From the Amgen Institute, Toronto, Ontario M5G 2C1,
Canada, the § Departments of Medicine and Biochemistry,
Howard Hughes Medical Institute, Duke University Medical Center,
Durham, North Carolina 27710, and the ¶ Department of
Pharmacology, University of Texas Southwestern Medical Center,
Dallas, Texas 75235-9041
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ABSTRACT |
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Regulator of G-protein signaling (RGS) proteins
increase the intrinsic guanosine triphosphatase (GTPase) activity of
G-protein subunits in vitro, but how specific
G-protein-coupled receptor systems are targeted for down-regulation by
RGS proteins remains uncharacterized. Here, we describe the GTPase
specificity of RGS12 and identify four alternatively spliced forms of
human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ
(PSD-95/Dlg/ZO-1) domain and the
RGS domain, are expressed in most tissues, with highest levels observed
in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3' end encoding a putative C-terminal PDZ
domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which
lack the PDZ domain and encode the RGS domain with and without the
alternative 3' end, respectively, are most abundantly expressed in
brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPase-activating protein for Gi class
subunits. Biochemical and interaction trap
experiments suggest that the RGS12 N terminus acts as a classical PDZ
domain, binding selectively to C-terminal (A/S)-T-X-(L/V)
motifs as found within both the interleukin-8 receptor B (CXCR2) and
the alternative 3' exon form of RGS12. The presence of an alternatively
spliced PDZ domain within RGS12 suggests a mechanism by which RGS
proteins may target specific G-protein-coupled receptor systems for
desensitization.
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INTRODUCTION |
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The mammalian "regulators of G-protein signaling"
(RGS)1 gene family
was first identified by sequence and functional similarity to fungal
and nematode genes captured in genetic screens for negative regulators
of specific G-protein-coupled receptor (GPCR) signals (1-3). In
vitro biochemical analyses soon established that this gene family
encodes potent accelerators ("GAPs") of the intrinsic GTP
hydrolysis activity of G-protein subunits, revealing a molecular mechanism by which RGS proteins drive G-proteins into their inactive GDP-bound form and hence down-regulate GPCR signal transduction in vivo (reviewed in Refs. 4 and 5). However, the mechanisms by which individual RGS proteins desensitize pathways activated by
particular GPCRs remain to be elucidated. Tightly regulated transcription has been described for RGS1 (3),
RGS2 (6), and RGS3-RGS11 (7), and palmitoylation
of the cysteine-rich N terminus of G
-interacting protein (GAIP) has
also been observed (8); however, transcriptional regulation and
post-translational modifications of particular RGS family members can
each only be expected to afford a gross level of intracellular control
over the temporal and spatial expression of G
-directed GAP
activity.
We and others have hypothesized that regions outside the RGS fold
contribute to regulation of G GAP activity and/or targeting of
individual RGS proteins to particular receptor signaling pathways (4,
5, 9, 10). Here, we report the GAP activity of RGS12 and identify a
PDZ-like N-terminal sequence within two splice forms. PDZ domains are
protein-protein interaction modules that bind to three or four amino
acid motifs at the extreme carboxyl termini of target proteins
(reviewed in Ref. 11). PDZ domains of many proteins localize enzymatic
activities and other protein-protein interaction domains to specific
submembranous regions. Members of the PSD-93/95 protein family cluster
NMDA receptors, nitric oxide synthase activity, and Shaker
K+ channels at neuronal synapses (12). The protein tyrosine
phosphatase FAP-1 interacts via a PDZ domain with the C terminus of
CD95, receptor of the apoptosis-inducing Fas ligand (13). Most
recently, the five-PDZ domain-containing protein InaD was shown to
organize the G-protein-coupled phototransduction machinery in
Drosophila, with individual PDZ domains binding to
phospholipase C, transient receptor potential ion channel, and protein
kinase C subunits (14). Using yeast two-hybrid, surface plasmon
resonance, and protein overlay analyses, we demonstrate specific
binding of the RGS12 PDZ domain to C-terminal
(A/S)-T-X-(L/V) motifs. Our findings suggest that, by virtue
of an alternatively spliced N terminus, specific isoforms of RGS12 may
localize in vivo to site(s) of specific G-protein-coupled
signaling complexes. In addition, affinity of the RGS12 PDZ domain for
an alternatively spliced C-terminal motif within RGS12 itself presents
the possibility of RGS12 autoregulation by intra- and/or intermolecular
association.
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EXPERIMENTAL PROCEDURES |
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Cloning of Human RGS12-- Oligonucleotides flanking both the open reading frame (sense primer 5'-ATATGGCTCCAAGGGAACAATGAGACG-3' and antisense primer 5'-TACGGGGCCAAGGTGGAGGGATCAG-3') and 3'-UTR of hRGS12 (sense primer 5'-ATCCCTCCACCTTGGCCCCGTAAGC-3' and antisense primer 5'-CTGCTGGGAGCCTCGCCTCAGTTTC-3') were designed based on cosmid sequences (9) and used to amplify the hRGS12 cDNA from 0.5 ng of Marathon-ReadyTM human brain cDNA (CLONTECH) using the ExpandTM long template PCR system (Boehringer Mannheim) as described previously for hRGS16 (15). The resulting PCR products were then cloned and sequenced as described previously (15).
Protein Expression and Purification--
cDNA fragments from
rat RGS12 encoding amino acids 1-94 and 664-885, and from
human RGS12 encoding amino acids 1-110, were each amplified
by PCR, cloned into the GST fusion vector pGEX4T3 (Amersham Pharmacia
Biotech), sequenced, and transformed into Escherichia coli
strain BL21 (Stratagene). Expression of GST-rRGS12-(1-94), GST-rRGS12-(664-885), and GST-hRGS12-(1-110) fusion proteins was induced with 0.8 mM
isopropyl--D-thiogalactopyranoside for 4 h at
37 °C in bacterial cultures at an OD600 nm of 0.7. Cells were lysed by lysozyme treatment and sonication in lysis buffer (50 mM sodium phosphate, pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.5% Nonidet P-40, and protease inhibitors) and
cleared by centrifugation at 27,000 × g for 30 min.
Supernatant was passed through a glutathione-Sepharose column (Amersham
Pharmacia Biotech) pre-equilibrated with lysis buffer and the column
was washed with lysis buffer and eluted with 10 mM
glutathione in 50 mM Tris-HCl, pH 8.0. Eluted proteins were
dialyzed against storage buffer (25 mM HEPES, pH 7.9, 2 mM EDTA, 2 mM DTT, 150 mM NaCl),
concentrated by ultrafiltration, and stored at
80 °C.
GTPase Assays--
G-protein subunits Gs
,
myr-Gi
1, and myr-Go
were
expressed and purified as described elsewhere (16). A point mutant of
Gq
, Arg-183 to Cys, which reduces without abolishing the
intrinsic GTPase activity, was purified from Sf9 cells
co-infected with Gq
(R183C), G
1, and G
2
baculoviruses (17). Recombinant RGS4 was purified from E. coli as described previously (18). Purification of recombinant
RGS2 from Sf9 cells will be described
elsewhere.2
Northern Blot Analyses-- Human MTN blots (CLONTECH), containing 2 µg of poly(A)+ RNA from multiple adult human tissues and brain regions, were hybridized consecutively with a 686-bp hRGS12 3'-UTR cDNA probe (nucleotides 4185-4870 of GenBank AF035152), a 330-bp cDNA probe spanning the entire PDZ domain (nucleotides 55-384 of GenBank AF035152), and a 350-bp alternative 3'end probe (nucleotides 2441-2790 of GenBank AF030109). Blots were then hybridized with a 1.3-kb fragment of human GAPDH cDNA to correct for RNA loading. Probe labelings and blot hybridizations were performed as described previously (15).
Overlay Assay--
GST-h2AR tail (80 amino acids)
and GST-hCXCR2 tail (40 amino acids) fusion proteins were produced via
PCR amplification of the tails and insertion of the PCR products into a
pGEX-2T vector (Amersham Pharmacia Biotech). Mutation to the hCXCR2
tail was induced by the use of a mutant sequence oligonucleotide during PCR and confirmed by sequencing. GST fusion proteins were expressed and
purified as described above. GST and GST fusion proteins were resolved
by SDS-polyacrylamide gel electrophoresis, electroblotted onto
nitrocellulose, and incubated with recombinant
thioredoxin-rRGS12-(1-440) fusion protein. Bound protein was detected
using an anti-thioredoxin mouse monoclonal
antibody,3 Horseradish
peroxidase-conjugated goat anti-mouse secondary antibody, and enhanced
chemiluminescence (Amersham Pharmacia Biotech).
Biosensor Measurements--
Surface plasmon resonance
measurements were performed on the BIAcore 2000 (Biacore Inc.,
Piscataway, NJ). N-terminally biotinylated, synthetic polypeptides were
bound to a streptavidin-coated sensor surface (Sensor Chip SA) as per
manufacturer's instructions to a density of 300 response units (RU).
GST and thioredoxin fusion proteins were 2-fold serially diluted in
running buffer (10 mM HEPES, pH 7.4, 150 mM
NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P20) and injected
at a 10 µl/min flow rate; surface regeneration was performed by
10-µl injections of 1 M NaCl in 50 mM NaOH at a 20 µl/min flow rate. Relative binding to test peptides (test) was
calculated based on the increase in response units relative to
simultaneous measurement of binding to wild-type CXCR2 peptide (wt) and
negative control peptide (ctrl) surfaces at 286 s after the start
of a 300 s total protein injection: relative binding = (RU286stest RU286sctrl)/(RU286swt
RU286sctrl).
Yeast Two-hybrid Analysis-- Yeast strain PJ69-4A and the plasmids pAS1 and pACTII have been described (19, 20). Rat RGS12 cDNA (amino acids 1-94) was amplified from rat brain cDNA using PCR, cloned into pCR2.1 (Invitrogen), sequenced, and subcloned in-frame downstream of the Gal4p DNA binding domain (DBD) (amino acids 1-147) in pAS1 using the EcoRI and SalI sites. cDNA encoding amino acids 322-359 of the rat high affinity interleukin-8 receptor B (rCXCR2; Swiss-Prot no. P35407) was amplified from rat brain cDNA using PCR, cloned and sequenced in pCR2.1, and subcloned in-frame downstream of the Gal4p activation domain (AD) (amino acids 768-881) in pACTII using the EcoRI and XhoI sites. Nonsense and missense mutations to the rCXCR2 C-terminal tail were engineered within the pCR2.1 plasmid using QuikChangeTM site-directed mutagenesis (Stratagene, La Jolla, CA), sequenced, and subcloned into pACTII as above.
Yeast strain PJ69-4A was co-transformed with pAS1- and pACTII-based plasmids using the Gietz lab transformation kit (Bio/Can Scientific, Mississauga, Ontario, Canada) and plated on synthetic complete drop-out medium (SC) lacking tryptophan and leucine (BIO 101, Vista, CA). Six independent yeast colonies from at least two independent transformations were then plated onto SC lacking tryptophan, leucine, histidine, and adenine, supplemented with 2 mM 3-aminotriazole. ![]() |
RESULTS AND DISCUSSION |
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We recently described the cloning of Rgs12 from rat
brain using a degenerate PCR strategy directed toward conserved regions of the RGS domain (9). To ascertain whether RGS12 is capable of
stimulating the GTPase activity of G-protein subunits, the conserved RGS domain of rRGS12 (amino acids 664-885) was expressed as
a GST fusion protein in E. coli and purified by
glutathione-Sepharose chromatography. The ability of
GST-rRGS12-(664-885) protein to enhance G
GTP hydrolysis was
measured in single turnover assays using
[
-32P]GTP-loaded, recombinant
Gi
1, Go
, Gq
,
and Gs
proteins. The slow rate of GDP dissociation and
GTP binding observed for wild-type Gq
protein relative
to its intrinsic GTPase activity makes it difficult to prepare
GTP-Gq
for use in single turnover studies (21). Thus, a
GTPase-deficient mutant of Gq
(R183C) was employed; this
protein serves as an adequate substrate for detection of the GAP
activities of RGS2 and RGS4.2 The GTPase activities of
myr-Gi
1 and myr-Go
proteins
were increased by the addition of GST-rRGS12-(664-885) protein, albeit
modestly in comparison to recombinant, full-length RGS4 (Fig.
1, A and B). In
contrast, Gs
and Gq
(R183C) GTPase
activities were not enhanced by RGS12 (Fig. 1, C and
D). Partially purified preparations of
thioredoxin-rRGS12-(1-1387) and thioredoxin-rRGS12-(440-1108) fusion
proteins also demonstrated Gi
1 and
Go
GAP activity (data not shown). Based on these
in vitro results, we conclude that RGS12 is a bona
fide member of the RGS family, in that it acts as a GAP for at
least Gi class
subunits.
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In our initial report of the cloning of rat Rgs12, we also identified the human ortholog (9) within cosmid sequences mapping to human chromosome 4p16.3. PCR was used to amplify both the predicted open-reading frame and 3'-UTR of hRGS12 from human brain cDNA. These two cDNA clones overlapped to form a contiguous sequence encompassing 4870 nucleotides and encoding 1376 amino acids (GenBank AF035152). Comparison of rat and human RGS12 protein sequences previously revealed nearly identical RGS domains and a highly conserved N terminus with similarity to a C-terminal region of mouse rhophilin (9); subsequent sequence and biochemical analyses (see below) indicate that the latter region encodes a PDZ domain. During the cloning of rRgs12 cDNA, we observed an alternatively spliced variant lacking the PDZ domain (GenBank AF035151); an alternative exon 1 with a unique 5'-UTR was found to replace exons 1 and 2 of the longer rRgs12 cDNA (GenBank U92280). Sequence of several human expressed sequence tags suggests that alternative exon usage also occurs in the same position in human RGS12 (e.g. GenBank N31659 and AA455449).
To explore the tissue distribution of human RGS12 mRNA isoforms containing or lacking the PDZ domain region, Northern blot analyses were performed with probes specific to either the 3'-UTR or the PDZ domain. Hybridization of the 3'-UTR cDNA probe to poly(A)+ RNA prepared from various human tissues (Fig. 2A) and brain regions (Fig. 2B) revealed two major hRGS12 transcripts of 6.3 and 3.7 kb. The 6.3-kb transcript was expressed abundantly in spleen, testis, ovary, cerebellum, and caudate nucleus, whereas the 3.7-kb transcript was most abundant in whole brain, kidney, thymus, and prostate, and detected at lower levels in all other tissues examined. Within specific brain regions, the 3.7-kb mRNA was most abundant in the cerebral cortex, occipital pole, frontal lobe, temporal lobe, putamen, amyglada, caudate nucleus, and hippocampus. Hybridizing the same Northern blots with a PDZ domain-specific probe revealed two hRGS12 transcripts of 6.3 and 5.7 kb (Fig. 2, A and B); the absence of hybridization to lower molecular weight mRNA indicates that the 3.7-kb hRGS12 transcript corresponds to an isoform lacking the PDZ domain. The 6.3-kb mRNA detected by the PDZ domain-specific probe corresponds to the size and tissue distribution of the 6.3-kb transcript detected by the 3'-UTR probe. The additional, 5.7-kb transcript was observed in both the same tissue distribution and abundance as seen for the 6.3-kb transcript.
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To determine how the 5.7- and 6.3-kb isoforms differed, the sequence of human RGS12 was compared with the GenBank data base. Alignments to the predicted amino acid sequences of human expressed sequence tags (e.g. GenBank AA523013) and several other hRGS12 cDNA clones (e.g. GenBank AF030109 and AF030111) revealed several isoforms of RGS12 possessing an alternative 3' end. Alternative splicing removes the last four amino acids of RGS12 (GenBank AF035152) and inserts an additional 75 amino acids of open-reading frame and an alternative 3'-UTR. Hybridization of Northern blots with a probe specific for the alternative 3' end detected hRGS12 mRNA of 5.7 and 3.1 kb (Fig. 2A and B), with the size and tissue distribution of the 5.7-kb transcript identical to that detected with the PDZ domain-specific probe. The 3.1-kb isoform was observed to be expressed with the same tissue distribution and abundance as the 3.7-kb isoform. These data indicate the presence of at least four alternatively spliced isoforms of hRGS12, containing or lacking an N-terminal PDZ domain, and encoding one of two alternative C termini (Fig. 2C).
Alternative splicing of the N terminus of human and rat RGS12 suggests
that this region may be functionally important. Sequence analysis
revealed highest homology of this region to the PDZ domains of the
Na+/H+ exchanger regulatory factor (NHERF; Fig.
3A), a protein that has
recently been shown to bind the C-terminal tail of the
2-adrenergic receptor (22). Thus, we examined whether
RGS12 might also bind the tail of
2AR or other
G-protein-coupled receptors (GPCRs). Full-length and truncated forms of
human and rat RGS12 were expressed as GST or thioredoxin fusion
proteins and their binding to various peptides was investigated by
surface plasmon resonance (SPR) analysis. Streptavidin-coated biosensor
surfaces were pre-adsorbed with N-terminally biotinylated, synthetic
peptides encompassing the last 12 amino acids of various GPCRs. Neither
thioredoxin-rRGS12-(1-1387), thioredoxin-rRGS12-(1-440), nor
GST-rRGS12-(1-94) fusion proteins bound appreciably to a biosensor
surface coated with rat
1- or
2-adrenergic receptor tail peptides. However, the RGS12
fusion proteins did bind the C terminus of the rat interleukin-8
receptor B (rCXCR2; Fig. 3B and data not shown). GST and
thioredoxin proteins alone did not bind any peptide surface tested,
whereas full-length NHERF and NHERF domain 1 proteins bound both the
rat
2AR and CXCR2 tail peptides (data not shown).
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We tested the C-terminal-specific nature of this interaction in a
protein blot overlay using a point mutant of the CXCR2 C-terminal motif. Thioredoxin-rRGS12-(1-440) protein bound a GST-hCXCR2 tail fusion protein in blot overlay experiments but failed to interact with
either GST alone or GST-2AR tail (Fig. 3C);
mutation of Leu to Ala at the terminal position within the hCXCR2 tail
(L360A) abolished all binding. Specificity for the extreme C terminus of CXCR2 was also shown by yeast two-hybrid analysis. Rat RGS12 PDZ
domain fused to the Gal4p transcription factor DBD and wild-type or
mutated rCXCR2 tails fused to the Gal4p AD were expressed together in a
yeast strain containing HIS3 and ADE2 reporter
genes under the control of the GAL1 and GAL2
promoters, respectively (19). Growth on selective media lacking both
histidine and adenine was compared with a positive control strain
co-expressing the strongly interacting yeast proteins Snf1p and Snf4p
(23) and a negative control strain co-expressing solely the Gal4p DBD
and AD proteins. Co-expression of rRGS12 PDZ domain with wild-type
rCXCR2 receptor tail allowed prototrophic growth (Table
I); however, truncation of the last five
C-terminal amino acids of the rCXCR2 tail (
355-359), or mutation of
the
2 or terminal residues, abolished the interaction. Several
missense mutations at the
1 position within the rCXCR2 tail did not
disrupt the interaction, consistent with previous reports of PDZ domain
binding specificity (24).
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We were intrigued by the selectivity shown by RGS12 for threonine at
the 2 position of the CXCR2 tail, as removal of the C
methyl group
by mutation of threonine to serine was observed to abolish completely
the interaction in yeast (Table I). To confirm this specificity, we
tested three different rat metabotropic glutamate GPCR tails with
Ser/Thr-rich carboxyl termini ending in S-(S/T)-L, of which MGR1
and
MGR5 interact with the PDZ domain of the neuron-specific protein Homer
(25). The lack of binding of GST-rRGS12-(1-94) to these three MGR tail
peptides, as measured by SPR (Table II,
A), confirms the preference of RGS12 for the motif T-X-L
versus S-X-L. While necessary, the C-terminal
T-X-L motif alone is not the sole determinant of RGS12 PDZ
domain binding, as interaction with phospholipase C
1 and
3
carboxyl termini, both possessing the T-X-L motif, was at
least 4-fold weaker than with the CXCR2 tail (Table II, A and B).
Alanine/serine scanning mutagenesis of the last six residues of human
CXCR2 tail peptide revealed the most important residues for hRGS12 PDZ
binding specificity as the
3,
2, and 0 positions
(S-T-X-L), with lesser contributions from the
4 and
5
positions (Table II, B). In addition, the failure of
GST-hRGS12PDZ-(1-110) to bind amide-blocked C-terminal CXCR2 peptides
confirms a critical role for the free carboxylic acid moiety in the
binding interaction, as previously observed for other PDZ domains
(26).
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The predicted C terminus of the alternative 3' end splice isoform of hRGS12 is similar to that of human CXCR2, with both tails conforming to a consensus of (T/S)-S-(A/G)-H-X-(A/S)-T-X-(L/V). We therefore tested binding of the human RGS12 PDZ domain to synthetic peptides encompassing the last 12 amino acids of the two, alternative carboxyl termini of RGS12. Although no binding was detected to the T-S-R-F C terminus conserved between human and rat RGS12 (hRGS12Tail1; Table II, B), GST-hRGS12PDZ-(1-110) protein was shown to bind the A-T-F-V C-terminal RGS12 peptide (hRGS12Tail2; Table II, B). While a pattern search of the Swiss-Prot data base identified only CXCR2 as terminating with a polypeptide sequence conforming to the (T/S)-S-(A/G)-H-X-(A/S)-T-X-(L/V) motif, several other GPCRs, membrane-spanning proteins, and intracellular proteins with GAP or other enzymatic activities were identified as terminating in the shorter (A/S)-T-X-(L/V) motif. These potential RGS12 PDZ domain-binding proteins and their C-terminal sequences are summarized in Table III. It must be noted, however, that a terminal (A/S)-T-X-(L/V) motif alone is clearly not sufficient to specify RGS12 PDZ domain binding, as no appreciable binding has been observed to the A-T-N-V C-terminal tail of the human neuropeptide Y receptor type 2 (Table II, B). We are currently evaluating other proteins from Table III for their ability to bind RGS12.
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Binding of the RGS12 PDZ domain to the RGS12 A-T-F-V tail was somewhat
weaker than binding to the CXCR2 tail. However, among those form(s) of
RGS12 containing both the N-terminal PDZ domain and the A-T-F-V C
terminus, the interaction could be kinetically favored given its
intramolecular nature. Such a scenario is reminiscent of the mechanism
of reversible autoinhibition of Src family kinases, which adopt a
"closed" conformation upon intramolecular association of their
N-terminal SH2 and SH3 domains with C-terminal motifs (27).
Intramolecular association of the N and C termini within RGS12
isoform(s) may serve as a source of regulation, either of its
Gi-directed GAP activity or of PDZ-mediated binding to
other proteins. The ability of the alternative C terminus to serve as a
docking site for PDZ domain-containing proteins also presents the
possibility of in vivo concatemerization and/or organization of RGS12 within multi-component signaling complexes.
Compartmentalization by virtue of the N- and/or C-terminal domains of
RGS12 could thereby associate desensitizing GAP activity within
specific receptor complexes, analogous to inclusion of the negative
regulatory function of protein kinase C within the
Drosophila phototransduction complex by InaD (14).
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ACKNOWLEDGEMENTS |
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We thank J. Krumm, B. Sutton, and L. Antonio for sterling sequencing support, M. Jarosinski and T. Zamborelli for precise peptide production, L. Harrington and M. Tyers for interaction trap tips, D. Sawutz for suggesting various G-protein-coupled receptor tails for analysis, and J. McGlade and V. Stambolic for critical appraisal.
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FOOTNOTES |
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* This work was supported in part by Amgen, Inc. (to D. P. S.), National Institutes of Health Grants HL16037 (to R. J. L.) and GM34497 (to A. G. G.), and by the Raymond and Ellen Willie Distinguished Chair in Molecular Neuropharmacology (to A. G. G.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF035151 and AF035152.
To whom correspondence should be addressed: Quantitative
Biology Laboratory, Amgen Institute, 620 University Avenue, Suite 706, Toronto, Ontario M5G 2C1, Canada. Fax: 416-204-2277; E-mail: dsiderov{at}amgen.com.
1 The abbreviations used are: RGS, regulator of G-protein signaling; AD, activation domain; AR, adrenergic receptor; bp, base pair(s); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DBD, DNA binding domain; DTT, dithiothreitol; GAP, GTPase-activating protein; GPCR, G-protein-coupled receptor; GST, glutathione S-transferase; GTPase, guanosine triphosphatase; h, human; kb, kilobase(s); myr, myristoylated; NHERF, Na+/H+ exchanger regulatory factor; PCR, polymerase chain reaction; PDZ, PSD-95, Disc-large, and ZO-1; r, rat; RU, response unit(s); SPR, surface plasmon resonance; Trx, thioredoxin; UTR, untranslated region.
2 T. Ingi, A. M. Krumins, P. Chidiac, G. M. Brothers, S. Chung, B. E. Snow, C. A. Barnes, A. A. Lanahan, D. P. Siderovski, E. M. Ross, A. G. Gilman, and P. F. Worley, submitted for publication.
3 G. M. Brothers and D. P. Siderovski, unpublished data.
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REFERENCES |
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