(Received for publication, November 5, 1996, and in revised form, January 7, 1997)
From the Regulatory Biology Laboratory, Institute of Molecular and
Cell Biology, National University of Singapore, 10 Kent Ridge
Crescent, Singapore 119260, Republic of Singapore and the
Department of Immunology, The Scripps Research
Institute, La Jolla, California 92037
Genetic studies of molecules that negatively
regulate G-coupled receptor functions have led to the identification of
a large gene family with an evolutionarily conserved domain, termed the RGS domain. It is now understood that RGS proteins serve as
GTPase-activating proteins for subfamilies of the heterotrimeric
G-proteins. We have isolated from mouse pituitary a full-length
cDNA clone encoding a novel member of the RGS protein family,
termed RGS16, as well as the full-length cDNA of mRGS5 and mRGS2.
Tissue distribution analysis shows that the novel RGS16 is
predominantly expressed in liver and pituitary, and that RGS5 is
preferentially expressed in heart and skeletal muscle. In contrast,
RGS2 is widely expressed. Genetic analysis using the pheromone response
halo assay and FUS1 gene induction assay show that
overexpression of the RGS16 gene dramatically inhibits yeast response
to -factor, whereas neither RGS2 nor RGS5 has any discernible effect
on pheromone sensitivity, pointing to a possible functional diversity
among RGS proteins. In vitro binding assays reveal that
RGS5 and RGS16 bind to G
i and G
o subunits
of heterotrimeric G-proteins, but not to G
s. Based on
mutational analysis of the conserved residues in the RGS domain, we
suggest that the G-protein binding and GTPase-activating protein
activity may involve distinct functional structures of the RGS
proteins, indicating that RGS proteins may exert a dual function in the
attenuation of signaling via G-coupled receptors.
RGS1 proteins, regulators of G-protein
signaling, belong to a large gene family, whose members regulate the
G-protein-mediated signaling pathway in organisms ranging from yeast to
mammals (1, 36). The first mammalian RGS family members, RGS1/BL34 and
RGS2/G0S8, were isolated from activated B-lymphocytes, and stimulated
monocytes, respectively (2-5). More recently, genetic studies of genes
that take part in neurotransmitter-modulated behaviors such as
egg-laying and locomotion in Caenorhabditis elegans, have
also led to the isolation of a new gene, referred to as
EGL-10 (6). The protein product of EGL-10 has an
opposing effect of a G protein encoded by GOA-1 (7),
implying that EGL-10 exerts an inhibitory role on G-protein signaling.
Subsequent cDNA library screening under low stringency, degenerate
PCR reactions, as well as data base search of expressed sequence tags,
have identified over 15 different cDNA species of mammalian RGS
members (6, 8, 9). In addition, a two-hybrid screen has isolated
another mammalian RGS member, known as GAIP (G
interacting protein)
that binds to G
i3 known to play a part in protein
trafficking in the Golgi apparatus (10). They all share sequence
similarity to a yeast gene, SST2, the founding member of the
RGS family, isolated by genetic analysis from a strain that exhibits
supersensitivity to the yeast mating pheromone (11, 12). It has been
demonstrated that introduction of mammalian RGS family members (RGS 1 to 4) can substitute the yeast SST2 gene product in blunting
pheromone signaling (8, 9), emphasizing a functional significance of
the sequence similarity among RGS family members.
Strong genetic evidence has suggested that RGS proteins exert their
function through G-proteins (6, 13, 14). The biochemical nature of
their inhibitory effect remained unclear until the very recent
demonstrations that at least some of the RGS members (RGS1, -4, -10, and GAIP) can physically interact with Gi and
G
o proteins and accelerate the hydrolysis of GTP by
their intrinsic GTPase activity (10, 14-18). Whether this mode of
action is prototypic of other RGS functions remains to be seen. It is
also noteworthy that the mammalian RGS4 protein inhibits G-protein
function in yeast (8).
Tissue distribution analysis of RGS family members shows that they seem
to be differentially expressed. RGS4, for example, is mainly expressed
in the brain regions (8). It is likely that each RGS protein only
regulates the signal transduction pathway of a subset of receptors or a
subtype of G-proteins, either by temporal and spatial restriction of
expression or by having diverse intrinsic biochemical properties. We
have previously cloned the seven-transmembrane, Gs-coupled
receptor for the growth hormone releasing factor, which is expressed
exclusively in the anterior pituitary and is required for the cell
proliferation of somatotrophs (19, 20). We reasoned that a
tissue-specific RGS protein may exist in the anterior pituitary that
modulates certain aspects of endocrine activity and cell growth of the
gland. We present in this report the identification and cloning of a
novel RGS member, referred to as RGS16, from mouse pituitary. We have
also isolated a full-length cDNA for mouse RGS5, a mouse homologue
of human RGS2, and have analyzed the tissue distribution of these RGS
mRNAs. Functional studies based on their ability to inhibit
pheromone signaling in yeast and their affinity for different
G subunits point to functional diversity among different
RGS proteins. Furthermore, mutational analysis of conserved residues in
the RGS domain underscores the functional significance of the sequence
conservation, and suggests also that binding and GAP activity may
involve separable structures.
Mouse pituitary cDNA was generated by reverse
transcription using avian myeloblastosis virus reverse transcriptase
(Amersham). The reverse transcription was carried out in a 25-µl
reaction mixture using 5 µg of total cellular RNA and 0.1 µg of
oligo(dT) primer (Boehringer Mannheim), according to the instructions
of Life Technologies, Inc. Degenerate primers, synthesized based on
conserved amino acid residues in the RGS domain, were used in
polymerase chain reactions (PCR) according to Koelle and Horvitz (6).
The primers that generated the probe inserts for mRGS2, mRGS5, and
mRGS16 were: "5R",
G(G/A)IGA(G/A)AA(T/C)(A/T/C)TI(A/C)GIT(T/C)TGG and "3T",
G(G/A)TAIGA(G/A)T(T/C)ITT(T/C)T(T/C)CAT. Amplification reactions
were performed using 2 µl of mouse pituitary cDNA generated as
described above in a final volume of 50 µl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM each of dATP, dCTP, dGTP, dTTP, and 2.5 units of Taq DNA polymerase
(Boehringer Mannheim). Reaction cycles were as follows: following an
initial denaturation of reaction mixture at 94 °C for 3 min, 35 cycles of 94 °C for 45 s, 40 °C for 1 min, and 72 °C for
2 min. Subcloning of the PCR products into pBluescript (Stratagene) and
subsequent sequencing analysis were carried out using standard
techniques (21). The PCR fragments (240 base pairs) were then used as
probes to screen a mouse pituitary gt11 cDNA library (a gift
from Drs. B. Andersen and G. Rosenfeld, University of California San
Diego) for full-length cDNA clones according to manufacturer's
instructions (Stratagene).
A
Northern blot containing eight different mouse tissue
poly(A)+ RNAs (2 µg each lane, Clontech) was probed
successively with 32P-labeled RGS2, -5, and -16 cDNA.
The DNA probes were generated from full-length cDNA inserts using a
random priming reaction kit (Boehringer Mannheim) and
[-32P]dCTP (DuPont NEN) to specific activities of
approximately 2 × 109 cpm/µg. Northern
hybridization was carried out at 42 °C overnight using a buffer
containing 5 × SSPE, 10 × Denhardt's solution, 1% SDS,
0.5 mg/ml herring sperm DNA (Sigma), and 50% formamide. The blot was
then thoroughly rinsed and washed with 1 × SSC at 65 °C for 20 min, followed by washing with 0.2 × SSC at 65 °C for 30 min. A
-actin probe (Clontech) was used for RNA loading normalization.
For further analysis of the RGS16 expression in different tissues, RNase protection analysis was performed. An RGS16 SacI-EcoRI 157-base pair fragment (101 to 257 nucleotides downstream of the initiation ATG codon) was blunt-ended with Klenow polymerase and ligated into the SmaI site of pBluescript. The antisense strand RNA probe was transcribed using T7 RNA polymerase (Promega) and hybridized to total tissue RNAs, and the RNase protection assay was performed as described previously (19).
Yeast Pheromone Response AssaysA bioassay was used to
measure the sensitivity of pheromone response in yeast cells that
express different mammalian RGS proteins. Both bar1
mutant cells, US356 (MATa, ade2-1, trp1-1, can1-100, leu2-3,
his3-11, bar1
), and W303 cells were transformed with each RGS
cDNA in the pMW29 vector under a galactose-inducible promoter (22)
and selected on ura
dropout plates. Halo assays were
carried out as described (8, 23), except that transformants were grown
on ura
dropout plates containing 2% galactose. Pheromone
response was also monitored by measuring the pheromone-inducible levels
of the FUS1 transcript in cells transformed with different
RGS cDNA constructs. Cell culture, pheromone treatment, and
Northern hybridization with the FUS1 probe were carried out
as described (24). Briefly, a single colony of each yeast transformant
was grown at 30 °C in 40 ml of ura
dropout medium
supplemented with 2% galactose to an OD of 0.8. Each culture was then
divided into halves. The one half-culture was added with
-factor
(Sigma) to a final concentration of 5 µM and incubated
for an additional 20 min, while the other half was untreated. Cells
were then pelleted and subjected to RNA isolation as described (25).
Equal amounts of total cellular RNA (15 µg) isolated from each sample
was separated on a formaldehyde-denaturing agarose gel, transferred to
a nitrocellulose filter, and hybridized with 32P-labeled
FUS1 probe (gift from Dr. L Lim, Glaxo-IMCB), as described above.
To compare expression
levels of different wild-type RGS proteins and RGS16 mutants in yeast
cells, PCR was employed to generate an XhoI site immediately
upstream of the ATG start codon of all RGS cDNAs. A pBluescript
derivative vector containing a synthetic DNA fragment coding for the
flag tag (DYKDDDDKH) was constructed and used to fuse in-frame to the
translation initiation codon of each RGS cDNA. After confirmation
by sequencing analysis, the flag-tagged RGS cDNAs were released by
EcoRV and BamHI from pBluescript and ligated to
SmaI/BamHI-treated yeast expression vector pMW29. Flag-tagged RGS constructs in the pMW29 vector were separately introduced to the US356 yeast cells as described above. Single colonies
were inoculated into 2 ml of ura dropout medium
supplemented either with 2% galactose or 2% glucose, and were grown
at 30 °C to an identical density (A600 = 1.0). Cells (1.5 ml) were pelleted and lysed in 100 µl of SDS protein sample buffer (2% SDS, 50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 0.1% bromphenol blue, and 10%
glycerol). Protein was extracted by freezing in liquid nitrogen
followed by thawing and boiling for 5 min. The cell lysates were then
sonicated for 20 s and spun in a microcentrifuge for 5 min. The
protein concentration of each extract was determined using the Bio-Rad
DC Protein Assay (Bio-Rad). Proteins (150 µg each) were separated on
10% SDS-PAGE gels, transferred to a HybondTM-C Extra
filter (Amersham), and flag-tagged RGS proteins were detected using the
M2 anti-flag antibody precisely according to the manufacturer's
instructions (Kodak) and the ECL system (Amersham). In parallel, the
ability of flag-tagged wild-type RGS members and RGS16 mutants to
attenuate pheromone signaling was re-assessed by halo assays as
described above.
In vitro site-directed mutagenesis was performed using the TransformerTM Site-directed Mutagenesis Kit (Clontech). The oligonucleotides used to create RGS16 mutants, G74R, L82S, E85G, EN89/90GA, I116D, LM161/162SK, RF169/170SC, were TGAACAGTAAAAATcGGGTGGCTGCCTTCC, CTTCCATGCCTTCtcAAAGACGGAATTCAG, TTCCTAAAGACGGgATTCAGTGAGGAGAAC, GAATTCAGTGAGGgGgcCCTGGAGTTCTGGTTG, GGGCTCACCACgaCTTTGACGAGTACATCC, AGACCCGCACATcGAaGGAGAAGGACTCCT, and GACTCCTATCCGaGCTgCCTCAAGTCACCA, respectively. The mutant oligonucleotide for the XbaI site of the pBluescript vector was GGGATCCACTAGTtCTAGAGCGGCCGCCAC. Double-stranded RGS16 cDNA carrying an XhoI site immediately upstream of the translation initiation site in pBluescript was heat-denatured and annealed to each of the specific mutant oligonucleotides and the common mutant primer for the XbaI site. The mutagenesis procedures were carried out according to the manufacturer's instructions (Clontech). The resulting mutants were confirmed by sequencing analysis.
RGS-G-protein Binding AssaysWild-type and mutant RGS
cDNAs were fused in-frame to XhoI site of a derivative
of the bacterial expression vector pGex2TK (Pharmacia); the fusion
proteins were expressed in the Escherichia coli strain
BL21(DE3)-pLysS. Transformant cells were grown to an OD of 1.0 and
induced with 1 mM
isopropyl-1-thio--D-galactopyranoside (Life
Technologies, Inc.) at 37 °C for 2 h to express fusion RGS proteins. Cells were then pelleted and lysed in a lysis buffer containing 0.4 M NaCl, 50 mM Tris-HCl (pH 7.6),
1 mM EDTA, 1% Triton X-100, and 10% glycerol,
supplemented before use with 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 4 mM benzamidine, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The
lysates were then sonicated for 3 × 30 s on ice and
centrifuged at 4 °C at 30,000 rpm for 20 min in an SW41 rotor. The
supernatants (from 400 ml of cell culture) were incubated at 4 °C
for 30 min with 1 ml of 1:1 suspension of glutathione-agarose beads
(Sigma). The beads were washed six times each with 10 volumes of lysis
buffer. The purified glutathione S-transferase fusion
proteins were stored in the beads in the same lysis buffer except 1%
Triton was omitted. Protein was eluted with the lysis buffer without
Triton containing 10 mM glutathione (Sigma) and quantified
by the Bradford method (Bio-Rad).
Full-length cDNAs encoding G-proteins, Gs,
G
i2 (26), and G
o (27) were obtained by
PCR amplification using Pfu DNA polymerase (Stratagene) and total
cDNA derived from mouse pituitary; and G
i3 (28) was
amplified from rat pituitary cDNA. The oligonucleotide sequences
for G
i2 were: CCATGGGCTGCACCGTGAGCGCC and
CCCTCAGAAGAGGCCACAGTCCTT; for G
i3,
CATATGGGCTGCACGTTGAGCGCCGA and AGTCGACTCAGTAAAGCCCACATTCCT; for
G
o: CATATGGGATGTACGCTGAGCGCA and
TAGGTTGCTATACAGGACAAGAGG. Amplified cDNA fragments were first
cloned into the pBluescript vector (Stratagene) and multiple
recombinant clones were isolated for each cDNA species for
analyzing PCR fidelity by restriction digestions and DNA sequencing.
For G
s, due to the high G/C content of its cDNA
sequence at the 5
-end, it took two steps to obtain the full-length
cDNA. The 3
-end oligonucleotide for PCR amplification of
G
s cDNA was: TGAATTCGGGTGTTCCCTTCTTAGAGC. The
5
-oligonucleotide was designed from 45 nucleotides downstream of
the ATG codon (GGCCCAGCGCGAGGCCAACAAAAA) to avoid the
G/C-rich region. The amplified fragment was treated with Klenow DNA
polymerase and polynucleotide kinase and ligated to Klenow-treated
(blunt-end) ApaI site of pBluescript. In this way, ligation
of the vector (providing a G nucleotide) to the insert (GGCCC) restored
the ApaI site (GGGCCC) at the 5
-end. The recombinant vector
was then cut with ApaI and ligated to the following
synthetic double-stranded DNA fragment encoding the remaining
N-terminal region. The two synthetic oligonucleotides were:
cATGGCGGCGCGGGGCGCGGCCGGGCTGCGGGGCGGCGGGGAGAAGGCC and
TTCTCCCCGCCGCCCCGCAGCCCGGCCGCGCCCCGCGCCGCCATGGGCC.
The G-protein cDNAs were then transferred to the pMet vector under
the control of T7 promoter (29); and the G-proteins were generated and
35S-labeled by in vitro transcription and
translation reactions using the TNT Coupled Reticulocyte Lysate Systems
(Promega) and [35S]methionine. Assays for RGS
interactions with different G-proteins were carried out essentially in
the same way as described (16). Briefly, 10 µl of the in
vitro translated G-proteins (approximately 20 ng) were incubated
for 30 min at room temperature by adding 90 µl of buffer A (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM MgSO4, 20 mM imidazole, 10 mM -mercaptoethanol, 10% glycerol) supplemented with
either GDP (10 µM) or GDP and
AlF4
(10 and 30 µM,
respectively). RGS fusion proteins (approximately 1 µg) and
glutathione S-transferase (3 µg), as a control, bound to
agarose beads, were equilibrated with buffer A, and then separately incubated with the G-protein mixture for 30 min at 4 °C. The agarose beads were washed 4 times with buffer A containing either GDP or GDP
with AlF4
. The bound G-protein and RGS
fusion protein were eluted in 2 × SDS protein sample buffer and
separated on SDS-PAGE gels. After electrophoresis, gels were stained
with Coomassie Brilliant Blue, dried, and exposed to x-ray film.
To identify new RGS
members that are enriched in the anterior pituitary, degenerate PCR
primers were designed corresponding to conserved amino acid residues
among previously identified RGS members and used to perform PCR
amplification using mouse pituitary cDNA as the template as
described previously (6). We identified cDNA fragments
corresponding to RGS2, -4, -5, -6, -7, -9, and -11 (members numbered
according to Ref. 10, data not shown), which had been previously
identified by other groups or as an expressed sequence tag in the data
base (4, 6). In addition, we obtained a novel one, named clone 16 (Fig.
1). We used the novel clone 16 cDNA fragment,
together with those of RGS2 and RGS5 to screen the mouse gt11
pituitary cDNA library. In previous limited tissue distribution
analysis (5), RGS2 was found to be absent in all tissues, and we thus
wondered if it might be enriched in the pituitary. As for RGS5, only a
partial sequence was previously identified as both an EST partial
sequence (human) and a degenerate PCR product (rat) (6).
Sequence Analysis and Comparison to Known RGS Members
Nucleotide sequences and conceptually translated amino acid sequences derived from the isolated cDNA inserts were aligned with all other known RGS members. It was found that the cDNAs of RGS2, -5, and -16 all contained an initiation Met codon in a position corresponding to that of many previously characterized RGS proteins. Furthermore, RGS5 has an in-frame upstream stop codon. The cDNAs for RGS2, -5, and -16, encode open reading frames of 211, 181, and 201 amino acids, respectively. These sizes are in good agreement with those of other RGS proteins such as RGS1, hRGS2, and GAIP. Together with the presence of stop codons at the end of each open reading frame, these new cDNAs are full-length clones. The three mouse RGS full-length protein sequences, together with the human full-length RGS2 and the partial rat RGS5 sequences, are aligned as shown in Fig. 1A. The most conserved region, the RGS domain, is underlined (Fig. 1A). Sequence alignment for the RGS domains from various known full-length RGS proteins are aligned in Fig. 1B. The mouse RGS2 shares 95% overall identity with its human homologue (Fig. 1A), with only one conservative change (Thr156 to Ser156, from human to mouse) in the RGS domain. When the mouse RGS5 was compared with the rat counterpart (6), which contained only a large portion of the RGS domain (Fig. 1B), it was found that this region has one amino acid variation (Arg149 to His55, from mouse to rat). A BLAST search indicated that RGS16 is a novel RGS member of the RGS protein family, sharing the highest degree of similarity to RGS3 (63%) between their RGS domains, and is highly divergent outside this region from all known RGS members.
Tissue Distributions of RGS2, RGS5, and RGS16As an initial
step toward understanding the biological roles of our newly
characterized RGS proteins, we analyzed the tissue distribution of the
three RGS members by Northern hybridization and RNase protection assay.
A Northern blot containing poly(A)+ RNA from eight
different tissues was successively hybridized to the RGS2, RGS5, and
RGS16 probes. The RGS2 probe hybridized to two transcripts, 1.5 and 1.8 kb, in length, present in all tissues except liver (Fig.
2). RGS5 and RGS16 probes hybridized to a single RNA
species of 4.4 and 2.4 kb, respectively (Fig. 2). The RGS5 mRNA was
expressed abundantly in heart and skeletal muscle, and at low levels in
brain, liver, and kidney. In contrast, RGS16 was present predominantly
in liver, and at low levels in heart and brain (Fig. 2). Since RGS16
expression was very tissue-restricted, we further performed an RNase
protection assay using a collection of RNA samples from 14 mouse
tissues. The RNase protection assay showed that RGS16 was only present
in brain, pituitary, and liver (Fig. 3). This is in
total agreement with the Northern hybridization results indicating that
RGS16 is present in a limited number of tissues. The same RNase
protection experiment was also carried out with the RGS5 probe, showing
that it is expressed in all the tissues examined except liver, spleen,
intestine, fat, and adrenal (data not shown).
Inhibition of Pheromone Signaling by Mouse RGS16
Previous
work has shown that mammalian RGS members can attenuate the pheromone
response pathway in yeast (8, 9). To ascertain that we had cloned
functional RGS members, those RGS cDNAs were inserted into the
yeast expression vector pMW29 under a galactose-inducible promoter, and
were transformed into yeast strains US356 and W303. The former strain
harbors a deletion mutation in the bar1 gene, which encodes
a secreted proteinase capable of cleaving -factor (30). Results
obtained from the halo assays showed that in the bar1
mutant overexpression of RGS16 almost completely blocked the pheromone
response (Fig. 4A). On the other hand, RGS2
and RGS5 had very little effect on the pheromone response and were
phenotypically indistinguishable from that of the mutant cells to which
only the blank pMW29 vector was introduced (Fig. 4A).
Similar inhibitory effects on
-factor response were obtained with
yeast strain W303 (not shown). The efficacy of the three RGS proteins
in functionally resembling the endogeneous Sst2 protein was also
assessed by measuring the mRNA levels of the pheromone-inducible marker, Fus1, in the presence or absence of the
-factor.
As shown in Fig. 4B, the RGS16 protein almost entirely
abolished FUS1 transcript induction, whereas RGS2 and RGS5
had no effect on FUS1 induction. To compare the expression
levels of different RGS proteins in the yeast transformants, the
flag-tag was introduced to the N terminus of each RGS. Expression
levels of each RGS protein in yeast cells grown in galactose were
determined by Western blotting analysis using the monoclonal antibody
specifically against the flag epitope. Comparable levels of RGS2, RGS5,
and RGS16 proteins were detected (Fig. 4A, right lower
panel). The antibody specificity was tested by also using protein
extracts from different transformants grown in glucose (data not
shown).
Differential Binding of RGS Proteins to G
It has
been demonstrated that many RGS proteins bind to the -subunits of
heterotrimeric G-proteins (10, 14-18). We carried out biochemical
binding assays to determine the binding efficiencies of RGS2, RGS5, and
RGS16 to different G
subunits. Assays were carried out using
bacterially expressed glutathione S-transferase fusion RGS
proteins and in vitro translated G
subunits in the absence or presence of GTP
S, GDP, or GDP plus
AlF4
. In the presence of GTP
S
alone, none of the RGS proteins bound to any G-protein tested (data not
shown). RGS5 and RGS16 strongly interacted with G
i3,
G
i2, and G
o that had been preincubated with both GDP and AlF4
(Fig.
5). Interestingly, as previously reported with RGS1
(16), RGS5 and RGS16 also interacted, albeit to lower degrees, with the
GDP-bound forms of G
i2 and G
o. RGS2 did
not bind to any of the G-proteins (Fig. 5). RGS2/G0S8 was first
isolated as a cDNA specifically expressed in monocytes (4).
Although we have shown that it is also expressed in other tissues at
low levels, it is conceivable that RGS2 protein may only recognize
G-proteins of a hematopoietic origin. Our results showed that all of
the RGS proteins did not bind to the stimulatory subunit
G
s (data not shown), confirming the previous findings
(15-17).
Conserved Amino Acid Residues in the RGS Domain Are Critical for RGS Function
As the RGS domains among all the RGS proteins share
significant identity in amino acid sequence, we generated seven RGS16 mutations within the RGS domain to alter residues that are absolutely conserved (Fig. 1B). These mutants will be informative in
understanding whether those conserved residues are critical for either
G-protein binding or the inhibition of G-protein signaling, or both.
Halo assays showed that alterations of Gly74 to Arg,
Glu85 to Gly, Glu-Asn89/90 to Gly-Ala,
Ile116 to Asp, Leu-Met161/162 to Ser-Lys,
Arg-Phe169/170 to Ser-Cys, respectively, abolished the
inhibiting effect on pheromone signaling (Fig.
6A, and data not shown), although these mutants were expressed at a comparable level to that of the wild type
and to that of L82S (Fig. 6B, and data not shown). The
substitution of Leu82 with Ser, however, had no effect on
either the inhibition of pheromone signaling (Fig. 6), or its binding
to the putative cognate G-proteins (Fig. 7). However,
replacements of Glu-Asn89/90 with Gly-Ala, or
Arg-Phe169/170 with Ser-Cys, eliminated binding to any
G-protein (Fig. 7). Unexpectedly, the mutant G74R and I116D proteins,
which are no longer functional in the attenuation of pheromone
signaling in yeast, had strong binding to the G-proteins in the
presence of GDP and AlF4, but did not
bind to the G-proteins preincubated with GDP only (Figs. 6 and 7, and
data not shown). Binding of mutant E85G and LM161/162SK proteins to
G-proteins were not tested. These results indicate that the conserved
residues are critical for RGS function, and yet the RGS domain may
comprise distinct three-dimensional functional structures that are
separately important for binding to cognate G-proteins and for GAP
activity.
Employing a degenerate PCR approach, we have isolated a novel RGS member from the pituitary gland, as well as full-length cDNA clones encoding the mouse RGS2 and rat RGS5. Tissue distribution analysis showed that these three genes are differentially expressed. RGS2 is most widely expressed, present in all the eight tissues examined except liver. RGS5 exhibits a very interesting expression pattern, mainly in heart and skeletal muscle, both of which are muscle tissues. However, extended analysis using the RNase protection assay shows that RGS5 is also present in pituitary, which correlates well with the fact that we obtained over 100 clones, out of one million screened, which hybridized to the RGS5 probe. In addition, it is also expressed in other tissues including all the 14 tissues shown in Fig. 3 except spleen, intestine, fat, and adrenal. In drastic contrast, RGS16 is only expressed abundantly in liver and pituitary, and at low levels in heart and brain, as assayed by both Northern blotting and RNase protection experiments.
It is therefore clear that the mouse RGS members have overlapping yet distinct patterns of tissue distributions. It has been well established that each eukaryotic cell possesses different subsets of receptors, G-proteins, and effectors, responding to distinct signals in the form of chemicals, hormones, and cytokines (reviewed in Refs. 31-33). The differential tissue distribution of various RGS members suggests that they each may only regulate a certain aspect of biological function.
Based on pheromone response halo assays and measurement of
FUS1 induction, we showed that only RGS16 has an inhibitory
effect on the yeast pheromone response pathway, whereas mouse RGS2 and RGS5 do not, although previous results show that RGS2 has a minor desensitizing effect on the sst2 AG57 mutant cells (8). Functional selectivity has been shown for RGS1-4 in their ability to interfere with interleukin-8 receptor signaling (8). The simplest explanation for
the functional difference among various RGS proteins is that they are
divergent outside the RGS domain, especially in their N terminus
regions and may thus have different functional targets. However, when
the N termini of the RGS2 and RGS16 were exchanged for each other, the
resulting N2/C16 (RGS2 N terminus replacing the corresponding N
terminus of RGS16 at amino acid residues 98 and 79, respectively) and
N16/C2 chimeras did not inhibit pheromone signaling as does the
wild-type RGS16 (data not shown). In fact, the N2/C16 chimera had some
residual inhibiting activity, indicating that functional specificity
may be conferred by the conserved RGS domain. Nevertheless, deletion of
the N-terminal 13-amino acid residues completely abolished RGS16
function in the attenuation of pheromone signaling in yeast, suggesting
at least that the N terminus is critical for a functional conformation
(data not shown).
RGS5 and RGS16 bind with high affinity to all of the three G-proteins
when they are incubated with GDP and
AlF4, but not when they are in the
active (GTP
S bound) form. These results are consistent with what has
been observed with RGS1, RGS4, and GAIP (15-18). As previously
suggested, RGS proteins bind and stablize G
proteins in their
transition state leading to GTP hydrolysis (15, 16, 18, 34, 35). It is
therefore likely that RGS5 and RGS16 both act as a GAP for their
cognate G-proteins. However, it is also clear that RGS proteins do not only bind to G-proteins in the GTPase transition state, as one study
has shown that RGS10 binds strongly to a GTPase-inactive form of
G
i3 (17), in which Gln204 necessary for
transition state stablization (34, 35) is changed to Leu. However, the
binding assays did not indicate which form(s) of G
i3
(Q204L), GDP-bound or GTP-bound, associates with RGS10. Similarly to
what has been observed with RGS1 (16), RGS5 and -16 also significantly
interact with G
proteins that are GDP-bound, which is particularly
true for their binding to G
i2 or G
o. It is noteworthy that RGS proteins can bind to some G-proteins either in
the transition state (GDP/AlF bound) or in the inactive state (GDP
bound), but not to G-proteins in the active state (GTP
S bound).
While binding to the transition state is a prerequisite for GAP
activity, binding of RGS to GDP-bound G-proteins may be of as yet
unidentified physiological importance. One possible function is that
RGS proteins, in addition to enhancement of GTP hydrolysis, may
decrease dissociation of GDP from G-protein to prolong the inactive
state. It is equally possible that RGS binding may render the
GDP·G
complex inaccessible to G-protein-coupled receptors.
Mutational analysis showed that most of the conserved amino acid
residues in the RGS domain are critical for RGS functions. It is
perplexing, however, that while mutations in EN89/90GA and RF169/170SC
abolish both G-protein binding and attenuation activity of pheromone
signaling, G74R and I116D mutations retain their ability to bind
G-proteins. Interestingly, binding of G74R and I116D to
Gi2 and G
o is strictly
GDP/AlF4
-dependent. They
no longer bind to the GDP-bound form of the G-proteins. This finding
suggests there may be several separable, yet not mutually exclusive,
functional structures that are critical for either the physical
interaction of RGS proteins with G-proteins or GAP activity on
G-proteins.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) mRGS2, U67187[GenBank]; mRGS5, U67188[GenBank]; and mRGS16, U67189[GenBank].
We acknowledge our colleagues Dr. Mingjie Cai for providing the yeast expression vector pMW29, yeast strain W303, and the act1 probe, Dr. Uttam Surana for the gift of the US356 strain, and Drs. Z. Zhao and L. Lim for providing the FUS1 probe as well as for their technical instructions. We also thank Drs. B. L. Tang, M. Cai, and P. Li for critical discussions on the manuscript. Technical assistance from Yi Zhang, X. Wang, and Michelle Tan is appreciated.