From the Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425
Received for publication, August 18, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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AGS3, a 650-amino acid protein encoded by an
~4-kilobase (kb) mRNA enriched in rat brain, is a
G G-proteins serve as primary transducers for propagation of signals
initiated by the superfamily of G-protein-coupled receptors. The basal
activity of such G-protein signaling systems varies among cell
types and at different stages of development. Several factors influence
the basal activity of these systems, including constitutively active
receptors and the type or amount of G-proteins and downstream signaling
molecules expressed in the cell as well as proteins that directly
influence the activation state of G-protein independent of
G-protein-coupled receptors. Such receptor-independent regulators of
G-protein activity include RGS proteins, caveolins, GAP43, and AGS
proteins (1-10). Such regulators may allow G-proteins to subserve
cellular functions independent of the typical, seven-membrane span
receptor motif. AGS1-3, which do not share any apparent conserved sequences, were identified in a functional screen as
receptor-independent activators of G-protein signaling (5-10). AGS1 is
a member of the Ras family of small G-proteins and apparently acts as a
guanine nucleotide exchange factor to increase GTP binding to
G AGS3 (650 amino acids) is encoded by an
~4-kb1 mRNA enriched in
brain (6). AGS3 has two distinct domains separated by a linker region
in the middle of the protein. The amino-terminal half of the protein
contains seven tetratricopeptide repeats (TPRs), whereas the
carboxyl-terminal half of the protein contains four G-protein
regulatory (GPR) motifs,2
each of which is capable of interacting with G Materials--
[32P]UTP (800 Ci/mmol) was
purchased from PerkinElmer Life Sciences. Tissue culture supplies were
obtained from JRH Biosciences (Lenexa, KS). Acrylamide, bisacrylamide,
protein assay kits, and SDS were purchased from Bio-Rad. Guanosine
diphosphate was obtained from Roche Molecular Biochemicals.
Polyvinylidene difluoride membranes were obtained from Pall Gelman
Sciences (Ann Arbor, MI). Polyclonal G RNA Blot Analysis--
Rat brain and heart RNAs were isolated
from 220-g Harlan Sprague-Dawley rats by extraction with RNAzol
(TEL-TEST, Inc.). Total RNA (50-70 µg) was electrophoresed on
3% formaldehyde-containing 1% agarose gel and transferred to a
nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Buckinghamshire,
United Kingdom) under vacuum. The membrane was baked for 2 h at
80 °C in a vacuum oven and prehybridized and hybridized in phosphate
buffer containing 0.5 M Na2HPO4, pH
7.2, 1% bovine serum albumin, 7% SDS, and 1 mM EDTA as
previously described (16). 32P-Labeled cDNA probes were
generated by random priming using the MultiPrime kit (Amersham
Pharmacia Biotech). Blots were hybridized with the probe for 12 h
at 65 °C, and the membrane was washed twice (40 mM
Na2HPO4, pH 7.2, 5% SDS, and 1 mM
EDTA) at 65 °C and exposed to XAR-5 film for 2-4 days at
Library Screening--
A rat heart cDNA library
(Uni-ZAP XR, lot 937512, Stratagene) was screened with a
32P-labeled 40-mer oligonucleotide probe
(5'-GTGGCCCTACCATGCCTGATGAGGATTTCTTCAGCCTTAT-3') corresponding to
sequence located 1695 nt 3' to the translational start codon of
AGS3-LONG. Procedures for hybridization, phage propagation, and plasmid
excision were performed as previously described (16) and in accordance
with the manufacturer's instructions. Positive phagemids were excised
from the Uni-ZAP XR vector, and the nucleotide sequence of the insert
was determined in the DNA Sequencing Facility at the Medical University
of South Carolina.
5'-RACE--
mRNA was isolated from rat heart
ventricle using the Oligotex mRNA kit (QIAGEN Inc., Chatsworth,
CA). First-strand cDNA was synthesized from 4 µg of mRNA by
reverse transcription (3 units of Superscript II, 30 min, 42 °C)
using a gene-specific primer (5'-TTGGGAGGATTTGGCTTAC-3') derived
from the 3'-UTR. An anchor sequence was then added to the 5'-end of
the cDNA using terminal transferase and dCTP, and the cDNA was
amplified by polymerase chain reaction using anchoring primers
(5'-CUACUACUACUAGGCCACGCCTCGACTAGTACGGGIIGGGIIGGGIIG-3' and
5'-CUACUACUACUAGGCCACGCGTCGACTAGTAC-3') and nested AGS3-specific primers (nucleotides from the translational start codon of AGS3-LONG: nt 1670, 5'-GTTGTTGAGGGTGATGCGAAGC-3'; nt 1539, 5'-TCTGTGGTGAAGCTGTTACAGAGGG-3'; nt 1509, 5'-GTAGCCTCAGCAGCCCCAGC-3';
and nt 1476, 5'-CAGGGGACAGCGTTGGTCATC-3') in accordance with the
manufacturer's instructions. Several series of experiments were done
to optimize the tailing step and conditions for cDNA amplification.
The identification of 5' termini was also addressed by a similar
general strategy using Marathon-Ready cDNA
(CLONTECH, CA) and forward anchoring primers
(5'-CCATCCTAATACGACTCACTATAGGGC-3' and 5'-ACTCACTATAGGGCTCGAGCGGC-3')
and reverse AGS3-specific primers (nt 225 3' to the translational stop
codon of AGS3-LONG, CCCTCCCCATACAGCAGCTCTA-3'; and nt 1941 in the
protein coding region of AGS3-LONG, 5'-TTAGCTGGCACCTGGCGGACAT-3'). After four and two rounds of amplification with nested primers for the
dC-tailed cDNA (kit from Life Technologies, Inc.) and Marathon-Ready cDNA, respectively, the amplified products were electrophoresed on agarose gels, and membrane transfers were
hybridized with a 32P-labeled oligonucleotide (nt 1421 in
the protein coding region of AGS3-LONG,
5'-CCCCGTCCTCTGACGAGGAGTGTTTCTTCGATCTG-3'). Positive DNA fragments were
eluted from the gel and subcloned into the TA cloning vector
pCR-TOPO (Invitrogen, San Diego, CA). Bacterial colonies obtained from
these transformants were again screened by hybridization with the same
AGS3 oligonucleotide. Plasmids from positive colonies were
purified, and the nucleotide sequence of the insert was determined in
the DNA Sequencing Facility at the Medical University of South Carolina.
RNase Protection Assays--
Tissue and cellular mRNAs were
isolated as described above. Riboprobes were labeled with
[32P]UTP (800 Ci/mmol), and RNase protection assays were
performed using the Ambion series of pre-made reagents as described
(15). RNA probes were generated from fragments of AGS3-SHORT cDNAs
that spanned the region of sequence divergence from AGS3-LONG, thus allowing the detection of both AGS3-LONG and AGS3-SHORT transcripts in
the same sample simply based upon size differences. To generate constructs for probe generation, an AGS3-SHORT-1 cDNA obtained by
5'-RACE was isolated from pCR-TOPO by restriction with NaeI and EcoRI and subcloned into the EcoRI and
SmaI sites of pBluescript SK. For AGS3-SHORT-2, the
longest cDNA isolated by library screening was restricted with
EcoRI and SmaI and subcloned into the
EcoRI and SmaI sites of pSK.Bluescript. The
vectors were linearized by XhoI, and 32P-labeled
antisense riboprobes were made using T3 RNA polymerase. 1 µg of heart
or brain mRNA or 0.5 µg of CHO total RNA was hybridized with
probe for 12 h at 45 °C, followed by RNase A/RNase T1
digestion. Protected fragments were separated on an 8 M
urea and 5% polyacrylamide gel and visualized by exposure to X-Omat
film at Cell Transfection and Tissue Fractionation--
CHO and COS-7
cells were grown in Ham's F-12 medium and Dulbecco's modified
Eagle's medium, respectively, supplemented with 10% fetal bovine
serum, penicillin (100 units/ml), streptomycin (100 µg/ml), and
Fungizone (0.25 µg/ml). For transient expression, CHO and COS-7 cells
at 70-80% confluency (100-mm dish) were transfected with 10 µg of
AGS3-LONG or AGS3-SHORT cDNA using 30 µl of LipofectAMINE. Cells
were harvested after 48 h and resuspended in lysis buffer (5 mM Tris-HCl, 5 mM EDTA, 5 mM EGTA,
pH 7.4, 1 mM phenylmethylsulfonyl, 0.2 µg/ml soybean
trypsin inhibitor, and 10 µg/ml aprotinin) with a 26 Immunofluorescence Microscopy--
CHO and COS-7 cells were
plated onto coverslips (18-mm round no. 1) precoated with 0.01%
polylysine and transfected with AGS3-SHORT-1, AGS3-LONG, or AGS3-TPR in
pcDNA3. The AGS3-TPR construct consisted of the first 454 amino
acids of AGS3-LONG. AGS3-TPR was generated by PCR using primers
5'-GGGGATCCATGGAGGCCTCCTGTCTGG-3' (forward) and
5'-GGAATTCTTTAATCAGCACTGTCCAGTG-3' (reverse), in which amino acid codon
455 of AGS3-LONG was mutated to a stop codon. 48 h after
transfection, cells were briefly washed with cell washing solution
(CWS; 137 mM NaCl, 2.6 mM KCl, 1.8 mM KH2PO4, and 10 mM
Na2HPO4) and fixed for 5 min in CWS containing
3% paraformaldehyde. Fixed cells were rinsed with CWS (3 × 3 ml/coverslip) and incubated with 25 mM NH4Cl in
CWS for 10 min to quench free aldehyde groups. Fixed cells were
incubated with 0.05% Triton X-100 for 5 min at room temperature and
washed twice with CWS (3 ml/coverslip). The coverslips were then
incubated with CWS containing 1% fetal bovine serum for 30 min at
24 °C, followed by incubation with a 1:100 dilution of
affinity-purified AGS3 PEP22, PEP32, or PEP98 antibody in CWS
for 14 h at 4 °C. Coverslips were washed three times with CWS
(3 ml/coverslip) and incubated with secondary antibody (fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody, 5 µg/ml) for
60 min at 24 °C. Coverslips were washed three times (3 ml/coverslip) and mounted with Slow-Fade anti-fade reagent (ICN). A Leica DMLB fluorescence microscope and an Olympus confocal fluorescence microscope (FV5-PSU laser, BX50) were used for immunofluorescent microscopy. The
cell nucleus was identified by propidium iodide staining. Multiple
series of experiments (different fixation procedures, incubation times,
and amounts of antibody) were performed to determine the optimal
conditions for signal detection and to verify the specificity of
observed signals. No immunofluorescent signal was detected in control
cells transfected with the pcDNA3 control vector or in the absence
of primary antibody. Each field of cells evaluated by microscopy
contained both transfected and non-transfected cells, providing
additional internal controls for observed signals.
Protein Interaction Assays--
The binding of AGS3-SHORT to
G-proteins was assessed by protein interaction experiments using both
rat heart lysates and purified G
For protein interaction studies with purified G-protein, GST or
GST-AGS3-SHORT-1 fusion proteins (300 nM) were incubated
with 100 nM G Nucleotide Binding Assays--
GTP Tissue Distribution of AGS3 mRNA--
AGS3 was initially
defined as a 650-amino acid protein encoded by an ~4-kb cDNA (6).
To determine the tissue distribution of the AGS3 mRNA, we performed
mRNA blot analysis with two probes derived from the upstream and
downstream regions of the ~4-kb mRNA coding region (Fig.
1A). Both probes identified an
~4-kb mRNA enriched in brain (Fig. 1B). The 3'-probe
identified an additional ~2-kb mRNA enriched in heart (Fig. 1,
B and C). The ~2-kb mRNA was also
identified with hybridization probes derived from the 3'-UTR of the
~4-kb cDNA.3 The
relative expression of the ~4- and ~2-kb AGS3 mRNAs in heart was developmentally regulated. In rat heart, the ~4-kb mRNA was enriched at birth, whereas the ~2-kb mRNA predominated in the adult rat (Fig. 1C). Within the adult rat heart, the ~2-kb
mRNA was clearly enriched in ventricular tissue. Although a
full-length human AGS3 cDNA is not available, we obtained a partial
human AGS3 expressed sequence tag clone and used this sequence to
perform a similar RNA blot analysis in human tissue (Fig.
2). The human AGS3 probe (344 nt)
corresponded to the same region of AGS3 used to generate PROBE
B in Fig. 1 and thus would detect both AGS3-SHORT and AGS3-LONG.
In human, as in rat, there were two AGS3 transcripts of ~2 and 4 kb
that were differentially enriched in heart and brain (Fig. 2). As in
rat heart, the ~4-kb AGS3 mRNA in human heart predominated at
birth, whereas both mRNAs were expressed in the adult heart (Fig.
2). The ~4-kb mRNA and its encoded protein were termed AGS3-LONG,
whereas the ~2-kb mRNA was termed AGS3-SHORT, and we initiated
experiments to define the smaller mRNA and its encoded protein.
Identification of AGS3-SHORT cDNAs--
To define the
AGS3-SHORT transcript, we screened a rat heart cDNA library,
identified the 5' terminus by RACE, and performed RNase protection
experiments. A rat heart cDNA library was screened with an
oligonucleotide derived from the 3'-UTR of AGS3-LONG. 11 individual
cDNAs were isolated and sequenced. The sizes of the cDNAs
ranged from 1509 to 2134 nt, and each contained a 3'-UTR (~1.3 kb)
identical to that of AGS3-LONG. Three identical cDNAs (AGS3-SHORT-1) contained 114 nt of 5'-terminal sequence that diverged from that of AGS3-LONG in the linker domain of the protein coding region of AGS3-LONG (Fig. 3). A fourth
cDNA (AGS3-SHORT-2) contained 46 nt of 5'-terminal sequence that
also diverged from that of AGS3-LONG in the linker region, and this
5'-sequence was distinct from that of AGS3-SHORT-1. The lengths of
AGS3-SHORT-1 and AGS3-SHORT-2 cDNAs (1979 and 2134 nt plus the
poly(A) tail) were similar to the size of the AGS3-SHORT mRNA
identified by RNA blot analysis. The remaining cDNAs isolated from
the rat heart library also terminated in the linker region, but did not
diverge from the AGS3-LONG sequence, likely representing incomplete
processing of the mRNA during reverse transcription.
To further define the population of AGS3-SHORT mRNAs in heart, we
performed 5'-RACE by different methods with two different sources of
rat heart mRNA. AGS3-SHORT-1 and AGS3-SHORT-2 were identified by
5'-RACE in both experimental methods. In the first method, mRNA was
isolated from rat ventricles and reverse-transcribed with an
AGS3-specific primer corresponding to sequence in the 3'-UTR of
AGS3-LONG, and the AGS3-SHORT cDNAs were identified by library
screening. The generated cDNAs were dC-tailed at the 5'-end and
amplified with an anchor primer and a series of nested primers
downstream from the 5' terminus of the AGS3-SHORT cDNAs identified
by library screening. The second method involved a similar
amplification strategy with different AGS3 nested primers, but used a
commercial source of double-stranded cDNAs from rat heart as
template. The two methods were selected to take advantage of the
different 5'-tailing procedures and to evaluate different sources of
mRNA. AGS3-SHORT-1 was represented by 11 of 14 cDNAs in the
first method and by 10 of 18 cDNAs in the second method. The
longest AGS3-SHORT-1 cDNAs identified by 5'-RACE contained 144 nt
of 5'-terminal sequence that diverged from that of AGS3-LONG (Fig. 3).
AGS3-SHORT-2 was identified (3 of 18) only in the second method. The
remaining cDNAs (3 of 14 in the first method and 5 of 18 in the
second method) contained various lengths of sequence that did not
diverge from AGS3-LONG and likely represented incomplete extensions of
the larger cDNA. Thus, AGS3-SHORT-1 and AGS3-SHORT-2 cDNAs were
identified in three procedures using different sources of RNA and
experimental strategies. In each case, AGS3-SHORT-1 cDNA was the
predominant species.
Finally, we confirmed the expression of AGS3-SHORT cDNAs in heart
by RNase protection assays using a probe design that allowed us to
identify the AGS3-LONG and AGS3-SHORT cDNAs in the same reaction
(Fig. 4A). To provide internal
controls for observed signals, CHO cells were transiently transfected
with AGS3-SHORT or AGS3-LONG cDNAs. As illustrated in Fig.
4B, RNase protection assays with the AGS3-SHORT-1 cDNA
probe yielded two protected fragments in heart of ~350 and ~230 nt,
corresponding to the predicted sizes of AGS3-SHORT-1 and AGS3-LONG
(Fig. 4B), respectively. Due to the design of the
AGS3-SHORT-1 probe, the signal in heart and brain actually represents
the sum total of both AGS3-LONG and AGS3-SHORT-2 mRNAs. RNase
protection assays with the AGS3-SHORT-2 cDNA probe (Fig.
4C) also yielded two protected fragments in heart and brain
of ~230 and ~190 nt, corresponding to the predicted sizes of
AGS3-SHORT-2 and AGS3-LONG, respectively. The two protected species
corresponding to AGS3-SHORT and AGS3-LONG were the two major fragments
detected in the assay, indicating the absence of any other
heterogeneity in this region of the transcript. The two major protected
species were also observed with both probes using brain RNA, although
the AGS3-LONG fragment clearly predominated in brain tissue. Based upon
the relative intensities of the AGS3-SHORT and AGS3-LONG protected
species in Fig. 4 (B and C), it is also clear
that the AGS3-SHORT-1 cDNA is the major AGS3-SHORT transcript, as
suggested by the results obtained with library screening and 5'-RACE.
These data indicate that the AGS3-SHORT and AGS3-LONG transcripts
contain identical 3'-UTRs and share a segment of the AGS3-LONG coding
region, but differ in their 5' termini. The 5'-end of AGS3-SHORT mRNAs terminates in the linker domain of the protein coding region of AGS3-LONG and as such may encode a "truncated" version of the protein encoded by AGS3-LONG. This truncated version would be of
particular interest as it would contain the GPR motifs that regulate
G Identification of the Protein Encoded by AGS3-SHORT
cDNAs--
Analysis of the open reading frames for AGS3-SHORT
cDNAs indicated that the longest open reading frame corresponded to
the carboxyl-terminal third of the protein encoded by AGS3-LONG (Fig. 5) and that the divergent 5'-sequence of
the AGS3-SHORT cDNAs was not in frame with the AGS3 protein coding
sequence. For AGS3-SHORT-1, the major transcript in heart, the longest
open reading frame began at nt 217, encoding a protein of 166 amino
acids with a calculated Mr of 18,117. AGS3-SHORT-2 contains a slightly longer portion of AGS3-LONG at its
5'-end, and the longest open reading frame would begin at nt 189, encoding a protein of 227 amino acids with a calculated
Mr of 25,076. The longest open reading frames for the AGS3-SHORT transcripts would initiate at methionines within the
AGS3-LONG sequence itself. Neither of these methionines is placed in
the context of an optimal Kozak consensus sequence for initiation of
translation (17).
To define the encoded protein, we transiently expressed
AGS3-SHORT cDNAs in COS-7 and CHO cells and performed immunoblot
analysis with antisera generated against peptides derived from
different regions of AGS3-LONG (Fig.
6A). PEP22 and PEP98 antisera
would recognize AGS3-LONG and any AGS3-related proteins encoded by the AGS3-SHORT transcripts. PEP32 would recognize AGS3-LONG, but would not
recognize any AGS3-related proteins encoded by the AGS3-SHORT-1 transcripts. The first in-frame methionine for AGS3-SHORT-2 is in the
middle of the peptide sequence used to generate PEP32 and thus may
or may not be recognized by PEP32 depending upon the epitope (Fig.
6A). Immunoblot analysis indicated that both AGS3-SHORT cDNAs encoded a major Mr ~ 23,000 polypeptide (Fig. 6B). The Mr ~ 23,000 peptide was not observed in immunoblots with PEP32 (Fig. 6B). The Mr ~ 23,000 peptide was
not observed following transfection of COS-7 cells with
pcDNA3-AGS3-SHORT-1M*, in which the first in-frame methionine was
eliminated by site-directed mutagenesis (Fig. 6C). The start
methionine for the Mr ~ 23,000 peptide
corresponds to the first in-frame methionine for AGS3-SHORT-1 (nt 217)
and the second in-frame methionine for AGS3-SHORT-2 (nt 372). Thus, the
Mr ~ 23,000 peptide likely reflects
translational initiation at the same methionine for both AGS3-SHORT
cDNAs.
The AGS3-SHORT-2 cDNA also encoded two additional
immunoreactive species (Mr ~ 32,000 and
34,000) upon COS-7 or CHO cell transfection. Both the
Mr ~ 32,000 and 34,000 peptides were
recognized by AGS3 PEP32, PEP22, and PEP98 antisera (Fig.
6B). The Mr ~ 32,000 peptide was
not observed following transfection of COS-7 cells with
pcDNA3-AGS3-SHORT-2M*, in which the first start methionine (nt 189)
in frame with the AGS3 coding sequence was eliminated by site-directed
mutagenesis (Fig. 6C). The immunoreactive signal for the
Mr ~ 32,000 peptide was weaker than that for
the Mr ~ 34,000 peptide, on the PEP32 immunoblot, which may reflect differences in the ability of the antibody to recognize the two proteins. As there are no additional in-frame codons for methionines upstream of nt 189, the
Mr ~ 34,000 peptide must initiate at a non-AUG
start codon in frame with the AGS3 protein coding sequence
(18-20). The immunoreactive proteins expressed by AGS3-SHORT cDNAs
in the transient transfection system were identical to those observed
by in vitro translation using [35S]methionine
to detect expressed proteins.3
As AGS3-SHORT mRNAs are enriched in heart, we then determined if
these mRNAs were indeed translated. Rat heart tissue was fractionated to generate a post-nuclear crude membrane fraction and a
100,000 × g supernatant for analysis by immunoblotting
with AGS3-specific antisera. A major Mr ~ 23,000 polypeptide was detected in heart by immunoblotting with either
PEP22 (Fig. 7) or PEP98 antisera.3 The Mr ~ 23,000 immunoreactive species comigrated with the Mr ~ 23,000 peptide observed following transient expression of
AGS3-SHORT cDNAs (Fig. 7). As observed for the
Mr ~ 23,000 peptide expressed in COS-7 or CHO
cells, the Mr ~ 23,000 immunoreactive peptide in heart was also enriched in the cytosol or 100,000 × g supernatant. In contrast to heart, the
Mr ~ 72,000 peptide encoded by AGS3-LONG was
the major immunoreactive peptide in brain. The
Mr ~ 34,000 immunoreactive peptide in brain
detected by PEP22 (Fig. 6) was not recognized by either PEP32 or PEP98
antibodies. The Mr ~ 32,000 and 34,000 peptides encoded by AGS3-SHORT-2 following transient transfection of
COS-7 or CHO cells were not observed in heart (Fig. 6A). The
lack of the larger Mr peptides likely reflects both the lower abundance of AGS3-SHORT-2 mRNA in heart as compared with AGS3-SHORT-1 mRNA as well as the preferential use of the second in-frame methionine (nt 372) as the translational start site in
AGS3-SHORT-2 (Fig. 6B).
Subcellular Distribution of AGS3-SHORT and AGS3-LONG--
Whereas
AGS3-LONG contains seven TPR and four GPR motifs, AGS3-SHORT lacks TPR
domains and contains 3.5 GPR motifs. The TPR regions of the
Drosophila AGS3 homolog PINS play a key role in the
positioning of the protein within the cell (21, 22). Thus, AGS3-SHORT
and AGS3-LONG may follow different trafficking patterns within the
cell. To more specifically address this issue, we performed immunofluorescent experiments to determine the subcellular location of
AGS3-SHORT and AGS3-LONG following transient expression in COS-7 and
CHO cells. Immunofluorescent analysis of AGS3 expression indicated
clear differences in the subcellular distribution of the long and short
AGS3 variants (Fig. 8). AGS3-SHORT was
found throughout the cell, with a diffuse distribution in the cytosol. AGS3-LONG exhibited more of a punctate distribution, non-homogeneously distributed in the cell cytoplasm. The subcellular distribution of
AGS3-TPR, which contained the first 454 amino acids of AGS3 and thus
lacked the GPR domains, was identical to that of full-length AGS3-LONG.
The subcellular distributions of AGS3-SHORT, AGS3-LONG, and AGS3-TPR
were similar in both COS-7 and CHO cells. Another construct in which
the AGS3-SHORT sequence was extended upstream to the carboxyl-terminal
end of TPR-VII exhibited a subcellular distribution identical to that
of AGS3-SHORT.3 These data indicate that the TPR domains
indeed account for the differences in the targeting of AGS3-SHORT and
AGS3-LONG within the cell.
Multiple experiments in our laboratory are consistent with this point,
particularly if one evaluates the relative distribution of the two
forms of AGS3 in a 100,000 × g pellet and supernatant following cell lysis. Subcellular fractionation indicated that AGS3-SHORT readily partitioned into a 100,000 × g
supernatant in rat heart and following transient expression in
mammalian cells (Figs. 6 and 7). In contrast, AGS3-LONG was found in
both the 100,000 × g pellet and supernatant following
transient expression in mammalian cells. The relative distribution of
AGS3-LONG in the 100,000 × g pellet versus
supernatant was sensitive to the method
of cell fractionation and varied in different cell
types.3-5 Repeated washing
of the 100,000 × g pellet releases the majority of
AGS3-LONG into the supernatant, as observed for preparations from rat
brain and primary cultures of cortical neurons and transfected cells
(8).3-5 In contrast, essentially all of the AGS3-SHORT was
found in the 100,000 × g supernatant independent of
pellet washing. These data suggest a poorly understood, loose
association of AGS3-LONG with a binding partner that retains AGS3-LONG
in a different cellular compartment compared with AGS3-SHORT.
Interaction of AGS3-SHORT with G-proteins Results in Inhibition of
Guanine Nucleotide Exchange and Movement of AGS3-SHORT to a Membrane
Fraction--
To define the functional influence of AGS3-SHORT on
G-protein, we first evaluated the interaction of AGS3-SHORT with
purified G
Although AGS3-SHORT and AGS3-LONG both bind G The response of the cell to external stimuli has
become progressively more intricate as higher organisms have evolved.
Nature has certainly found the seven-membrane span motif along with its "partner" G-proteins to be one of the most efficient mechanisms for
selective processing of spatially and temporally delineated signals by
the cell. As the cell found itself challenged by a number of diverse
stimuli, it evolved various mechanisms of integration to handle these
inputs. The seven-membrane span motif has become more and more diverse
in terms of the stimuli that it recognizes, and G-proteins have evolved
by subunit diversification, differences in guanine
nucleotide-binding/hydrolysis properties, and variations in
post-translational modifications (23). Mechanisms for signal integration also include stoichiometric considerations, signal cross-talk, and spatial restriction of signaling components within the cell.
Additional specialized mechanisms for fine-tuning signal processing
include receptor regulation and non-receptor proteins/lipids that
influence subunit interactions and the activation state of G-proteins.
Such entities include a number of proteins identified in protein
interaction or functional screens. AGS3 is one of three proteins
isolated as a G-protein regulator in a functional screen. This report
indicates the expression of two forms of AGS3 (AGS3-SHORT and
AGS3-LONG) that are differentially enriched in heart and brain. The two
forms of AGS3 essentially differ in the presence and absence of the TPR
domains found in the amino-terminal half of AGS3-LONG. The mechanism by
which AGS3 is generated as two forms is unclear. Although the
AGS3 (rat or human) gene is not defined, the AGS3-related protein in Caenorhabditis elegans is encoded by 14 exons
(GenBankTM/EBI accession number U40409) (8). Thus, the two
forms of AGS3 may be generated by alternative splicing,
trans-splicing or even alternative promoters. Data base
searches (BLAST) with the divergent 5'-ends of AGS3-SHORT cDNAs
indicated no matches with AGS3-SHORT-2; however, the
AGS3-SHORT-1-specific sequence was identical to a segment of the 5'-UTR
of OMP25 (24). Whereas AGS3-LONG is the major species in adult rat
brain, AGS3-SHORT is the major AGS3 protein expressed in adult rat
heart. The relative expression of the two forms of AGS3 is
developmentally regulated and apparently varies among the different
regions of the heart.
At a structural level, the major differences between the short and long
form of AGS3 are due to the absence in AGS3-SHORT of the seven TPR
domains found in AGS3-LONG. This likely has important implications in
terms of regulation of the function of AGS3-SHORT and AGS3-LONG. The
TPR domain of the AGS3-LONG-related protein in Drosophila,
PINS, is required for translocation of the protein to a specific
membrane domain of the neuroblast, and this is achieved by the binding
of the protein INSCUTEABLE to the TPR domains of PINS. The TPR
motif is a highly degenerate sequence found in a large number of
proteins, where it subserves various functions via protein
interactions. The absence of the TPR motif in the AGS3-SHORT protein
apparently accounts for the differences in subcellular distribution of
the two forms of AGS3. The generation of two forms of AGS3 that
apparently differ in subcellular trafficking and tissue distribution,
but yet both interact with G AGS3-LONG in rat brain (8) and the AGS3-LONG-related protein PINS in
Drosophila melanogaster (21, 22) are complexed with
Gi/G
t-binding protein that competes with G
for interaction with G
GDP and acts
as a guanine nucleotide dissociation inhibitor for heterotrimeric
G-proteins. An ~2-kb AGS3 mRNA (AGS3-SHORT) is enriched in rat
and human heart. We characterized the heart-enriched mRNA,
identified the encoded protein, and determined its ability to interact
with and regulate the guanine nucleotide-binding properties of
G-proteins. Screening of a rat heart cDNA library, 5'-rapid
amplification of cDNA ends, and RNase protection assays identified
two populations of cDNAs (1979 and 2134 nucleotides plus the
polyadenylation site) that diverged from the larger 4-kb
mRNA (AGS3-LONG) in the middle of the protein coding region.
Transfection of COS-7 cells with AGS3-SHORT cDNAs resulted in the
expression of a major immunoreactive AGS3 polypeptide
(Mr ~ 23,000) with a translational start site
at Met495 of AGS3-LONG. Immunoblots indicated the
expression of the Mr ~ 23,000 polypeptide in
rat heart. Glutathione S-transferase-AGS3-SHORT selectively
interacted with the GDP-bound versus guanosine
5'-O-(3-thiotriphosphate) (GTP
S)-bound conformation of
G
i2 and inhibited GTP
S binding to G
i2.
Protein interaction assays with glutathione
S-transferase-AGS3-SHORT and heart lysates indicated
interaction of AGS3-SHORT with G
i1/2 and
G
i3, but not G
s or G
q.
Immunofluorescent imaging and subcellular fractionation following
transient expression of AGS3-SHORT and AGS3-LONG in COS-7 and Chinese
hamster ovary cells indicated distinct subcellular distributions of the
two forms of AGS3. Thus, AGS3 exists as a short and long form, both of
which apparently stabilize the GDP-bound conformation of
G
i, but which differ in their tissue distribution and
trafficking within the cell.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i/G
o (5). In contrast, AGS3 actually
stabilizes the GDP-bound conformation of G
i, inhibits
dissociation of GDP from G
i, and competes with G
for interaction with G
i (6, 8-10). Thus,
AGS3 may function as a guanine nucleotide dissociation inhibitor for
G
i proteins and/or serve as a binding partner for
G
i independent of G
.
i. As part
of a continuing effort to understand the overall role of AGS3 and related proteins in G-protein function, we defined the tissue distribution of AGS3 mRNA. RNA blot analysis actually revealed two
major transcripts of ~2 and ~4 kb that were selectively enriched in
heart and brain. Further analysis of the AGS3 mRNA species indicated that the two transcripts potentially encoded related but
distinct AGS3 proteins. We report the identification of a short form of
AGS3 (AGS3-SHORT, 166 amino acids) that is encoded by the ~2-kb
heart-enriched mRNA. AGS3-SHORT lacks the TPR domains found in
AGS3-LONG, but contains three complete GPR motifs and a portion of the
fourth GPR motif found in AGS-LONG. GST-AGS3-SHORT selectively
interacted with G-proteins in heart cell lysates and inhibited
nucleotide exchange on purified G
i2. Immunofluorescent imaging of mammalian cells transiently expressing AGS3-SHORT or AGS3-LONG indicated distinct locations of the two forms of AGS3 within
the cell. AGS3 thus exists as short and long forms that differ in their
tissue distribution and trafficking within the cell.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i3 antisera
generated against the carboxyl-terminal 10 amino acids was kindly
provided by Dr. Thomas W. Gettys (Department of Medicine, Medical
University of South Carolina) (12). Superscript II, LipofectAMINE, and
the 5'-RACE kit were obtained from Life Technologies, Inc.
Marathon-Ready cDNA and multiple-tissue blots for rat and human
mRNAs were obtained from CLONTECH.
G
i2, expressed in and purified from Sf9 cells,
was kindly provided by Stephen G. Graber (Department of Pharmacology,
West Virginia University School of Medicine) (13). All other materials
were obtained as previously described (6, 14, 15).
70 °C.
90 °C for 12-24 h. The respective insert and probe
lengths for AGS3-SHORT-1 were 357 and 447 nt. The respective insert and
probe lengths for AGS3-SHORT-2 were 230 and 319 nt. The sizes of the
protected fragments resulting from annealing the AGS3-SHORT-1 and
AGS3-SHORT-2 probes with AGS3-LONG would be 228 and 184 nt, respectively.
Ile436)) from different areas of
AGS3-LONG were synthesized and conjugated for generation of rabbit
polyclonal antisera in the Peptide Synthesis and Antibody Production
Facility at the Medical University of South Carolina. Each of the
antisera was affinity-purified prior to use.
i2. The AGS3-SHORT
sequence encoding the Mr ~ 23,000 peptide was
amplified by polymerase chain reaction and fused in frame to
glutathione S-transferase in the pGEX-4T vector (Promega,
Madison, WI). The GST-AGS3-SHORT fusion protein was expressed in
bacteria (Escherichia coli BL21, Amersham Pharmacia Biotech)
and purified on a glutathione affinity matrix. The AGS3-SHORT fusion
protein was eluted from the resin, and glutathione was removed by
desalting as previously described (14) to allow a solution-phase
interaction assay. Rat heart lysates were prepared by Dounce
homogenization in buffer (5 ml/heart) containing 50 mM
Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1%
Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride. The
homogenates were gently rotated for 1 h on ice at 4 °C and then
centrifuged at 27,000 × g for 30 min. Supernatants
were collected and spun at 100,000 × g for 30 min to
generate a detergent-soluble fraction.
i2 (preincubated with 10 µM GDP or GTP
S and 10 mM MgCl2 for 10 min at 4 °C) in 250 µl of incubation buffer (20 mM Tris-HCl, pH 7.4, 0.6 mM EDTA, 1 mM dithiothreitol, 70 mM NaCl, 0.01% Lubrol, 10 µM GDP or GTP
S, and 10 mM
MgCl2) for 3 h at 4 °C with gentle rotation. 30 µl of a 50% slurry of glutathione-Sepharose was added, and
incubation was continued at 4 °C for 40 min. Samples were then
centrifuged, and the matrix was washed with 3 × 250 µl of incubation buffer prior to solubilization in Laemmli sample buffer. For
interaction assays with tissue lysates, GST-AGS3-SHORT (5 µg) was
incubated with 1-2 mg of lysate protein in buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM
EDTA, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride,
and 10 µM GDP for 12 h at 4 °C in a total volume
of 250 µl. 25 µl of a 50% slurry of glutathione-Sepharose was
added, and the mixture was rotated at 4 °C for 40 min, after which
the affinity matrix was pelleted and washed three times with 500 µl
of incubation buffer. Proteins retained on the matrix were solubilized
in Laemmli sample buffer. Solubilized samples were electrophoresed on
denaturing 10% polyacrylamide gels, and proteins were transferred to
polyvinylidene difluoride membranes for immunoblotting with G
antisera. For reprobing of membranes, the membrane transfers were
washed with immunoblot buffer containing 20 mM Tris-HCl, pH
7.6, 140 mM NaCl, and 0.2% Tween and then incubated with
preheated stripping buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM
-mercaptoethanol) for 20 min in a
55 °C water bath with gentle shaking. The membrane was washed with
immunoblot buffer and processed for immunoblotting. Each blot was also
incubated with AGS3 antisera and stained with Amido Black to provide
internal controls for protein loading.
S binding assays were
generally conducted as described (8, 9). G-proteins (100 nM) were preincubated for 20 min at 24 °C in the
presence and absence of increasing concentrations of GST or
GST-AGS3-SHORT. Reactions were initiated by addition of 0.5 µM [35S]GTP
S (4 × 104
dpm/pmol), and incubations (total volume of 50 µl) were continued for
30 min at 24 °C. Both preincubations and GTP
S binding assays were
conducted in binding buffer containing 50 mM Hepes-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, 2 mM MgCl2, 50 µM adenosine triphosphate, and 10 µg/ml bovine serum albumin. Reactions were terminated by rapid filtration through nitrocellulose filters with
3 × 4 ml of stop buffer (50 mM Tris-HCl, 5 mM MgCl2, and 1 mM EDTA, pH 7.4) at
4 °C. Radioactivity bound to the filters was determined by liquid
scintillation spectrometry. Nonspecific binding was defined with 100 µM GTP
S.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Tissue distribution of rat AGS3
mRNA. RNA blots in B and C were
hybridized with radiolabeled probe derived from different regions of
AGS3 as indicated in the schematic representation of AGS3 in
A. The hatched and solid bars within
the AGS3 coding region correspond to TPR and GPR motifs, respectively.
B, CLONTECH multiple-tissue blot from
rat containing 2 µg of poly(A)+ RNA/lane; C,
70 µg of total rat brain or heart RNA. Blots were stripped and
hybridized with a random-primed probe from the coding region of rat
-actin. ORF, open reading frame; bp, base
pairs; sm, skeletal muscle. In C, atria and
ventricle are from adult rat.
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Fig. 2.
Tissue distribution of human AGS3
mRNA. CLONTECH multiple-human tissue and
cardiovascular system blots containing 2 µg of poly(A)+
RNA/lane were hybridized with a 344-nt probe generated from a partial
human AGS3 cDNA (GenBankTM/EBI accession number AI272212;
2117 nt). The 344-nt probe corresponded to same region of AGS3 used to
generate PROBE B in Fig. 1 and thus would detect both
AGS3-SHORT and AGS3-LONG. Sm, skeletal muscle;
LA, left atrium; RA, right atrium; LV,
left ventricle; RV, right ventricle. Blots were stripped and
also hybridized with a random-primed probe from the coding region of
rat -actin.
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Fig. 3.
AGS3-SHORT cDNAs identified by library
screening and 5'-RACE. Two populations of heart AGS3 cDNAs
(AGS3-SHORT-1 and AGS3-SHORT-2) were identified as described under
"Experimental Procedures." Uppercase letters
indicate sequence identity to AGS3-LONG. The sequence unique to
AGS3-SHORT cDNAs is indicated by lowercase letters. The
arrows in the 3'-UTR and the coding region of AGS3-LONG
indicate nested primers used for PCR amplification in 5'-RACE in the
first and second experimental methods as described under
"Experimental Procedures." The hatched and solid
bars within the AGS3-LONG coding region correspond to TPR and GPR
motifs, respectively. The stippled and striped
segments of AGS3-SHORT cDNAs indicate sequence divergence from
AGS3. The sequences of the unmarked open segment of
AGS3-SHORT cDNAs and the 3'-UTR of AGS3-SHORT cDNAs were
identical to those of AGS3-LONG.
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Fig. 4.
RNase protection experiments with probes
derived from AGS3-SHORT cDNAs. A, the regions used
to generate riboprobes for AGS3-SHORT cDNAs are indicated by the
thick lines. The probes thus overlap the point at which the
sequence of AGS3-SHORT cDNAs diverged from that of AGS3-LONG as
described under "Experimental Procedures." Refer to the Fig. 3
legend for additional details. B and C,
AGS3-SHORT cDNAs were inserted into pcDNA3 and transiently
expressed in CHO cells as described under "Experimental Procedures"
to provide internal controls. 1 µg of mRNA from brain or heart
and 0.5 µg of total RNA from CHO transfectants were incubated with
~300,000 cpm AGS3-SHORT-1 (B) or AGS3-SHORT-2
(C) riboprobes at 45 °C for 12 h, and hybrids were
treated with RNases, followed by precipitation. The pellets were
resuspended in 20 µl of loading buffer and resolved on urea-5%
polyacrylamide gels. 3 µl of the CHO transfectant samples and 10 µl
of the tissue samples were loaded in B. 5 µl of the CHO
transfectant, 5 µl of the brain samples, and 15 µl of the heart
samples were loaded in C. In B, the AGS3-SHORT-1
cDNA used for CHO transfection actually lacked 15 nt at the 5'-end
found in the AGS3-SHORT-1 RNA used for probe generation and
thus resulted in the slightly smaller size observed with CHO
versus heart RNA. In C, the AGS3-SHORT-2 cDNA
used for CHO transfection and probe generation shared an extra 9 nucleotides from the vector, thus accounting for the slightly larger
size observed with CHO versus heart RNA.
i, but would lack the tetratricopeptide repeats
found in the amino-terminal half of the protein.
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Fig. 5.
Open reading frame for AGS3-SHORT-1 and
AGS3-SHORT-2. Uppercase letters indicate sequence
identity to AGS3. The sequence unique to AGS3-SHORT cDNAs is
indicated by lowercase letters. Amino acids are indicated
beginning with the first ATG start codon in AGS3-SHORT cDNAs that
is in frame with the AGS3-LONG protein coding sequence.
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Fig. 6.
Transient expression of AGS3-SHORT cDNAs
in COS-7 cells. A, schematic diagram indicating the
locations of peptides used to generate AGS3 antisera. B,
COS-7 cells were transfected with pcDNA3-AGS3-SHORT-1,
pcDNA3-AGS3-SHORT-2, or pcDNA3-AGS3-LONG, and cells were
harvested and processed 48 h later as described under
"Experimental Procedures." P, 100,000 × g pellet; S, 100,000 × g
supernatant. Membrane transfers were immunoblotted with antisera
generated against AGS3 peptides derived from different regions of
AGS3-LONG as indicated in A. LONG and
SHORT-1 lanes, 15 µg of protein loaded for the pellet and
supernatant; Vector and SHORT-2 lanes, 30 µg of
protein loaded for the pellet and supernatant. The migration positions
of size standards (×10 3; low molecular
weight standards, Bio-Rad) are indicated between the panels. The
arrowheads indicate the migration positions of
immunoreactive AGS3 peptides. Data are representative of four
independent experiments, and identical results were obtained with CHO
transfectants. C, shown are site-directed mutations in
putative translational start sites in AGS3-SHORT cDNAs. The first
in-frame methionines for AGS3-SHORT cDNAs were disrupted by
site-directed mutagenesis (ATG to GGG) and then transiently expressed
in COS-7 cells as described under "Experimental Procedures." After
48 h, cells were harvested and processed to generate a
100,000 × g supernatant fraction for evaluation by
SDS-polyacrylamide gel electrophoresis and immunoblotting with PEP22
antisera. SHORT-1 and SHORT-1M* lanes, 15 µg of protein
loaded; Vector, SHORT-2, and SHORT-2M*
lanes, 30 µg of protein loaded. Data are representative of two
independent experiments using different batches of transfected cells.
Refer to the Fig. 3 legend for additional details.
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Fig. 7.
Expression of AGS3-SHORT in heart.
Tissues were homogenized and processed to generate crude membrane and
cytosol fractions as described under "Experimental Procedures."
P, 100,000 × g pellet; S,
100,000 × g supernatant. Internal controls for protein
migration were generated by transient expression of AGS3-SHORT and
AGS3-LONG cDNAs in COS-7 cells. Protein was loaded as follows:
Heart and Brain lanes, 100 µg for the pellet
and supernatant; AGS3-LONG lane (COS-7), 5 µg of protein;
AGS3-SHORT-1 and AGS3-SHORT-2 lanes (COS-7), 10 and 20 µg of protein, respectively. The migration positions of size
standards (×10 3; low molecular weight
standards, Bio-Rad) are indicated to the right. Similar data were
obtained in five separate experiments with different cell and tissue
preparations.
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Fig. 8.
Immunofluorescent analysis of AGS3
distribution in mammalian cells. CHO (upper panels) and
COS-7 (lower panels) cells were transfected with
AGS3-SHORT-1, AGS3-LONG, or AGS3-TPR and processed as described under
"Experimental Procedures." AGS3-SHORT and AGS3-LONG were detected
with PEP22 antibody, whereas PEP32 antisera were used to detect
AGS3-TPR. Each field of cells was evaluated by confocal microscopy at
different planes of the cells, and the micrographs are images taken
from approximately the middle plane of the cells. The large
rounded area in the middle of the cell devoid of signal
corresponds to the cell nucleus as defined by propidium iodide
staining. This image was generated with an Olympus laser scanning
confocal imaging system at a magnification of ×60. This figure is
representative of images obtained in 5-10 different transfection
experiments. No immunofluorescent signal was detected in the absence of
primary antibody. Similar results were obtained with PEP98 antibody for
AGS3-SHORT and with PEP98 and PEP32 antibodies for AGS3-LONG.
i2. AGS3-SHORT was generated as a GST fusion
protein and incubated with purified G
i2 in the presence
of GDP or GTP
S. G-protein bound to AGS3-SHORT was isolated on a
glutathione affinity matrix and identified by immunoblotting (Fig.
9A). AGS3-SHORT selectively interacted with the GDP-bound conformation of G
i2. These
studies were then extended to determine the effect of AGS3-SHORT on the nucleotide-binding properties of G
i2. AGS3-SHORT
effectively inhibited the binding of GTP
S to G
i2
(Fig. 9B), consistent with the idea that the interaction of
AGS3-SHORT with G
i2 stabilizes the GDP-bound
conformation of G
i. Further studies with rat heart lysates indicated a preference of AGS3-SHORT for the Gi
family of proteins (Fig. 9C). GST-AGS3-SHORT bound
G
i1/2 and G
i3, but not G
s,
G
o, or G
q. Despite the presence of added
GDP, which would stabilize the heterotrimeric G-protein complex,
G
was not associated with the G
i bound to AGS3
(Fig. 9C).
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Fig. 9.
Interaction of AGS3-SHORT with
G-proteins. The region of AGS3-SHORT cDNA encoding the
Mr ~ 23,000 immunoreactive peptide was
generated as a GST fusion protein and used in protein interaction
studies with purified G i2 (A), guanine
nucleotide binding assays with purified G
i2
(B), or protein interaction assays with heart lysates
(C) as described under "Experimental Procedures." In
B, data are presented as the mean ± S.E. from three
experiments. In C, 2 mg of heart protein lysate was
incubated with 5 µg of GST or GST-AGS3-SHORT fusion protein.
AGS3-SHORT and proteins bound to AGS3-SHORT were isolated on a
glutathione affinity matrix (10). Following gel electrophoresis of
bound proteins, membrane transfers were probed with G-protein subunit
antisera with intervening stripping of the blots as described under
"Experimental Procedures." Experiments generating the data for
A and C were repeated twice with identical
results. The INPUT lanes in A and C
contain one-twentieth of the lysate or purified G
i2 used
in each individual interaction assay.
i, the
relative distribution of AGS3 and G-proteins within the cell differs, indicating that only a subpopulation of the two proteins may be complexed at any given time. A key question is what determines the
subcellular location of AGS3 and if the trafficking of AGS3 is
regulated by signals from inside or outside of the cell. As an initial
approach to this issue, we investigated if coexpression of AGS3-SHORT
or AGS3-LONG with G
i influenced the amount of AGS3 or
G
i2 found in the 100,000 × g pellet
following transient expression in COS-7 or CHO cells. Coexpression of
AGS3-SHORT with G
i2 resulted in the movement of the
AGS3-SHORT protein from the 100,000 × g supernatant to
the 100,000 × g pellet (Fig.
10). The already substantial portion of
AGS3-LONG found in the 100,000 × g pellet was not
altered by coexpression of G
i2. In cells transfected
with pcDNA3-G
i2, essentially all of the
G
i2 protein was in the 100,000 × g
pellet, and this was not altered by coexpression of AGS3-SHORT or
AGS3-LONG.3 The translocation of AGS3-SHORT by
G
i2 was specific, as there was only minimal
translocation of AGS3-SHORT following overexpression of
G
s (Fig. 10), which is consistent with the protein
interaction experiments in which GST-AGS3-SHORT selectively bound
G
i versus G
s in heart cell
lysates (Fig. 10).
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Fig. 10.
Influence of
G i2 and
G
s on subcellular distribution of
AGS3-SHORT and AGS3-LONG. COS-7 cells were transfected with
pcDNA3 (Vector), pcDNA3-G
i2 (7.5 µg), pcDNA3-G
s (7.5 µg), pcDNA3-AGS3-SHORT-1
(2.5 µg), pcDNA3-AGS3-LONG (2.5 µg), or pcDNA3-G-protein
plus pcDNA3, pcDNA3-AGS3-SHORT, or pcDNA3-AGS3-LONG. In
each transfection, the total amount of plasmid used was 10 µg. Cells
were harvested 48 h after transfection and processed as described
under "Experimental Procedures." A 100,000 × g
pellet and supernatant were prepared for each sample, and 20 µg of
membrane protein was loaded in each lane. Membrane transfers were
immunoblotted with PEP22 antisera. Blots were also stripped and
reprobed with G
i and G
s antisera to
confirm expression of the transfected G
protein. The migration
positions of size standards (× 10
3; low
molecular weight standards, Bio-Rad) are indicated to the left. Data
are representative of four independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i subunits, is of particular
interest. The two forms of AGS3 may play different roles in
G-protein-regulated events.
i, and AGS3-SHORT selectively interacts with
G
i proteins in heart. The regions of AGS3 interacting
with G-proteins are localized to the GPR motifs in the
carboxyl-terminal half of the protein (6, 8), which are present in both
AGS3-SHORT and AGS3-LONG. Each GPR motif in AGS3 appears capable of
binding G
i(GDP) (8). Indeed, protein interaction
experiments with a GST-AGS3 fusion protein containing four GPR motifs
indicate that multiple G
i subunits may be tethered to
AGS3, suggesting a role for the protein within a larger signal
transduction complex (8). The interaction of AGS3 GPRs with
G
i or G
t actually inhibits dissociation of GDP from G
i (8-10). The AGS3 GPRs also compete with
G
for interaction with G
i (8). In the context of
Gi heterotrimers, such an effect would lead to the
generation of a G
i(GDP)·AGS3 complex with
"release" of G
(6, 8). Indeed, in the protein interaction
assays using heart lysates, G
subunits were not associated with
the GST-AGS3-SHORT·G
i complex. The interaction of AGS3
with G
i/G
t may also play a regulatory
role in G-protein activation by G-protein-coupled receptors. The GPR
domains of AGS3 actually block rhodopsin activation of transducin and
also prevent the interaction of Gi proteins with
G-protein-coupled receptors (9, 10). Alternatively, the
G
i(GDP)·AGS3 complex may exist independent of G
and subserve unique regulatory functions within the cell, where it
might receive input from a putative guanine nucleotide exchange factor
other than a G-protein-coupled receptor. AGS3 is actually just one of
several proteins that regulate the activation state of G-protein
independent of the receptor. Such proteins may be key players in signal
integration and the dysfunctional signaling events observed in
pathophysiological settings.
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ACKNOWLEDGEMENTS |
---|
We thank Mary Cismowski and Emir Duzic for
their thoughts, suggestions, and encouragement. We thank John D. Hildebrandt (Medical University of South Carolina) for providing
Gs, G
o, and G
antisera; Thomas W. Gettys for G
i3 antisera; and Drs. Goldsmith, Shenkar, and Spiegel for Gq antibody. We thank Drs. George
Lindenmayer and Robert Thompson (Medical University of South Carolina)
for assistance with heart dissections. We thank Stephen G. Graber for purified G
i2 and Jane Jourdan for technical support.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants NS24821 and MH59931 (to S. M. L.).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.
Recipient of a Medical University of South Carolina Health
Sciences Foundation research fellowship and an Association de Recherche Contre le Cancer postdoctoral fellowship.
§ Supported in part by an Ikuei-Kai scholarship and a visiting graduate student from the Department of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan.
¶ To whom correspondence should be addressed: Dept. of Pharmacology, Louisiana State University Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112. Tel.: 504-568-4740; Fax: 504-568-2361; E-mail:slanie@lsuhsc.edu.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M007573200
2 The GPR motif was also termed the GoLoco motif (11).
3 N. Pizzinat and S. M. Lanier, unpublished observations.
4 M. Bernard and S. M. Lanier, unpublished observations.
5 J. Blumer, S. M. Lanier, and J. Chandler, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
kb, kilobase(s);
TPR, tetratricopeptide repeat;
GPR, G-protein regulatory;
GST, glutathione S-transferase;
RACE, rapid amplification of
cDNA ends;
nt, nucleotide(s);
UTR, untranslated region;
CHO, Chinese hamster ovary;
CWS, cell washing solution;
GTPS, guanosine
5'-O-(3-thiotriphosphate).
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REFERENCES |
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