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(Received for publication, November 1, 1995; and in revised form, January 16, 1996) From the
Peptides which stimulate the formation of inositol phosphates
(InoPs) in lymphocyte cell lines were identified by screening synthetic
peptide libraries composed of random sequences of hexapeptides. The
peptides containing the consensus sequence XKYX(P/V)M
were found to be most active in the phospholipase C (PLC)-mediated
formation of InoPs in a human B myeloma cell line, U266. The peptides
also stimulated the phosphoinositide hydrolysis and the release of
[Ca Many biological actions such as ligand-receptor interactions are
based on the specificity of proteins conferred by the primary sequence
of amino acids as well as the secondary and tertiary structures
dictated by the primary sequence. Of special importance is the
formation of local environments, such as active site and motif, which
play a key role in the function of a protein. Among the random
sequences of short peptides, there may be sequence(s) which can act on
such local environments, and these sequences could serve as the lead
for the development of effective new drugs. Recently various methods
have been developed for identification of the sequence(s) of interest
from vast mixtures of random peptide sequences or polymers (template)
with various side chain groups (chemical diversity libraries) within a
short period of time with minimal
effort(1, 2, 3, 4, 5) .
Successful screening of these libraries has been described not only for
epitopes recognized by monoclonal
antibodies(6, 7, 8) , but also for the
identification of the biologically active peptides such as
antibacterial and antifungal peptides(2) , human
immunodeficiency virus protease inhibitors(9) ,
substrate-analog trypsin inhibitors(10) , and
interleukin-8-specific antagonist(11) . Phosphoinositide-specific phospholipase C (PLC) ( Generally, in mature B lymphocytes, interaction of ligands with the
membrane-bound immunoglobulin triggers a series of metabolic events
including the rapid activation of a protein tyrosine kinase associated
with the B-cell receptor(23, 24, 25) . The
activation of these kinases leads to the rapid tyrosine phosphorylation
of a number of substrates including the phospholipase C (PLC)- A GTP-binding protein (G-protein) that has been
postulated as a modulator of the PLC- In this
study, we have identified peptides which stimulate the formation of
InoPs in cells from libraries of hexapeptides. The peptides appear to
have positive effects on the formation of InoPs and
[Ca
The testing of each of total 114 peptide pools of PS-SPCLs
permits the determination of the most effective amino acid at each of
the six positions in a hexapeptide. The results of the initial
screening of the peptide library are shown in Fig. 1. The
peptide mixtures, WXXXXX-NH
Figure 1:
Initial screening of the
PS-SPCLs for the peptides which stimulate the formation of InoPs. Each
panel represents the result obtained with the peptide pools with the
known amino acids at each of the six positions of hexapeptides. The six
positions are individually defined (O
The amino acids chosen for reiterative synthesis of
peptides were as follows: 1st, W, M, and R; 2nd, K and H; 3rd, Y and F;
4th, M, V, and I; 5th, P, V, and R; and sixth, M. The selected amino
acids at 4th, 5th, and 6th position were linked in all combinations,
and for the 1st, 2nd, and 3rd positions the mixtures of the selected
amino acids used were: X The reiterative synthesis generates
nine peptide pools containing 3
Figure 2:
Effect of the peptide pools synthesized
from the selected amino acids and the single selected peptide for their
ability to stimulate the InoPs formation in U266 cell line. A,
the first three positions consist of mixture (X) of defined
amino acid (X
IP
Figure 3:
Changes in
[Ca
In order to investigate if the
peptides have a general effect on other cell types, we examined the
effect of an active peptide (WKYMVM-NH
Figure 4:
Stimulation of InoPs formation by a
peptide in various cell lines. Subconfluent cultures of each type were
prelabeled with myo-[
PLC- To investigate the possible
involvement of a G-protein in the peptide-induced formation of InoPs,
U266 cells were treated with pertussis toxin (150 ng/ml) for 12 h prior
to the addition of WKYMVM-NH
Figure 5:
Effects of pertussis toxin on the
peptide-induced formation of InoPs in U266 cells. Subconfluent cultures
of each cell lines were prelabeled with myo-[
Figure 6:
Lack of additive effect on the formation
of InoPs by AlF
To
investigate the possible action of the peptide through the binding to a
cell-surface receptor, a fixed number of U266 cells was incubated for
90 min at room temperature in the presence of the various
concentrations of
Figure 7:
Binding of
Figure 8:
Lack of effect by fMLP on the formation of
InoPs in U266 cells. myo-[
In this study, we have found that the hexapeptides with the
following consensus sequence XKYX(P/V)M (where X is any amino acid) stimulate the formation of InoPs in the B
lymphocyte cell types through the action of PLC via a pertussis
toxin-sensitive G-protein coupled cell-surface receptor. These peptides
were identified by screening the Positional Scanning Synthetic Peptide
Combinatorial Libraries described by Houghten et al.(2) in which each position of hexapeptide is fixed with a
known amino acid and the rest of five positions are any of 20 amino
acids (in this study cysteine was omitted). Detailed studies with
one of the active peptides WKYMVM-NH It
is widely accepted that the hydrolysis of PIP In this report, we suggest that the pertussis
toxin-sensitive G-protein is directly involved in the peptide-induced
PI hydrolysis in B cells. This is evident from the observations that
pertussis toxin inhibits the formation of InoPs and the release of
[Ca Experiments with the peptide labeled with For further study,
the molecular characterization of the receptor, G-protein, and PLC
related to this peptide signaling could provide the insight into the
understanding of the ligand-triggered signal cascade in certain cell
types.
Volume 271,
Number 14,
Issue of April 5, 1996 pp. 8170-8175
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
]
in HL60
and U937 cell lines. On the other hand, these peptides showed no effect
in the following cell lines: NIH3T3, PC12, Daudi, Sp2, Jurkat, H9,
Molt-4, SupT-1, K562, and RBL-2H3. The result suggests the possibility
that the peptides may have cell type specificity. Experiments with one
of the active peptides, WKYMVM-NH
showed that its action
mimics the effect of AlF
which is a
G-protein activator in the InoPs generation, and pertussis toxin
partially blocked the InoPs accumulation and
[Ca] release
induced by the peptide in the U266 cells. Binding assays with the
peptide labeled with 
I showed that U266 cells have a
saturable number of binding sites for the peptide. Taken together,
these results suggest that the peptides could activate PLC-mediated
signal transduction via a pertussis toxin-sensitive G-protein coupled
receptor in certain cell types.
)plays a
pivotal role in the signal pathway for cell growth and
differentiation(12, 13) . The activated PLC catalyzes
hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP)
into two intracellular second messengers, inositol 1,4,5-trisphosphate
(IP
) and diacylglycerol (DAG). The IP induces
an increase in intracellular free calcium concentration
([Ca]), while DAG
directly activates protein kinase
C(14, 15, 16, 17) . On the basis of
amino acid sequence similarity, the PLCs in mammalian tissue have been
divided into three types (PLC-
, -
, and -
), each of which
comprises more than one
subtype(18, 19, 20, 21, 22) . 1
and PLC-2(26, 27, 28, 29) . It
is assumed that the phosphorylation of PLC- type will lead to
enzymatic activation. Therefore, the activation of protein tyrosine
kinase and PLC- appears to be essential for ligand-mediated B-cell
activation. activity has partially been
characterized at the molecular
level(30, 31, 32) . B cells that have been
treated with tyrosine kinase inhibitors (genistein, herbimycin,
tyrphostin) can still undergo phosphoinositide (PI) hydrolysis. The
cells also display increases in
[Ca] when stimulated
directly through G-proteins by
AlF(28, 33) . Similarly,
inactivation of the B-cell receptor abolishes subsequent tyrosine
kinase activation, although the cells continue to respond to the
G-protein-activating agent, mastoparan(34) . These observations
suggest that B-cell activation may involve PLC- types or another
isoform of PLC that can be mediated by G-proteins independently of
tyrosine phosphorylation of PLC- types. PLC- types are shown
to be activated by members of the G
or
G
subunit of hetertrimeric
G-protein(30, 35) . However, the implication of
G-protein in B-cell activation remains to be characterized.] release in
certain cell types such as U266 (human B myeloma), HL 60 (human
promyelocytic lymphoma), and U937 (human histiocytic lymphoma). These
effects appear to be mediated through the binding of the peptide to
cell-surface receptors.
Materials
Fmoc amino acids were obtained from
Millipore Co (Bedford, MA). Rapidamide resin was purchased from DuPont
(Boston, MA). Myo-[2-H]inositol and the ECL kit
were purchased from Amersham Corp. (Bucks, UK). Fura-2
pentaacetoxymethyl ester (Fura-2/AM) and 1,
2-bis(2-aminophenoxy)ethane-N,N,N`N`-tetraacetoxymethyl
ester (BAPTA/AM) were purchased from Molecular Probes (Eugene, OR).
RPMI 1640 was obtained from Life Technologies, Inc. Dialyzed fetal
bovine serum and supplemented bovine calf serum were purchased from
Hyclone Laboratories Inc. (Logen, UT).Cell Culture
The U266 B myeloma cell and other
cell lines were purchased from the ATCC, Rockville, MD, and maintained
in RPMI 1640 medium supplemented with 10% fetal calf serum. Cells were
maintained at densities of 2 
10
cells/ml at 37
°C in a humidified incubator supplied with 95% air and 5%
CO.Preparation of Positional Scanning-Synthetic Peptide
Combinatorial Libraries (PS-SPCLs)
Libraries were synthesized
according to the protocol of Houghten et al.(2) and
Pinilla et al.(5) . Briefly, PS-SPCLs, consisting of
six-residue peptide sequences having free N termini and amidated C
termini, were synthesized. A single position in each peptide mixture
was individually and specifically defined with 19 of the 20 natural L-amino acids (cysteine excluded), while the five remaining
positions consisted of mixtures of the same 19 amino acids. Defined
positions are represented by O and the positions with mixed
amino acids are represented by X. The six sets of PS-SPCLs
(total 114 pools) are represented by the formula; OXXXXX-NH, XOXXXX-NH, XXOXXX-NH, XXXOXX-NH, XXXXOX-NH, and XXXXXO
-NH. Libraries of peptides were
constructed on Rapidamide resin beads as described
elsewhere(36) . Fmoc chemistry was used. The resin beads were
distributed into different reaction vessels for each amino acid at each
coupling step; pooled, washed, and thoroughly mixed for randomization;
deprotected; and redistributed into the various vessels again for the
next coupling step and so forth. The amount of each amino acids used to
yield approximately equimolar coupling was determined empirically. The
completeness of each reaction was checked with ninhydrin(37) .
Side chains were deprotected with a mixture of trifluoroacetic acid
(ethanedithiol:water:thioanisole; 90:5:4:1, vol/vol). The 114 peptide
mixtures were individually extracted with water, lyophilized, and
dissolved in water at a final concentration of 27 nM for a
peptide sequence in each pool.Synthesis of Individual Peptides
The peptides with
known sequences were synthesized on a Rapidamide resin as carrier.
Briefly, peptides were assembled via standard Fmoc/t-butyl
strategy on an acid-labile linker. Upon treatment with trifluoroacetic
acid, the peptides were cleaved and released as amides at their
C-terminal position.Peptide Analysis
The efficiency of coupling was
determined by analyzing the peptide solution, before and after
coupling, by reversed-phase HPLC and by reading the absorbance at 215
nm. The peptides were characterized by chromatography on a C18
reversed-phase HPLC column (Vydac, 218TP1022, 22 300 mm). The
peptides were eluted with 0-40% gradient of CHCN in
0.05% trifluoroacetic acid. The composition of the peptides was
confirmed by amino acid analysis(38) , and the peptides were
sequenced by using an Applied Biosystems 473A protein/peptide
microsequencer.Measurement of Inositol Phosphates Induced by
Peptides
The cells grown in culture were harvested by
centrifugation, washed with inositol-free RPMI 1640 medium, and
resuspended at a density of 2 10 cell/ml in the
same medium. The cells were labeled with myo-[H]inositol (1 µCi/10 cell, Amersham) for 24 h at 37 °C and rinsed twice with
inositol-free RPMI 1640 medium containing 0.5% fetal bovine serum, 20
mM Hepes, pH 7.2, 20 mM LiCl, and bovine serum
albumin (1 mg/ml) and resuspended at a density of 2 10
cells/ml. A portion (0.1 ml) of the cell suspension was
transferred to a microcentrifuge tube and incubated at 37 °C for 15
min. Stimulation of PIP hydrolysis was initiated by
addition of peptides or solvents for indicated time, and the reaction
terminated by the addition of 0.1 ml of ice-cold 5% perchloric acid
(HClO). After 30 min in an ice bath, the tubes were
centrifuged, and the supernatants were diluted 5-folds with distilled
water and applied to Bio-Rad Dowex AG 1-X8 anion exchange columns. Each
column was then washed with 2 ml of distilled water followed by 10 ml
of 60 mM ammonium formate containing 5 mM sodium
tetraborate. Total inositol phosphates were eluted with a solution
containing 1 M ammonium formate and 0.1 M formic
acid. To determine IP, the columns was first washed with
0.4 M ammonium formate and 0.1 M formic acid, and
then IP was eluted with 0.8 M ammonium formate and
0.1 M formic acid. Radioactivity of
[H]inositol phosphates was determined by counting
in a scintillation counter (Tri-Packard, Meriden, CT).Measurement of
[Ca
The level of
intracellular Ca]
was determined using
fura-2/AM(39) . Briefly, cells were incubated in serum-free
RPMI 1640 medium with 3 µM fura-2/AM at 37 °C for 50
min with continuous stirring. The cells were washed with serum-free
RPMI 1640 medium. Sulfinpyrazone (250 µM) was included in
all solutions to prevent dye leakage from the cells. Before the
measurement, a small portion of the cells was aliquoted into a
centrifuge tube and centrifuged. The pelleted cells were resuspended in
Ca-free Locke's solution (158.4 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl, 5
mM Hepes, 10 mM glucose, and 0.2 mM EGTA, pH
7.3). Changes in fluorescence ratio were determined at dual excitation
wavelengths of 340 and 380 nm and emission wavelength of 500 nm. The
calibration of the fluorescence ratio in terms of
[Ca] was performed according to
Grynkiewicz et al.(40) .Cell Binding Experiments
Peptide
(MKYMPM-NH) was radiolabeled with I
(IODO-GEN, Pierce)(41) . U266 cells were suspended in
phosphate-buffered saline, pH 7.4 containing 0.1% bovine serum albumin.
Binding was initiated with the addition of various amounts of I-labeled peptide. Equilibrium binding was established at
room temperature for 90 min and terminated by rapid filtration through
multiscreen-FB filters (Millipore Co.) followed by five washes with
ice-cold binding buffer. The radioactivity of the punched filter
membrane was determined in a -counter. The level of specific
binding was determined after correction for the nonspecific binding
occurring in the presence of 250-fold excess unlabeled peptide.
, was found to strongly
stimulate the formation of InoPs in U266 cells. The amino acids with
slightly less active than tryptophan (W) at the first position were
methionine (M) and arginine (R). For the second position, several amino
acids appear to be active, lysine (K) and histidine (H) being slightly
more active than others. The active amino acids at the third position
were tyrosine (Y) and phenylalanine (F), tyrosine being slightly more
active. The most active amino acid at the fourth position were
methionine (M). Valine (V) and proline (P) appear to be more active
than other amino acids at the fifth position. The sixth position showed
marked contrast between the active amino acid (methionine, M) and other
amino acids.
and O . . .) with each of the 19 L-amino
acids. The remaining five positions consist of mixtures (X) of
19 L-amino acids (cysteine excluded). The library consists of
114 peptide pools; the PS-SPCL in total is made up of 47,045,881
different peptides. U266 cells (2 10) were
prelabeled with [H]inositol (1 µCi/10 cell) for 24 h in inositol-free medium. The cells were then
rinsed and incubated for 15 min in LiCl mixture and stimulated with
peptide mixture. [H]InoPs formations were
analyzed as described under ``Experimental Procedures.'' Each bar represents the inositol phosphates formation stimulated by
a peptide pool.
XXMPM-NH, XXXMVM-NH, XXXMRM-NH, XXXVPM-NH, XXXVVM-NH, XXXVRM-NH, XXXIPM-NH, XXXIVM-NH, XXXIRM-NH. X: W, M, R; X: K, H; X: Y, F. 2 2 3 3
1 = 108 individual hexapeptides. The nine peptide pools
were tested for stimulation of the formation of InoPs. Among these, XXXMPM-NH, XXXMVM-NH and XXXVPM-NH were found to be more active than others
for formation of InoPs (Fig. 2A). Each active peptide
pools was resolved and purified by using HPLC on a C18 column. Each
peak fraction from the C18 column was tested for effect on the
formation of InoPs, and the amino acid sequence of the active fractions
were determined. Fig. 2B shows the result obtained with
one of the active peptide WKYMVM-NH. The peptide showed the
half maximal activity at about 6 10
M. Also, most of the active peptides have a consensus of XKYX(P/V)M. A time course study showed that the
formation of IP reached a maximal level in 5 min and
returned to a basal level in 20 min after the addition of the peptide
to U266 cells. However, the formation of total InoPs was consistently
elevated throughout the 20 min incubation with the peptides (data not
shown).
: W, M, R; X: K,
H; X: Y, F). The remaining three positions were
individually defined with each of the selected amino acids. The partial
library consists of 9 mixtures; each mixture contains 12 single
peptides. The total number of peptides in 9 mixtures were 12 9
= 108. Error bars were omitted for clarity of the figure. B, A peptide WKYMVM-NH selected from the
experiment described above was tested for stimulation of InoPs
formation in U266 cells.
is one of the major second messengers that
triggers Ca release from the internal Ca pools in the cell. Generally, the elevation of
[Ca] is achieved by both
Ca release from the internal stores as well as by
influx from the extracellular environment. Fig. 3A shows
that there was peptide dose-dependent increase of
[Ca
]. In order to determine the
peptide-induced Ca release from the internal stores,
we measured the Ca mobilization of U266 cell in
Ca-free medium containing 0.2 mM EGTA. In
addition, depleting intracellular free Ca by
preloading U266 cells with the Ca-buffering agent
BAPTA completely inhibited the change in
[Ca] at maximal effective
concentration of the peptide (Fig. 3B). These results
demonstrate that the [Ca] increase induced by the peptide was not due to mobilization from
the extracellular Ca, but from the intracellular
Ca reservoir.
] after treatment
with peptide in the absence of external Ca. A, [Ca] was
determined fluorometrically using fura-2/AM as described
under``Experimental Procedures.'' Before measurement, U266
cells (2 10) were suspended in
Ca-free Locke's solution containing 0.2 mM EGTA. Cells were treated with various concentrations of
WKYMVM-NH where indicated by the arrow. B, U266
cells in upper trace were stimulated with excess
WKYMVM-NH. Cells used in the lower trace (+
BAPTA label) had been preincubated in RPMI containing 60 µM BAPTA acetoxymethyl ester for 30 min at 37
°C.
) on the formation of
InoPs in NIH3T3 (NIH Swiss mouse embryo fibroblast) and PC12 (rat
adrenal pheochromocytoma) cells. It showed no effect on either cells (Fig. 4). Daudi (human Burkitt lymphoma), Sp2 (mouse myeloma),
Jurkat (human acute T-cell leukemia), H9 (human T-cell lymphoma),
Molt-4 (human peripheral blood T cell), SupT-1 (human T-cell
lymphoblastic lymphoma), K562 (human chronic myelogenous leukemia),
RBL-2H3 (rat mast cell), U937 and HL60 cells were tested for the effect
of the peptide in a variety cell types originated from hemopoietic
lineage. Among these cells, the peptide stimulated the formation of
InoPs only in HL60 and U937 cell lines (Fig. 4). The results
suggest that the peptides increase the formation of InoPs only in cell
type-specific manner.
H]inositol (1
µCi/10 cell) for 24 h in serum-free RPMI media. Cells
were then treated with peptides (WKYMVM-NH, 1
µM) in RPMI containing 20 mM Hepes, pH 7.2, 20
mM LiCl, and 1 mg/ml bovine serum albumin for 10 min at 37
°C, and [H]InoPs were extracted and separated
by Dowex AG 1-X8 column as described under ``Experimental
Procedures.'' Results are presented as the total InoPs and
expressed as mean ± S.D. from three independent experiments done
in duplicate.
is activated by phosphorylation of
specific tyrosine residues. The activated PLC- catalyzes
hydrolysis of PIP into two intracellular second messengers,
IP and DAG(19) . It is assumed that the
phosphorylation of PLC- will lead to its activation. Experiments
were therefore performed to investigate whether tyrosines of PLC-
were phosphorylated in U266 cells in response to the peptides and
whether the time course of the phosphorylation was compatible with the
changes in the InoPs formation. However, there were no appreciable
changes in the level of tyrosine phosphorylation of PLC 1 and
2 enzymes within the time period examined, although PLC 1 and
2 were clearly detectable in U266 cells (data not shown). This
result suggests that the peptides may not induce PI hydrolysis and
[Ca] release through the
activation of PLC- type.. The peptide-dependent
formation of InoPs was reduced by 70% (Fig. 5).
AlF as a G-protein activator induced the
InoPs formation in U266 cells. In addition, increasing amounts of
WKYMVM-NH in the presence of a fixed amount of
AlF showed no additive effect on the
formation of InoPs (Fig. 6). These results support that
pertussis toxin-sensitive G-proteins may be involved in the PLC-
mediated PI hydrolysis in response to the peptides in U266 cells.
H]inositol (1 µCi/10 cell) for 24 h in serum-free RPMI media. U266 cells were treated
with indicated concentration of WKYMVM-NH in the absence
(
) or presence (
) of 150 ng/ml of pertussis toxin for 12 h,
and [H]InoP formations were analyzed as described
under ``Experimental Procedures.'' Results are expressed as
means ± S.D. from three independent
experiments.
and
WKYMVM-NH. myo-[H]Inositol-labeled cells were
incubated with the indicated concentrations of WKYMVM-NH in
the presence of AlF (20 µM AlCl3/10 mM NaF) after then, perchloric acid added and
InoPs formation was quantitated as described under ``Experimental
Procedures.'' The data are the mean ± S.D. of triplicate
determinants.
I-labeled peptide. After washing the
cells, we determined the amount of bound I-labeled
peptide using multiscreen binding assay system (Millipore). A
representative result of these assays is shown in Fig. 7. As the
concentration of I-labeled peptide increased, there was a
corresponding increase in the amount of the peptide bound to the cells
until it reached a plateau, suggesting that there is a saturable number
of binding sites for the peptide on the surface of U266 cells. On the
other hand, fMLP is known as the peptide that stimulates the formation
of InoPs through the cell-surface receptor which is coupled with a
G-protein in neutrophils(42, 43, 44) . Thus,
it was possible that the peptides we have identified in this study act
on the fMLP receptor. However, fMLP showed no effect on the formation
of InoPs in U266 cells (Fig. 8). Therefore, it appears that our
peptide does not bind to the fMLP receptor.
I-MKYMPM-NH to U266 cells. U266 cells (10) in 0.2 ml of
phosphate-buffered saline containing 0.1% bovine serum albumin were
incubated with an increasing concentrations of I-MKYMPM-NH for 90 min at room temperature.
Each data point represents specific binding, which was computed by
subtracting nonspecific binding in the presence of excess unlabeled
peptide from total binding. Data are presented as means ± S.E.
of the separate experiments, each performed in triplicate. Error bars
were omitted for clarity of the figure.
H]Inositol-labeled cells were
incubated with the indicated concentrations of WKYMVM-NH or
fMLP, then perchloric acid was added, and InoP formation was
quantitated as described under ``Experimental Procedures.''
The data are the mean ± S.D. of triplicate
determinants.
suggest that the
peptide stimulates the formation of InoPs only in certain cell types
such as U266, HL60, and U937 cells. Up to now we do not know exactly
what induced the differential effect on each cell types because of the
limited information in cell expression patterns for putative receptors,
G-proteins, and PLC isozymes. The pertussis toxin-sensitive G-proteins
are inactivated by ADP-ribosylation on the 
subunit and include
members of G or G
family(45) . In
NIH3T3 and Sp2 cells, an increase in IP and a subsequent
transition increase in [Ca] were elicited through a pertussis toxin-sensitive manner by
lysophosphatidic acid(46) . Also, C3a and
lysophosphatidylserine stimulated the generation of IP that
was inhibited by pertussis toxin-sensitive G-protein signal pathway in
mast cell(47, 48) . Nevertheless, our peptide have no
effect on the formation InoPs in these NIH3T3, Sp2, and mast cells.
Thus, the cell type-specific action of our peptide might be due to the
existence of putative receptor specific for the peptide, rather than
the difference of the downstream components which need for the
pertussis toxin-sensitive InoPs formation. One of the major goals of
future work is to identify the putative receptor for the peptide. by PLC and
subsequent formation of DAG and IP is a major signaling
pathway employed by a variety of hormones, growth factors, and various
neurotransmitters(14, 15, 16, 17) .
IP stimulates the release of free Ca ions
from intracellular Ca stores. Ca leads to the activation of one or more isozymes of protein kinase
C, which may participate in the induction of various immediately early
genes such as c-fos and c-myc(49) .
Accordingly, peptide-induced IP formation and the
subsequent release of Ca is very important for B-cell
activation. As in T lymphocytes, antigen receptor interaction in B
cells leads to tyrosine phosphorylation of PLC-1 (28) ,
but also of PLC-2 that appears to be the major isozyme in B
cells(50) . Recently, tyrosine phosphorylation of PLC-2
was reported to be induced in murine B cells upon cross-linking of
membrane immunoglobulins (29, 30) and in the HL60
granulocytes stimulated with pervanadate(51) . In most cases,
the tyrosine phosphorylation of PLC- is accompanied by changes in
PI turnover. Despite this correlation, however, the peptides do not
affect phosphorylation of the tyrosines of PLC-1 and -2
enzymes. These results suggest that another isoform of PLC may be
involved in the formation of InoPs in the B cells treated with the
peptides.] by WKYMVM-NH (data not shown). Furthermore, the peptide-induced PI hydrolysis
mimics the stimulatory effect by AlF (G-protein activator). These data provide strong support for the
contention that the peptide-mediated activation of PLC in U266 cell
requires a G-protein, although an isoform of G-proteins are not
presently identified. It is clear that the pertussis toxin-insensitive
mechanism is mediated by -subunits of the recently discovered
G
family of G-proteins(52, 53) . However,
the pertussis toxin-sensitive mechanism for activation of PLC is less
well understood. Several recent reports suggest that the pertussis
toxin-sensitive response can be reconstituted through receptor-mediated
release of subunits from members of the G class,
and the toxin apparently blocks the activation of PLC-2 by
interfering with the release of the subunits from the
trimeric G-proteins(45, 54, 55) . In
addition, we detected that the PLC-2 was preferentially expressed
more than other -isoforms in U266 cell (data not shown).
Therefore, PLC-2 may be potential mediator for the action of the
peptides.I
suggest that the cells have a saturable number of binding sites for the
peptide on the cell surface, possibly a receptor. Presently we have no
information on the nature of the receptor. It is likely that the
formation of InoPs in response to the peptide is mediated through the
interaction of the peptide to a cell-surface receptor. In addition, the
peptide receptors may be different from the fMLP receptors because fMLP
does not increase the level of InoPs in U266 cells. However, although
peptide receptors are distinct from the fMLP receptors, it is possible
that the two receptors share the same signal transduction pathways,
because both fMLP and the peptide we reported here all stimulate PI
hydrolysis in a pertussis toxin-sensitive manner.
), phosphatidylinositol 4,5-bisphosphate;
IP, inositol 1,4,5-trisphosphate; DAG, diacylglycerol;
G-protein, GTP binding protein; InoP, inositol phosphate; PI,
phosphoinositide; PS-SPCLs, positional scanning-synthetic peptide
combinatorial libraries; fMLP, N-formyl-methionyl-leucyl-phenylalanine; Fmoc, N-(9-fluorenyl)methyl)oxycarbonyl; Fura-2/AM, fura-2
pentaacetoxymethyl ester; BAPTA/AM,
1,2-bis(2-aminophenoxy)ethaneN,N,N`N`-tetraacetoxymethyl
ester.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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