(Received for publication, July 25, 1994; and in revised form, September 19, 1994)
From the
The binding of insulin-like growth factor II (IGF II) to the
mannose 6-phosphate (M6P)/IGF II receptor has previously been reported
to induce the activation of trimeric G proteins by
functional coupling to a 14-amino acid region within the cytoplasmic
receptor domain (Nishimoto, I., Murayama, Y., Katada, T., Ui, M., and
Ogata, E.(1989) J. Biol. Chem. 264, 14029-14038). In the
present study, we examined further the potential functional coupling of
G-proteins with the human M6P/IGF II receptor and mutant receptors
lacking the proposed G-protein activator sequence. IGF II treatment of
mouse L-cells expressing either wild type or mutant M6P/IGF II
receptors failed to attenuate the pertussis toxin-catalyzed
modification of a 40-kDa protein or enhance GTPase activity. In broken
L-cell membranes expressing wild type or mutant M6P/IGF II receptors,
30 nM IGF II also failed to affect the pertussis toxin
substrate activity. By using phospholipid vesicles reconstituted with
human wild type or mutant M6P/IGF II receptors and pertussis
toxin-sensitive G-proteins, no stimulation of GTP
S binding to or
GTPase activity of G
, G
, or
G
/G
mixtures were observed in response to 1
µM IGF II. Furthermore, in vesicles containing purified
wild type M6P/IGF II receptors and monomeric G
or
G
and
dimers no effects of IGF II on
GTP
S binding could be detected. However, when vesicles
reconstituted with M6P/IGF II receptors and G
proteins
were incubated with 100 µM mastoparan GTP
S binding
was stimulated and GTPase activity was increased significantly. These
results indicate that the human M6P/IGF II receptor neither interacts
with G-proteins in mouse L-cell membranes nor is coupled to G
proteins in phospholipid vesicles. This study suggests strongly
that the M6P/IGF II receptor does not function in transmembrane
signaling in response to IGF II.
The 300-kDa mannose 6-phosphate (M6P) ()receptor
medi-ates the transport of newly sythesized soluble acid hydrolases
from the Golgi to an endosomal/prelysosomal compartment where
dissociation of the ligands occurs. The released acid hydrolases are
delivered to lysosomes while the receptors recycle back to the Golgi or
move to the plasma membrane. About 10% of the 300-kDa M6P receptors are
localized at the cell surface where they function in binding and
internalization of exogenous lysosomal enzymes and the nonglycosylated
insulin-like growth factor II (IGF II) (see Refs. 1 and 2 for review).
Both classes of receptor ligand bind to distinct sites, and the
receptor can bind lysosomal enzymes and IGF II simultaneously (3, 4, 5) .
IGF II is functionally and
structurally related to IGF I, and both factors have been shown to act
in an autocrine/paracrine fashion regulating growth and
differentiation(6, 7, 8) . The use of various
antibodies against IGF I or M6P/IGF II
receptors(9, 10) , mutants of IGF II with reduced
affinities for the M6P/IGF II receptor (11, 12) and
targeted mutagenesis in mice (13) indicate that most of the
metabolic and mitogenic effects of IGF II are mediated by the IGF I
receptor. The IGF I receptor is composed of an extracellular
ligand-binding subunit and an intracellular
subunit that
contains an intrinsic ligand-activated tyrosine kinase. However, there
are reports suggesting that certain IGF II responses are mediated by
its binding to the monomeric M6P/IGF II receptor, which lacks tyrosine
kinase activity in its cytoplasmic domain.
IGF II stimulates the
Na/H
exchange and the production of
inositol trisphosphate and diacylglycerol in basolateral membranes of
renal proximal tubular cells(14, 15) . Other studies
showed that IGF II promotes amino acid uptake in myoblasts (16) and DNA synthesis and Ca
influx in
primed-competent BALB/c 3T3 cells(17) . The involvement of
G-proteins in the latter processes was suggested, since the effects of
IGF II were completely abolished by pretreatment of cells with
pertussis toxin. These data were confirmed by the demonstration of IGF
II-induced activation of isolated G
proteins via
functional coupling to purified M6P/IGF II receptors in phospholipid
vesicles and cell membranes(18, 19) . Furthermore, the
IGF II-induced activation of G
proteins could be inhibited
by M6P and M6P-containing lysosomal enzymes(19) .
Because of
structural similarities with mastoparan, a wasp venom peptide
activating G and G
proteins(20) ,
Nishimoto and co-workers (21) identified a region corresponding
to residues 123-136 of the cytoplasmic domain of the human
M6P/IGF II receptor, which they proposed may play a major role in the
receptor-activating function on G
proteins. In contrast,
the large family of known G-protein-coupled receptors is characterized
by a common sequence pattern with seven hydrophobic transmembrane
helices resulting in the same basic three-dimensional structure (for
review see (22) ). Binding of ligands to those receptors
stimulates the activation of trimeric G-proteins by catalyzing a
GDP/GTP exchange, which initiates either intracellular second messenger
formation or regulation of ion channel function(23) .
In the
present study we reevaluate the previously suggested participation of
M6P/IGF II receptors in a G-protein-dependent signaling pathway by
different experimental approaches, including the reconstitution of
purified truncated receptors and G-proteins in phospholipid vesicles.
Our data failed to provide evidence for IGF II-induced functional
coupling and activation of G proteins through the M6P/IGF
II receptor.
Human recombinant IGF I and IGF II were generously supplied
by Dr. W. Märki (Ciba Geigy, Basel) or were
purchased from GroPep (Adelaide, SA). Phosphomannan from Hansenula
hostii was a gift of Dr. M. E. Slodki (Northern Regional Research
Center, Peoria, IL). Pentamannosyl 6-O-phosphate-substituted
bovine serum albumin (PMP-BSA) was prepared and iodinated with the aid
of IODO-GEN (24) to a specific activity of 65 µCi/µg.
The following reagents were obtained commercially as indicated:
TranS-label (ICN, Biomedical; 1100 Ci/mmol);
[
S]GTP
S (1100-1400 Ci/mmol),
[
-
P]ATP,
[
P]phosphoric acid, and
C-labeled
molecular mass standard proteins (DuPont-NEN); carrier-free
Na
I, [
-
P]NAD (30 Ci/mmol),
prestained high molecular standards, and the ECL detection reagents
(Amersham Corp.); IODO-GEN and disuccinimidyl suberate (DSS) (Pierce),
chloramine T, azolectin, mastoparan (Vespula leviesii),
pertussis toxin, and CHAPS (Sigma); Pansorbin (10% staphylococcus
aureus cell suspension) (Calbiochem).
[
-
P]GTP was synthesized from
[
P]phosphoric acid as described(25) .
Oligonucleotides were synthesized on an Applied Biosystems model 381A solid phase synthesizer. The cDNA of human M6P/IGF II receptor and mouse L-cells deficient in M6P/IGF II receptor was kindly provided by Drs. William S. Sly (St. Louis University, St. Louis) and Stuart Kornfeld (Washington University, St. Louis).
Figure 1:
Immunoprecipitation of M6P/IGF II
receptors and affinity cross-linking of I-IGF II and
I-IGF I to nontransfected and transfected mouse L-cells. A, L-cells expressing wild type or mutant M6P/IGF II receptors
were metabolically labeled with [
S]methionine.
The receptors were immunoprecipitated by antibodies directed against
the luminal domain and analyzed by SDS-PAGE and fluorography. B, cells were incubated with 3 ng
I-IGF II or
I-IGF I in the absence or presence of unlabeled IGF II or
IGF I (0.5 µg), respectively. The receptor-ligand complexes were
cross-linked with DSS and electrophoresed on a 7.5% polyacrylamide gel
under reducing conditions. An autoradiogram of a representative
experiment is shown. The positions of the M6P/IGF II, the IGF I
receptor, and molecular mass markers (kDa) are
indicated.
The specificity of I-IGF II binding to mutant cell surface receptors was
investigated by affinity cross-linking. For this purpose, cells were
incubated with
I-labeled IGF II or IGF I, and then
treated with DSS followed by SDS-PAGE under reducing conditions.
Cross-linking with
I-IGF II resulted in exclusive
labeling of a 250-kDa band in wild type and D123-136 mutant
M6P/IGF II receptor-expressing L-cells, whereas no 250-kDa cross-link
product was observed in nontransfected L-cells (Fig. 1B). Under the conditions used, only faint
binding of
I-IGF II to the 135-kDa subunit of the IGF I
receptor was detectable in nontransfected cell lines. The binding of
I-IGF II to the receptor was inhibited by an excess of
unlabeled IGF II. In L-cells expressing the truncated M6P/IGF II
receptor mutants
I-IGF II bound specifically to
electrophoretically faster moving polypeptides than the wild type
receptor polypeptides (not shown) corresponding to the electrophoretic
pattern in Fig. 1A. After cross-linkage of
I-IGF I bound to cell surface receptors of nontransfected
and transfected mouse L-cells, in all cell lines one prominent labeled
band of apparent 135 kDa was detectable representing the IGF I receptor
subunit (Fig. 1B). No labeling of the M6P/IGF II
receptor with
I-IGF I was observed. The binding of
I-IGF I to the 135-kDa protein was completely inhibited
in the presence of an excess of unlabeled IGF I. These results indicate
that the wild type and mutant M6P/IGF II receptors expressed in
receptor-deficient cell lines are normally distributed and that the
binding properties of mutant receptors for both classes of ligands were
comparable with those of wild type receptors. Furthermore, IGF II bound
specifically only to the M6P/IGF II and not to the IGF I receptor.
Figure 2:
Effect of IGF II on pertussis toxin
substrate activity in nontransfected and transfected L-cells.
Nontransfected, wild type, and D123-136 mutant M6P/IGF II
receptor-expressing L-cells were incubated with 30 nM IGF II
alone or in combination with 5 mM M6P or G6P for 10 min. The
prepared membranes (100 µg of protein) were incubated with 40
µg/ml Ptx and 10 µM [P]NAD at
30 °C for 30 min. Proteins were electrophoresed by SDS-PAGE
containing 10-15% acrylamide. As a control, membranes were
prepared from cells preincubated for 16 h with Ptx (0.1 µg/ml). The
molar mass of the Ptx substrate activity is
indicated.
In a
second approach, membranes prepared from different transfected and
nontransfected L-cell lines were preincubated with 30 nM IGF
II followed by Ptx-catalyzed ADP-ribosylation of the 40-kDa protein.
This approach was carried out according to the experimental procedures
of Nishimoto et al.(18) examining the effect of IGF
II preincubation of BALB/c 3T3 membranes on the subsequent
Ptx-catalyzed modification of the 40-kDa protein. Fig. 3indicates that IGF II did not cause a significant
reduction in Ptx-substrate activity in any of the tested cell lines.
Reduced ADP-ribosylation of the 40-kDa protein in membranes from
L-cells expressing the truncated M6P/IGF II receptor StR 119 was
observed in only one out of three experiments. In contrast,
coincubation of membranes with GTPS, which activates G-proteins,
reduced incorporation of [
P]ADP-ribose into the
40-kDa proteins significantly by 60% (not shown). The pretreatment of
membranes with Ptx and unlabeled NAD for 16 h inhibited the subsequent
[
P]ADP-ribose incorporation in the 40-kDa
protein completely (Fig. 3). Whereas it was reported that in
experiments carried out with intact BALB/c 3T3 cells (45) or
with membranes prepared from these cells(18) , IGF II
attenuates the Ptx-catalyzed ADP-ribosylation of the 40-kDa protein by
30-50 and 80%, respectively, in the present study IGF II failed
completely to affect the ADP-ribose incorporation into the 40-kDa
protein. Alternatively, we measured GTPase activity in membranes of
nontransfected and wild type M6P/IGF II receptor-expressing L-cells
preincubated with and without pre- or concomitant incubation of IGF II.
None of the tested conditions revealed any significant stimulatory
effect of IGF II on GTPase activity (not shown).
Figure 3:
Effect of IGF II on pertussis
toxin-catalyzed ADP-ribosylation in membranes of nontransfected and
transfected L-cells. Membranes were prepared from nontransfected, wild
type, and mutant M6P/IGF II receptor-expressing L-cells. The membranes
were pretreated with IGF II (15 min) or Ptx (16 h), centrifuged, and
resuspended (20 mg/ml) before ADP-ribosylation in the presence of 40
µg/ml Ptx and 10 µM [P]NAD for
30 min at 30 °C. The proteins were separated by SDS-PAGE
(10-15% acrylamide) and visualized by
autoradiography.
Additionally, in
EGF ``primed'', PDGF ``competent'' L-cells (45) expressing the wild type M6P/IGF II receptor, IGF II did
not attenuate the Ptx-catalyzed ADP-ribosylation of the 40-kDa protein
(not shown). The 40-kDa pertussis toxin substrate in mouse L-cells was
recognized by the anti-G antibody AS 269 and
comigrated with purified ADP-ribosylated G
but not
with pertussis toxin-modified G
or G
(data not shown). All of these results indicate that in M6P/IGF
II receptor-expressing L-cells, IGF II failed to activate G-proteins.
Figure 4: Purification of wild type and truncated M6P/IGF II receptors. A, a Triton X-100 extract of mouse L-cells expressing the human wild type or truncated M6P/IGF II receptor was passed over a phosphomannan-Sepharose column. An aliquot of the M6P eluate (1 µg of protein) was separated on SDS-PAGE followed by silver staining. B and C, a portion of the M6P-eluted receptor fraction was immunostained after Western blotting by means of antibodies directed against the luminal domain (B) or against C-terminal peptide 15C (C).
Figure 5:
Identification and pertussis
toxin-mediated ADP-ribosylation of G and G
.
Aliquots of fractions containing purified G
- (A)
and G
-proteins (B) were precipitated with acetone
in the presence of BSA (10 µg) and loaded on gels (6 M urea, 9% acrylamide). After blotting, nitrocellulose filters were
incubated with antibodies AS 6 (anti-
serum,
diluted 1:300), AS 248 (anti-
antibodies,
affinity-purified), AS 266 (anti-
antibodies,
affinity-purified), AS 269 (anti-
serum, diluted
1:150). For ADP-ribosylation, G-protein
-subunits
(G
, 600 ng; G
, 340 ng) were treated
with pertussis toxin in the presence of unlabeled and
P-labeled NAD (lanes 5 and 6) and brain
subunits (680 ng) as described under ``Materials and
Methods.'' Proteins were subsequently resolved on gels (6 M urea, 9% acrylamide). After blotting, nitrocellulose-filters were
exposed to x-ray films. Corresponding
-subunits were detected by
incubating nitrocellulose filters with antibody AS 6
(anti-
serum diluted 1:300, A, lanes 1-3) or AS 266 (anti-
antibodies, affinity-purified; B, lanes
1-3). The ECL system was used for detection of filter-bound
antibodies. A, lane 1, heterotrimeric
G
/G
-pool; lane 2, heterotrimeric
G
; lanes 3-6, G
. B, lane 1, heterotrimeric
G
/G
-pool; lane 2, heterotrimeric
G
; lanes 3-6, G
.
Molecular masses (in kDa) of marker proteins are indicated. It should
be noted that pertussis toxin-mediated ribosylation of G
(A, lanes 5 and 6) was incomplete,
resulting in two immunoreactive bands (lane 5) of unmodified (lower band) and modified (upper band)
protein.
Figure 6:
Characterization of G protein
and M6P/IGF II receptors reconstituted into phospholipid vesicles. A, [
S]GTP
S (500 nM,
100,000 cpm/tube) binding to G
in different phospholipid
vesicle containing fractions eluted from the Sephadex G-50 column. B, GTPase activity. Reconstituted vesicles were incubated with (black bars) or without (open bars) 100 µM mastoparan for 15 min at 25 °C prior to determination of GTP
hydrolysis as described under ``Materials and Methods.'' C,
I-IGF II affinity cross-linkage followed by
receptor immunoprecipitation. Reconstituted vesicles were incubated
with
I-IGF II in the presence and absence of unlabeled
IGF II (0.5 µg) for 16 h at 4 °C. After cross-linkage with DSS
and immunoprecipitation using antibodies directed against the 15
C-terminal amino acids of the cytoplasmic M6P/IGF II receptor domain,
the immunocomplexes were analyzed by SDS-PAGE (5% acrylamide) and
autoradiography. The position of the 200-kDa molecular mass standard is
indicated. Panels A-C show the data of one
representative experiment out of three to five for each of the tested
trimeric G-proteins. The data for panels A and C were
obtained from the same preparation.
Figure 7:
Effect of IGF II and mastoparan on
GTPS binding to G
or G
in reconstituted
vesicles. Wild type (A, B, and C), StL 75 (D and E), or StR 119 mutant (F) M6P/IGF II
receptors reconstituted with trimeric G
(A and D) or G
(B, C, E, and F) were incubated for the indicated period with
[
S]GTP
S in the absence (
) or presence
(
) of 1 µM IGF II or 100 µM mastoparan
(
). Each point represents the mean of duplicates. The GTP
S
binding to G-proteins of one representative experiment out of two to
four is shown. GTP
S binding values were corrected by the
unspecific binding in the presence of 100 µM unlabeled
GTP
S.
Figure 8:
Effects of IGF II on GTPS binding to
G
monomers and
dimers reconstituted vesicles. Wild type
M6P/IGF II receptors were reconstituted with G
or
G
monomers and
dimers in an
:
ratio of 1:2. The vesicles were incubated for the
indicated period with [
S]GTP
S in the
absence (
) or presence (
) of 1 µM IGF II. Each
point is the mean of duplicates.
Figure 9:
Effects of IGF II and mastoparan on GTPase
activity of G in reconstituted vesicles. Wild type (n = 3) or StL 75 mutant (n = 2) M6P/IGF II
receptors reconstituted with trimeric G
were incubated for
15 min at 25 °C with [
P]GTP in the
absence or presence of IGF II (1 µM) or mastoparan (100
µM). All values represent means of two to three
determinations carried out in triplicate. The data are expressed in
relation to untreated controls that account for 236 ± 43 fmol of
P
released per fraction and
min.
Taken together, in reconstituted vesicles
containing different G-proteins and wild type or truncated M6P/IGF II
receptors devoid of the putative G-protein binding site, IGF II
increased the GTPS binding only slightly (1.2-1.3-fold)
independent of the subtype of G-protein used (G
or
G
) and the presence of the proposed G-protein-coupling
sequence in the cytoplasmic receptor tail. In addition, in
reconstituted vesicles containing wild type M6P/IGF II receptors and
purified G
and G
monomers and
-dimers, IGF II did not alter the GTP
S binding. Finally,
in wild type or mutant M6P/IGF II receptor/G
vesicles, IGF
II did not induce a stimulation of GTPase activity. These results fail
to support the proposal that IGF II binding to M6P/IGF II receptors
induces the functional coupling and activation of G
proteins via amino acid residues 123-136 of the cytoplasmic
receptor tail.
Whereas the IGF I receptor is capable of mediating the
transmembrane signaling of both IGF I and IGF II ligands by activation
of its intrinsic tyrosine kinase, it was previously proposed that the
binding of IGF II to the M6P/IGF II receptor activates G proteins. The M6P/IGF II receptor does not share structural
properties with conventional seven-transmembrane G-coupled receptors
but instead contains a sequence of 14 amino acids in its cytoplasmic
domain which was proposed to activate G
proteins
directly(18, 21) . The experiments presented here
examine interactions between wild type and mutant M6P/IGF II receptors
with G-proteins in response to IGF II. In this work we followed the two
main experimental approaches that were initially used (18, 19, 45) to implicate the direct coupling
of M6P/IGF II receptors with G
proteins: i) pertussis
toxin-catalyzed ADP-ribosylation of a 40-kDa protein in cell membranes
and ii) GTP
S binding to and GTPase activity of purfied G-proteins
in reconstituted phospholipid vesicles containing purified M6P/IGF II
receptors in response to IGF II.
In the first approach, we
investigated the effect of IGF II to reduce the ability of pertussis
toxin to modify G proteins in intact mouse L-cells
expressing the human wild type M6P/IGF II receptor, nontransfected
L-cells deficient for the M6P/IGF II receptor, or L-cells expressing a
mutant receptor lacking the proposed G-protein activator sequence
motif. Pertussis toxin modifies only trimeric GDP-bound G
proteins by ADP-ribosylation, whereas the dissociated GTP-bound
subunits formed after ligand-induced G-protein activation do not
function as toxin substrates(44) . The 40-kDa pertussis toxin
substrate of L-cells is G
, as demonstrated by the
pertussis toxin-catalyzed ADP-ribosylation of L-cell membranes in
comparison with the ADP-ribosylation of purified G
,
G
, and G
proteins as well as
anti-G
antibody reactivity. This is in accordance
with other reports that assume that G
is a major pertussis
toxin-sensitive G-protein in non-neuronal
cells(43, 47, 48, 49) . In all of
the tested cells, neither IGF II nor IGF I was able to reduce pertussis
toxin-catalyzed ADP-ribosylation of G
over a wide
range of concentrations. In addition, no attenuation of pertussis toxin
substrate activity was observed in broken L-cell membranes containing
the human wild type or D 123-136 M6P/IGF II receptor mutant.
Neither in our present study nor in studies using BALB/c 3T3 cells were
positive controls tested, i.e. the ability of ligands of known
G
protein-coupled receptors (such as the m2 muscarinic
receptor) to attenuate the Ptx-catalyzed ADP-ribosylation. First, it is
difficult to test such controls in L-cells and second, it is
questionable whether ligand-induced activation of G
proteins can diminish the amount of Ptx substrate activity. Thus
it is rather unlikely that in the presence of GTP a ligand-induced
transient dissociation of G-proteins might affect the extent of the
Ptx-catalyzed ADP-ribosylation, which rapidly takes place whether or
not the G- protein is undergoing dynamic cycling.
We used additional
criteria to confirm the inability of IGF II to induce the activation
and dissociation of a pertussis toxin substrate: (i) inability of IGF
II to stimulate GTPase activity in nontransfected or M6P/IGF
II-overexpressing L-cells, (ii) inability of IGF II to attenuate the
pertussis toxin-catalyzed ADP-ribosylation in PDGF/EGF-treated (primed
competent) L-cells expressing the wild type M6P/IGF II receptor, and
(iii) receptor-independent GTPS-induced attenuation of pertussis
toxin substrate activity. All of these data indicate that the binding
of IGF II to the M6P/IGF II receptor expressed in mouse L-cells does
not initiate transmembrane signaling via activation of G
.
This is in contrast to reports that incubation of BALB/c 3T3 cell
membranes with either IGF II or IGF I (5-10 nM) led to a
reduction in the pertussis toxin substrate activity by 70-80%
within 10 min(18) . Additionally, in intact BALB/c 3T3 cells,
low concentrations of IGF II and IGF I (1 nM) reduced the
pertussis toxin substrate activity of G
by 50% but with
slightly differing time courses(45) . However, both IGFs did
produce changes in pertussis toxin substrate activity of G
but only in primed competent BALB/c 3T3 cells, i.e. those pretreated for 3 h with PDGF followed by a 20-min EGF
treatment or in v-ras p21-transformed BALB/c 3T3 cells.
The
authors postulate that (i) in quiescent cells the M6P/IGF II receptors
are uncoupled from G resulting in nonresponsiveness to IGF
II and (ii) competence growth factors or v-ras p21 activation restores
functional coupling between the M6P/IGF II receptor and G
,
which enables both IGFs to stimulate calcium influx in these cells. If
the mouse L-cells have some mechanism for coupling the M6P/IGF II
receptor with G
, it must be different from that observed
in BALB/c 3T3 cells since coupling could not be obtained by treatment
with PDGF/EGF. However, it remains unclear why intact BALB/c 3T3 cells
should require pretreatment with competence growth factors to detect
attenuation of pertussis toxin substrate activity in response to IGF
II, whereas IGF II would be capable of activating G
proteins directly in membranes prepared from quiescent BALB/c 3T3
cells via the M6P/IGF II receptor. Finally, a different cellular
composition of
dimers in M6P/IGF II receptor-deficient mouse
L-cells used in our mutational analysis could not be excluded, which
could affect the response to IGF II (see below).
To circumvent these
problems, we turned to a second approach, which utilize a controlled
reconstituted system containing purified components. In these
experiments potential M6P/IGF II receptor-G-protein interaction in
phospholipid vesicles was followed by measuring GTPS binding and
GTPase activity in response to IGF II. The human M6P/IGF II receptors
used in these experiments were purified from overexpressing mouse
L-cells lacking the M6P/IGF II receptor. During purification under
optimized conditions, no proteolytic cleavage of the cytoplasmic
receptor tail occurred, as monitored by binding of antibodies directed
against the carboxyl-terminal 15 amino acids. We demonstrated that the
wild type M6P/IGF II receptors and the G-proteins were functionally
active in reconstituted vesicles. However, in vesicles containing the
wild type M6P/IGF II receptor, neither G
, G
,
nor purified G
/G
mixtures yielded a significant
stimulation of GTP
S binding or GTPase activity in response to IGF
II. Furthermore, similar findings were observed in experiments in which
truncated receptor proteins lacking the proposed G-protein activator
sequence of the cytoplasmic receptor domain were reconstitued with
G
or G
proteins or mixtures of all
G
/G
proteins. IGF II failed to increase
significantly the binding of GTP
S or GTPase activity. In contrast,
100 µM mastoparan yielded an approximately 2-fold increase
in GTP
S binding and 9-fold increase in GTPase activity in vesicles
reconstituted with wild type or StL 75 receptor and G
trimers. These results differ from those of Nishimoto and
colleagues(18, 19) , who reported that IGF II induced
a functional coupling and activation of G
with
purified M6P/IGF II receptors reconstituted in phospholipid vesicles
(2-fold increase in the GTP
S binding rate and GTPase activity),
which were completely inhibited by M6P and the M6P-containing
glucuronidase.
Although we have no obvious explanation for the
discrepancies between their findings and ours, we must consider
possible differences in the G-proteins or M6P/IGF II receptors
employed. Nishimoto's group used G-proteins purified from a
variety of sources (bovine spleen, lung, brain, or porcine
brain)(18, 19, 50) , although a similar
purification procedure was used in both studies. Furthermore,
differences in composition of the G-protein preparation used
in the two laboratories, which might facilitate or modulate stability,
localization, and activation of subunits or might directly interact
with receptors (51, 52) can also be excluded because
the purified
complexes used here represent a mixture of
dimers most likely identical with
complexes
Nishimoto employed. Therefore it seems more likely that the M6P/IGF II
receptor preparations of the two laboratories were different. Whereas
proteolytic cleavage of the cytoplasmic domain of M6P/IGF II receptors
was prevented during our purification procedure, Nishimoto did not
characterize the employed receptor preparations in great detail. This
might be particularly important with regard to proposed suppressor
sequences within the cytoplasmic receptor domain that could attenuate
G-protein activation in the absence of IGF II (53) . The
removal of such a suppressor element, localized C-terminal to the
proposed G-protein-activating sequence by proteolytic cleavage during
preparation, could facilitate the IGF II-induced G-protein activation.
The loss of parts of the cytoplasmic domain of M6P/IGF II receptors
during purification from bovine liver was previously reported (54) . Alternatively, reversible posttranslational
modifications of the cytoplasmic M6P/IGF II receptor domain, such as
phosphorylation/dephosphorylation, might modulate the functional
responsiveness to IGF II. This could explain the requirement of
PDGF/EGF pretreatment or ras p21 transformation of quiescent
BALB/c 3T3 cells to restore coupling of receptors to G
(45, 17) . It has been shown that following
treatment of serum-deprived BALB/c 3T3 cells with different growth
factors, the cellular cAMP levels increased (55) resulting in
protein kinase A activation. There are several reports of typical
G-protein-coupled receptors phosphorylated in an agonist-dependent
manner by receptor kinases resulting in desensitization of these
receptors(50, 56, 57) . Thus, the loss of
phosphate groups from distinct sites during the purification procedure
could contribute to a preformed IGF II-responsive M6P/IGF II receptor
conformation.
The physiological relevance of the proposed IGF
II-induced activation of G is still a matter of
speculation. Whereas some reports observed the generation of
intracellular signals in response to IGF II, such as the activation of
phospholipase C, calcium influx, and DNA
synthesis(15, 17) , which are also stimulated by IGF I
at low concentrations(45, 58, 59) , other
studies failed to demonstrate changes in intracellular levels of
inositol phosphates, diacylglycerols, cAMP, or calcium after incubation
with IGF II (60, 61, 62, 63) .
Furthermore, analysis of mutant mouse embryos indicated that only the
IGF I receptor and an additional as yet unknown receptor that is not
the M6P/IGF II receptor mediate the mitogenic signaling of IGF
II(7, 13) .
At the moment we cannot exclude the possibility that the M6P/IGF II receptor coupling to G-proteins and the stimulation of signal transduction pathways in response to IGF II are cell type-specific and require additional cellular activation processes. However, the localization of only 5-15% of total M6P/IGF II receptors at the cell surface and their rapid replacement within 2.8 min (24) as compared with typical G-protein-coupled signaling receptors argues against a role of M6P/IGF II receptors in signaling. Alternatively, trimeric G-proteins might be involved in regulation of cellular trafficking of M6P/IGF II receptors (for review see (64) ).
Activation of G proteins has been
shown to inhibit vesicle budding from the TGN(65) . It is not
known whether the M6P/IGF II receptor functions as an activator of
G-proteins in the TGN and thereby affects budding and vesicular
transport to endosomes. There are no data showing that precursor or
mature forms of IGF II bind in the TGN to M6P/IGF II receptors, and the
binding of M6P-containing newly synthesized lysosomal enzymes does not
activate G-proteins(19) . It remains to be determined whether
phosphorylation of the M6P/IGF II receptor cytoplasmic domain by a
TGN-associated receptor kinase (66) or the receptor association
with cytosolic/membrane-bound proteins (27) may contribute to
G-protein activation.
In conclusion, the present study fails to support a model in which the M6P/IGF II receptor functionally couples to G-proteins in cell membranes or in phospholipid vesicles.