulation of IP
receptor-mediated internal Ca
release during lymphocyte activation.