From the Department of Cell and Molecular Biology,
Northwestern University Medical School, Chicago, Illinois 60611 and the
§ Department of Cell Biology, University of Virginia Health
Sciences Center, Charlottesville, Virginia 22908
Received for publication, October 12, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have investigated the
participation of endogenous ADP-ribosylation factor (ARF)
nucleotide-binding site opener (ARNO) in desensitization of the
luteinizing hormone/choriogonadotropin (LH/CG) receptor, independent of
receptor internalization, using a cell-free plasma membrane model. We
recently showed that the addition of recombinant ARNO promotes binding
of The binding of saturating concentrations of agonist to guanine
nucleotide-binding (G) protein-coupled receptors initially results in
productive coupling to an effector, resulting in increased effector
activity. Thereafter, effector activity often declines or becomes
desensitized as a result of the uncoupling of the agonist-bound receptor from its cognate G protein (1). We have used a cell-free plasma membrane model to investigate the cellular mechanism of homologous luteinizing hormone/choriogonadotropin
(LH/CG)1 receptor
desensitization independent of LH/CG receptor internalization (2,
3).
We have recently shown that desensitization of the LH/CG receptor
requires the binding of membrane-delimited Based on our evidence that the addition of exogenous ARNO in the
presence of 1 µM GTP frees a pool of All chemicals were from previously described sources (4, 5).
Recombinant ARNO and E156K ARNO were expressed and purified as
described previously (7). A sucrose gradient-purified membrane fraction
enriched in adenylyl cyclase (AC) activity was isolated from
preovulatory-size porcine ovarian follicles and stored at We first determined whether ARNO is detectable in follicular
membranes enriched in AC activity. Results (Fig.
1A, lane 4) show
that a band reactive with affinity purified anti-ARNO antibody (7) is
readily detectable in porcine ovarian follicular membranes and migrates
on SDS-polyacrylamide gel electrophoresis somewhat faster than
His6-tagged recombinant human ARNO (lanes 5-8).
Quantitation of the amount of ARNO in partially purified ovarian
follicular membranes by Western blotting, using the signal generated by
recombinant ARNO as the standard, yielded a concentration of ~1.5
µg of ARNO protein (or 32 nmol)/mg of membrane protein. The basis for
the ARNO doublet (see Fig. 1A, lanes 1-3) is not
known but might correspond to phosphorylated and unphosphorylated ARNO
(13, 14). We additionally determined whether the amount of ARNO present
in unpurified 10,000 × g membrane and supernatant
fractions is regulated by follicular maturation. Results (Fig.
1B) show that whereas ARNO levels are relatively low and
unregulated in the supernatant fractions of small (1-2 mm) compared
with large (8-10 mm) follicles, the levels of ARNO in the membrane
fraction are higher (~6-fold) in large preovulatory follicles
enriched in LH/CG receptors compared with levels in small immature
follicles that do not contain LH/CG receptors (15). These results show
that the expression of ARNO is increased with development of
porcine follicles from an immature to a preovulatory phenotype and that
the majority of ARNO is localized to the pellet fraction.
-arrestin1 to the third intracellular (3i) loop of the active
LH/CG receptor, thereby reducing the ability of the receptor to
activate the stimulatory G protein and signal to adenylyl
cyclase. In the present report we determined whether ARNO is
detectable in follicular membranes and whether the catalytically
inactive E156K ARNO mutant, containing a mutation in the Sec7 domain,
can act in a dominant negative manner to block LH/CG receptor
desensitization. Results show that ARNO is readily detected in
follicular membranes and that levels of membrane-associated ARNO
increase with follicular maturation. The addition of catalytically
inactive E156K ARNO blocks both the release of
-arrestin1 from its
membrane docking site, based on Western blot analysis, and development
of LH/CG receptor desensitization. We also investigated whether a point
mutation in the pleckstrin homology (PH) domain of ARNO (R280D),
which blocks binding of phosphoinositides like phosphatidylinositol
3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate
(PIP2) but not catalytic activity, disrupts LH/CG receptor
desensitization. R280D ARNO neither promotes nor inhibits LH/CG
receptor desensitization, consistent with a requirement of the PH
domain of ARNO for its association with the plasma membrane. LH/CG
receptor activation of ARNO is not mediated by activation of
phosphatidylinositol 3-kinase (PI 3-kinase) or by G protein
subunits. Taken together, these results suggest that LH/CG receptor
promotes
-arrestin1 release from its membrane docking site to bind
to the 3i loop of the LH/CG receptor via activation of membrane
delimited endogenous ARNO. As ARNO activation is independent of PI
3-kinase and G
, our results are consistent with a role for
PIP2 in receptor-stimulated ARNO activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin1 (Arrestin 2)
to the third intracellular loop (3i) of the LH/CG receptor, resulting
in reduced cAMP production (4, 5). This conclusion is based on evidence
that preincubation of membranes with neutralizing anti-arrestin
antibodies prevents development of LH/CG receptor desensitization (5),
that
-arrestin1 binds directly and selectively to a synthetic
peptide corresponding to the 3i loop of the LH/CG receptor (4), and
that preincubation of membranes with a synthetic peptide corresponding
to the 3i loop of the LH/CG receptor completely blocks development of
receptor desensitization (4). We have also recently shown that LH/CG
receptor activation not only exposes a binding site for
-arrestin1
at the 3i loop on the receptor but also leads to the apparent
activation of the small G protein ADP-ribosylation factor 6 (ARF6),
resulting in the release of a pool of
-arrestin1 from its membrane
docking site (6). These conclusions are based on the following results.
(a) ARNO (25 nM), a guanine nucleotide exchange
factor (GEF) for ARFs 1 and 6 (7, 8), promotes desensitization of the
LH/CG receptor in the presence but not the absence of GTP concomitant
with the release of
-arrestin1 from its membrane docking site (6).
(b) Catalytically dead E156K ARNO mutant (at 25 or 50 nM) does not cause LH/CG receptor desensitization (6).
(c) Preincubation of membranes with synthetic N-terminal
ARF6 peptide, but not with the corresponding ARF1 peptide, prevents the
release of
-arrestin1 from its membrane docking site and thereby
prevents development of LH/CG receptor desensitization (6). LH/CG
receptor-dependent activation of the small G protein ARF6
is consistent with earlier studies showing that LH/CG receptor desensitization exhibits an obligatory requirement for GTP (3, 9-11).
-arrestin1 to
bind to the 3i loop of the activated LH/CG receptor to block receptor interaction with Gs (6), we hypothesized that LH/CG
receptor-dependent activation of ARF6 might be mediated by
activation of endogenous, membrane-delimited ARNO. In the present
report we therefore sought to determine whether endogenous
membrane-delimited ARNO participates in LH/CG receptor desensitization.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C (6). The two-stage desensitization reaction (4) is summarized in the Fig. 2 legend. Supernatant and pellet fractions of
porcine ovarian follicles were obtained by homogenizing tissue in 10 mM TRIS-HCl, pH 7.0, 1.0 mM EDTA with a
glass-glass Dounce homogenizer (~10 strokes) followed by
centrifugation at 1,000 × g for 5 min and then at
10,000 × g for 30 min. The final pellet was
resuspended in 1 volume of the original homogenate, and an aliquot of the final supernatant and pellet fractions was obtained for
protein determination. SDS-STOP was then added, and samples were boiled
for 10 min and stored at
70 °C. Western blotting with
anti-
-arrestin antibody (Transduction Laboratories, Lexington, KY),
the pan-arrestin antibody F4C1, and affinity-purified anti-ARNO antibody (7) was performed as described previously (6, 7). The results
were analyzed using Student's t test (p < 0.05) (12).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (12K):
[in a new window]
Fig. 1.
ARNO is detectable by Western blot in porcine
ovarian follicular membranes and increases with follicular
maturation. A, indicated amounts of recombinant
His6-tagged human ARNO and porcine ovarian follicular membrane proteins
were loaded onto an SDS-acrylamide gel. The resulting blot was probed
with affinity-purified anti-ARNO antibody. B, proteins
(~75 µg) in 10,000 × g supernatant and pellet
fractions of porcine ovarian follicles measuring 1-2 mm (small) or
8-10 mm (large, preovulatory) were separated by SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose, and probed with
affinity-purified anti-ARNO antibody. The results are representative of
two separate experiments.
Based on evidence that ARNO is readily detected in membranes of
preovulatory-size follicles, we next sought to determine whether catalytically inactive ARNO could compete with endogenous ARNO to block
agonist-dependent LH/CG receptor desensitization. The E156K
mutation in the Sec7 domain of ARNO blocks the ability of ARNO to
promote GTP exchange at ARF6 but does not disrupt its ability to bind
phosphoinositides via its pleckstrin homology (PH) domain (14).
Although preincubation of membranes with 50 (not shown) or 100 nM (Fig. 2A) E156K
ARNO did not affect the extent of LH/CG receptor desensitization
(hCG/hCG conditions), preincubation of membranes with 200 nM E156K ARNO significantly reduced (p < 0.05) the extent of LH/CG receptor desensitization. Thus, when
membranes were preincubated with 200 (Fig. 2A), 300, or 400 (not shown) nM E156K ARNO, hCG-stimulated AC activity
measured with hCG in stage 1 (i.e. desensitization
conditions) was no longer significantly different from values measured
with BSA in stage 1. The capability of E156K ARNO to block LH/CG
receptor desensitization was lost when E156K ARNO was boiled (Fig.
2B). The addition of E156K ARNO directly to the 5-min AC
assay did not affect basal or hCG-, forskolin-, or aluminum
fluoride-stimulated AC activities (Fig. 2C). This result
shows that E156K ARNO does not have a nonspecific effect on AC activity
or on the ability of the LH/CG receptor or Gs to activate AC. To
ascertain whether E156K ARNO indeed blocks development of LH/CG
receptor desensitization by preventing ARF6 activation, we determined
whether E156K ARNO blocks the release of -arrestin1 from its
membrane docking site. In the absence of LH/CG receptor activation,
-arrestin1 was readily detectable in follicular membranes (Fig.
2D, lane 4), presumably bound at a docking site
distinct from the LH/CG receptor (6). Incubation of membranes with hCG
resulted in the retention of a fraction of
-arrestin1 at the
membrane (Fig. 2D, lane 3). This fraction of
-arrestin1 was competed away with a synthetic peptide corresponding to the 3i loop of the LH/CG receptor (lane 2), consistent
with the premise that LH/CG receptor activation results in the binding of a fraction of
-arrestin1 to the 3i loop of the activated LH/CG receptor (4, 6). However, when E156K ARNO was included in the membrane
incubation mix with hCG and 3i peptide, this fraction of
-arrestin1
was retained in the membrane (lane 1), presumably at its
membrane docking site as it was no longer competed away by the 3i
peptide. These results suggested that exogenous catalytically dead
E156K ARNO effectively competes with endogenous ARNO to block the
ability of the activated LH/CG receptor to promote
-arrestin1 release and consequent LH/CG receptor desensitization. Based on these
results we can therefore conclude that endogenous ARNO is activated to
promote GTP exchange at ARF6 in response to LH/CG receptor
activation.
|
ARNO contains a C-terminal PH domain that can bind phosphatidylinositol
3,4,5-trisphosphate (PIP3), the product of
PI(4,5)bisphosphatate 3-kinase (PI 3-kinase), with high selectivity and
affinity (Kd~85 nM (16)). A point
mutation at R280D in the PH domain of ARNO results in a mutant protein
that retains its catalytic activity to promote GTP exchange on ARF6 but
cannot bind phosphoinositides (14). We have shown that, unlike
catalytically active wild-type ARNO (at 25 nM), which
promotes LH/CG receptor desensitization, R280D ARNO at 50 nM does not promote LH/CG receptor desensitization (6).
This result suggests that the binding of phosphoinositides to the PH
domain of ARNO is obligatory for LH/CG receptor to promote activation
of ARNO. We therefore sought to determine whether R280D ARNO could
function like E156K ARNO in a dominant negative manner to block
development of LH/CG receptor desensitization in response to receptor
activation. Preincubation of follicular membranes with 200 (Fig.
3A) or 300 nM (not
shown) R280D ARNO does not affect the ability of receptor activation to
promote LH/CG receptor desensitization. Consistent with this result,
preincubation of membranes with the PI 3-kinase inhibitor wortmannin at
100 nM does not reduce the extent of LH/CG receptor
desensitization (Fig. 3B). As some PH domains also bind the
subunits of activated G proteins (17), we tested whether a
inhibitor peptide (18), corresponding to residues 956-982 of
AC2, could block development of LH/CG receptor desensitization. The
results (Fig. 3C) show that preincubation of follicular
membranes with 10 µM
-inhibitor peptide QEHA
(QEHAQEPEROYMCHIGTMVEFAYALVGK), which blocks
-dependent stimulation of AC2,
-adrenergic
receptor kinase, and phospholipase C (18), does not affect LH/CG
receptor desensitization. Higher concentrations of the QEHA peptide
inhibited basal and hCG- and forskolin-stimulated AC activities (Fig.
3C and not shown). Taken together, these results indicate
that PIP3 or
signaling through the PH domain of ARNO
does not appear to be obligatory for the activated LH/CG receptor to
promote GTP exchange at ARF.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ARNO is detected in a purified membrane fraction enriched in AC
activity obtained from preovulatory-size porcine ovarian follicles at a
concentration of ~32 nmol/mg of membrane protein. Moreover, the
expression of ARNO appears to be regulated with follicular maturation.
Higher levels of ARNO are detected in membranes of mature preovulatory
(8-10 mm) porcine follicles, which express LH/CG receptors, compared
with small (1-2 mm) follicles, which do not express LH/CG receptors
(15). That increased expression of ARNO correlates with the induction
of LH/CG receptors is very interesting and suggests that the same
stimulus that induces LH/CG receptors, namely follicle-stimulating
hormone (19), might also increase expression of ARNO. These results
further suggest that ARNO, in an apparently inactive conformation, is
constitutively associated with the plasma membrane of preovulatory
ovarian follicles. The conclusion that ARNO is present in an inactive
conformation and is activated upon engagement of the LH/CG receptor is
based not only on results presented in Fig. 2, showing that LH/CG
receptor desensitization is blocked by E156K ARNO, but also on evidence that ARF activation, measured as cholera toxin-catalyzed
ADP-ribosylation of the long form of Gs, is negligible in the
absence of LH/CG receptor activation
(20).2 The basis for the
apparently constitutive membrane association of ARNO is not clear.
However, in other cellular models there is also evidence that a large
portion of total cellular ARNO can be associated constitutively with
the plasma membrane in the absence of directed membrane-receptor
activation both on overexpression in various cell lines (7) and in
chromaffin cells isolated from bovine adrenal glands (21).
We have shown with the porcine follicular membrane model that
catalytically inactive E156K ARNO acts in a dominant negative manner to
selectively block the development of LH/CG receptor desensitization in
response to LH/CG receptor activation. Catalytically inactive E156K
ARNO blocks the obligatory release of -arrestin1 from its membrane
docking site. As a result, the LH/CG receptor remains active to signal
to Gs and AC despite the continued presence of saturating
concentrations of receptor agonist. This result suggests that LH/CG
receptor desensitization requires the activation of endogenous ARNO.
However, based on the recent identification of the exchange factor for
ARF6 (EFA6) (22), in which the guanine nucleotide exchange activity is
also insensitive to brefeldin A, we cannot rule out the possibility
that the apparent dominant negative effect of E156K ARNO is
attributable to its potential ability to sequester available ARF6, thus
indirectly blocking LH/CG receptor desensitization potentially mediated
by EFA6.
ARNO, along with GRP1 and cytohesin-1, comprise a subfamily of guanine nucleotide exchange factors for the ARFs in which the guanine nucleotide exchange activity is not inhibited by the fungal metabolite brefeldin A (7, 8, 23-25). Each of these factors contains an N-terminal coiled-coil domain, a central Sec7 domain, that is sufficient for guanine nucleotide exchange activity on ARF (26) and is highly homologous with the SEC7 gene in yeast which encodes a GEF required for protein secretion (reviewed in Ref. 27), a C-terminal PH domain, and an adjacent cluster of basic residues (c domain) (8).
The PH domain of the proteins in this subfamily appears to be necessary
both for their plasma membrane association and for subsequent guanine
nucleotide exchange activity toward membrane-localized ARFs (14, 23,
24, 28, 29). These guanine nucleotide exchange proteins are often
localized to the cytosolic fraction of unstimulated cells and have been
shown to translocate to the plasma membrane in response to receptor
activation by insulin or epidermal growth factor in 3T3 L1 adipocytes
and Chinese hamster ovary cells (16, 30). Consistent with these
results, transfection of cells with ARNO or cytohesin-1 containing a
mutation in the PH domain disrupts the plasma membrane association of
these proteins and consequent biological responses, namely
cytohesin-1-dependent 2 integrin adhesion to
adhesion molecule 1 in Jurkat E6 leukemia cells (23) and
ARNO-dependent actin reorganization in HeLa cells (14).
The predominant ligands that have been shown to bind to the PH domains of these GEFs are phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP3 (31). Although there have been seemingly contradictory results on the affinities of the PH domains of these proteins for PIP2 or PIP3, recent evidence from the Czech laboratory (32) has resolved this apparent controversy by showing that ARNO, GRP1, and cytohesin-1 can exhibit either high selectivity for PIP3 or equivalent selectivity for PIP3 and PIP2 depending on whether the PH domain contains a diglycine or triglycine motif, respectively. Moreover, isoforms of each of the proteins in this subfamily appear to exist that express either the diglycine or triglycine motif even in the same tissue (7, 8, 33, 34). Consistent with this result, recruitment and activation of these GEFs can depend on either PIP2 or PIP3, contingent upon whether the diglycine or triglycine motif in the PH domain is present (8, 13, 24, 28, 35). In those cases in which GEF recruitment and activation rely on PIP3, these responses can be readily blocked by PI 3-kinase inhibitors like wortmannin (16, 23, 24, 30). There is also evidence for ARNO and cytohesin-1 that the adjacent C-terminal polybasic c domain cooperates with the PH domain to bind these GEFs tightly to the membrane, possibly by binding a phospholipid with a negative charge (8, 13).
LH/CG receptor desensitization is not inhibited by a point mutation in the PH domain of ARNO, which blocks its binding to all phosphoinositides. One interpretation of these results is that LH/CG receptor-dependent activation of ARNO is independent of the PH domain of ARNO. If phosphoinositide binding to the PH domain is not necessary for ARNO activation, then one would predict that R280D ARNO, like wild-type recombinant ARNO, should promote LH/CG receptor desensitization, since R280D ARNO retains its GTP exchange activity (14). Yet, R280D ARNO does not promote LH/CG receptor desensitization (Ref. 6 and present report). The inability of R280D ARNO to promote LH/CG receptor desensitization might be attributable to its inability to appropriately associate with the plasma membrane because of the mutation in its PH domain. A similar argument can be made for the inability of R280D to block desensitization in a dominant negative manner. Our results showing that R280D ARNO neither stimulates nor inhibits LH/CG receptor desensitization indirectly suggest that the PH domain of ARNO is indeed necessary at least for its membrane association. However, the ineffectiveness of the PI 3-kinase inhibitor wortmannin to block desensitization indicates that ARNO activation is not dependent on PIP3 generated by PI 3-kinase activation. As PIP3 does not appear to participate in the activation of ARNO leading to LH/CG receptor desensitization, it is likely that the predominant ARNO present in follicle membranes contains the triglycine motif in its PH domain, which does not selectively bind PIP3 but rather binds PIP2 with equivalent affinity (32, 36). The recombinant E156K ARNO and ARNO proteins that we have used contain the triglycine motif and are regulated by PIP2 rather than PIP3 (13). Our results therefore suggest that membrane PIP2, either locally synthesized or unmasked, is required for LH/CG receptor to activate ARNO. LH/CG receptor activation has been shown to promote PIP2 synthesis (37, 38). There is also evidence that ARF6 in a GTP- and phosphatidic acid-dependent manner can activate PI 4-phosphate 5-kinase (39) and PI 4-kinase (40) to increase PIP2 synthesis. Future studies are needed to determine how the ability of ARNO to activate ARF is regulated downstream of the LH/CG receptor.
LH/CG receptor desensitization is also not inhibited by the
inhibitor peptide QEHA. The ineffectiveness of the
inhibitor peptide QEHA is supported by our earlier evidence that neither transducin
(at 1 µM) nor rat liver
(at 400 nM) stimulates LH/CG receptor desensitization and that the
scavenger GST-
-adrenergic receptor kinase-peptide fusion
protein (at 14 µM) does not inhibit LH/CG receptor
desensitization (41). These results suggest that G
binding to the
PH domain of ARNO does not mediate LH/CG receptor activation of ARNO.
These results, however, do not rule out the possibility that ARNO is
anchored to the membrane via G
proteins.
In conclusion, these studies show that endogenous
membrane-delimited ARNO appears to be obligatory for the LH/CG receptor to promote -arrestin1 release from its membrane docking site and
receptor desensitization based on the ability of catalytically inactive
ARNO to block both of these responses. Although ARNO activation by the
LH/CG receptor is independent of PIP3 and G
, ARNO
activation could well be dependent on PIP2. The PH domain of ARNO also appears to be required for its membrane association, but
the ligand that interacts with this domain to promote membrane binding
is unlikely to be the same one that leads to its activation.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the gifts of anti-arrestin antibody F4C1 from Dr. Larry Donoso of the Wills Eye Hospital Research Division, Philadelphia, PA, and QEHA peptide from Dr. Ravi Iyengar, Dept. of Pharmacology, Mount Sinai School of Medicine, City University of New York, NY. We also thank Dr. Evelyn Maizels for critically reading this manuscript and Dr. Subhendu Mukhopadhy for purifying recombinant E156K and R280D ARNO.
![]() |
FOOTNOTES |
---|
* This work was funded by National Institutes of Health Grants R01 HD/DK 38060 (to M. H.-D.), and R01 AI 32991 (to J. E. C.) and by a Lalor Foundation fellowship (to S. M.). Preliminary results were presented at the 82nd Annual Meeting of the Endocrine Society, Toronto, Canada, June 2000.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Ave., Chicago, IL 60611. Tel.: 312-503-8940; Fax: 312-503-0566; E-mail: mhd@northwestern.edu.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.C000725200
2 L. M. Salvador, S. Mukherjee, R. A. Kahn, M. L. Lamm, M.-F. Bader, H. Hamm, M. M. Rasenick, J. E. Casanova, and M. Hunzicker-Dunn, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: LH/CG, luteinizing hormone/choriogonadotropin; hCG, human choriogonadotropin; ARF, ADP ribosylation factor; ARNO, ARF nucleotide binding site opener; BSA, bovine serum albumin; 3i, third intracellular loop; Gs, stimulatory guanine nucleotide-binding protein; GEF, guanine nucleotide exchange factor; AC, adenylyl cyclase; PI, phosphatidylinositol; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PH, pleckstrin homology.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hausdorff, W. P., Caron, M. G., and Lefkowitz, R. J. (1990) FASEB. J. 4, 2881-2889[Abstract] |
2. | Bockaert, J., Hunzicker-Dunn, M., and Birnbaumer, L. (1976) J. Biol. Chem. 251, 2653-2663[Abstract] |
3. | Ekstrom, R. C., and Hunzicker-Dunn, M. (1989) Endocrinology 124, 956-963[Abstract] |
4. |
Mukherjee, S.,
Palczewski, K.,
Gurevich, V. V.,
and Hunzicker-Dunn, M.
(1999)
J. Biol. Chem.
274,
12984-12989 |
5. |
Mukherjee, S.,
Palczewski, K.,
Benovic, J. L.,
Gurevich, V. V.,
and Hunzicker-Dunn, M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
493-498 |
6. |
Mukherjee, S.,
Gurevich, V. V.,
Jones, J. C. R.,
Casanova, J. E.,
Frank, S. R.,
Maizels, E. T.,
Bader, M.-F.,
Kahn, R. A.,
Palczewski, K.,
Aktories, K.,
and Hunzicker-Dunn, M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5901-5906 |
7. |
Frank, S.,
Upender, S.,
Hansen, S. H.,
and Cassanova, J. E.
(1998)
J. Biol. Chem.
273,
23-27 |
8. | Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S., Jackson, C. L., and Chabre, M. (1996) Nature 384, 481-484[CrossRef][Medline] [Order article via Infotrieve] |
9. | Ekstrom, R. C., and Hunzicker-Dunn, M. (1989) Endocrinology 125, 2470-2474[Abstract] |
10. |
Ezra, E.,
and Salomon, Y.
(1980)
J. Biol. Chem.
255,
653-658 |
11. |
Ezra, E.,
and Salomon, Y.
(1981)
J. Biol. Chem.
256,
5377-5382 |
12. | Bender, F. E., Douglass, L. W., and Kramer, A. (1982) Statistical Methods for Food and Agriculture , AVI Publishing Co., Inc., Westport, CT |
13. | Santy, L. C., Frank, S. R., Hatfield, J. C., and Casanova, J. E. (1999) Curr. Biol. 9, 1173-1176[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Frank, S. R.,
Hatfield, J. C.,
and Casanova, J. E.
(1998)
Mol. Biol. Cell
9,
3133-3146 |
15. | Channing, C. P., and Kammerman, S. (1974) Biol. Reprod. 10, 179-198[Medline] [Order article via Infotrieve] |
16. | Venkateswarlu, K., Oatey, P. B., Tavare, J. M., and Cullen, P. J. (1998) Curr. Biol. 8, 463-466[Medline] [Order article via Infotrieve] |
17. | Bottomley, M. J., Salim, K., and Panayotou, G. (1998) Biochim. Biophys. Acta 1436, 165-183[Medline] [Order article via Infotrieve] |
18. | Chen, J., DeVivo, M., Dingus, J., Harry, A., Li, J., Sui, J., Carty, D. J., Blank, J. L., Exton, J. H., Stoffel, R. H., Inglese, J., Lefkowitz, R. J., Logothetis, D. E., Hildebrandt, J. D., and Iyengar, R. (1975) Science 268, 1166-1169 |
19. | Hsueh, A. J. W., Adashi, E. Y., Jones, P. B. C., and Welsh, T. H., Jr. (1984) Endocr. Rev. 5, 76-110[Medline] [Order article via Infotrieve] |
20. |
Rajagopalan-Gupta, R. M.,
Rasenick, M. M.,
and Hunzicker-Dunn, M.
(1997)
Mol. Endocrinol.
11,
538-549 |
21. |
Caumont, A.-S.,
Vitale, N.,
Gensse, M.,
Galas, M.-C.,
Casanova, J. E.,
and Bader, M.-F.
(2000)
J. Biol. Chem.
275,
15637-15644 |
22. |
Franco, M.,
Peters, P. J.,
Boretto, J.,
van Donselaar, E.,
neri, A.,
D'Souza-Schorey, C.,
and Chavrier, P.
(1999)
EMBO J.
18,
1480-1491 |
23. |
Nagel, W.,
Zeitlmann, L.,
Schilcher, P.,
Geiger, C.,
Kolanus, J.,
and Kolanus, W.
(1998)
J. Biol. Chem.
273,
14853-14861 |
24. |
Klarlund, J. K.,
Guilherme, A.,
Holik, J. J.,
Virbasius, J. V.,
Chawla, A.,
and Czech, M. P.
(1997)
Science
275,
1927-1930 |
25. |
Franco, M.,
Boretto, J.,
Robineau, S.,
Monier, S.,
Foud, B.,
Chardin, P.,
and Chavrier, P.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9926-9931 |
26. | Roth, M. G. (1999) Cell 97, 149-152[Medline] [Order article via Infotrieve] |
27. |
Moss, J.,
and Vaughan, M.
(1998)
J. Biol. Chem.
273,
21431-21434 |
28. |
Paris, S.,
Beraud-Dufour, S.,
Robineau, S.,
gay, J.,
Antonny, B.,
Chabre, M.,
and Chardin, P.
(1997)
J. Biol. Chem.
272,
22221-22226 |
29. |
Nagel, W.,
Schilcher, P.,
Zeitlmann, L.,
and Kolanus, W.
(1999)
Mol. Biol. Cell
9,
1981-1994 |
30. |
Langille, S. E.,
Patki, V.,
Klarlund, J. K.,
Buxton, J. M.,
Holik, J. J.,
Chawla, A.,
Corvera, S.,
and Czech, M. P.
(1999)
J. Biol. Chem.
274,
27099-27104 |
31. | Jackson, C. L., and Casanova, J. E. (2000) Trends Cell Biol. 10, 60-67[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Klarlund, J.,
Tsiaras, W.,
Holik, J. J.,
Chawla, A.,
and Czech, M. P.
(2000)
J. Biol. Chem.
275,
32816-32821 |
33. |
Ogasawara, M.,
Kim, S.-C.,
Adamik, R.,
Togawa, A.,
Ferrans, V. J.,
Takeda, K.,
Kirby, M.,
Moss, J.,
and Vaughan, M.
(2000)
J. Biol. Chem.
275,
3221-3230 |
34. | Kim, H. S., Chen, Y., and Lonai, P. (1998) FEBS Lett. 433, 312-316[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Kharlund, J. K.,
Rameh, L. E.,
Cantley, L. C.,
Buxton, J. M.,
Holik, H. H.,
Sakelis, C.,
Patki, V.,
Corvera, S.,
and Czeck, M. P.
(1998)
J. Biol. Chem.
273,
1859-1862 |
36. | Cullen, P. J., and Chardin, P. (2000) Curr. Biol. 10, R876-R878[CrossRef][Medline] [Order article via Infotrieve] |
37. | Dimino, M. J., Snitzer, J., and Noland, T. A., Jr. (1987) Biol. Reprod. 36, 97-102[Abstract] |
38. |
Davis, J. S.,
Weakland, L. L.,
Farese, R. V.,
and West, L. A.
(1987)
J. Biol. Chem.
262,
8515-8521 |
39. | Honda, A., Nogami, M., Yokozeki, T., Yamazaki, M., Nakamura, H., Watanabe, H., Kawamoto, K., Nakayama, K., Morris, A. J., Frohman, M. A., and Kanaho, Y. (1999) Cell 99, 521-532[Medline] [Order article via Infotrieve] |
40. | Godi, A., Pertile, P., Meyers, R., Marra, P., DiTullio, G., Iurisci, C., Luini, A., Corda, D., and De Matteis, M. A. (1999) Nat. Cell Biol. 1, 280-287[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Rajagopalan-Gupta, R. M.,
Mukherjee, S.,
Zhu, X.,
Ho, Y.-K.,
Hamm, H.,
Birnbaumer, M.,
Birnbaumer, L.,
and Hunzicker-Dunn, M.
(1999)
Endocrinology
140,
1612-1621 |