An N-terminal region of C. elegans RGS proteins EGL-10 and ...functions and Gα target specificities...

An N-terminal region of C. elegans RGS proteins EGL-10 and EAT-16 directs inhibition of

Gαo versus Gαq signaling*

Georgia A. Patikoglou and Michael R. Koelle!

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520

Running title: An RGS N-terminal region directs inhibition of Gα signaling

Correspondence: Michael R. Koelle

Department of Molecular Biophysics and Biochemistry

Yale University School of Medicine

333 Cedar Street, SHM C-E30

New Haven, CT 06520-8024

Tel: 203-737-5808

Fax: 203-785-6404

E-mail: [email protected]

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Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 26, 2002 as Manuscript M208186200 by guest on A

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SUMMARY

Regulators of G protein signaling (RGS proteins) contain an RGS domain that inhibits

Gα signaling by activating Gα GTPase activity. Certain RGS proteins also contain a G gamma-like

(GGL) domain and a poorly-characterized but conserved N-terminal region. We assessed the

functions of these subregions in the C. elegans RGS proteins EGL-10 and EAT-16, which

selectively inhibit GOA-1 (Gαo) and EGL-30 (Gαq), respectively. Using transgenes in C.

elegans, we expressed EGL-10, EAT-16, their subregions, or EGL-10/EAT-16 chimeras. The

chimeras showed that the GGL/RGS region of either protein can act on either GOA-1 or EGL-

30 and that a key factor determining Gα target selectivity is the manner in which the N-terminal

and GGL/RGS regions are linked. We also found that coexpressing N-terminal and GGL/RGS

fragments of EGL-10 gave full EGL-10 activity, while either fragment alone gave little activity.

Biochemical analysis showed that coexpressing the two fragments caused both to increase in

abundance and also caused the GGL/RGS fragment to move to the membrane, where the N-

terminal fragment is localized. By coimmunoprecipitation we found that the N-terminal

fragment complexes with the C-terminal fragment and its associated Gβ subunit, GPB-2. We

conclude that the N-terminal region directs inhibition of Gα signaling by forming a complex

with the GGL/RGS region and affecting its stability, membrane localization and Gα target

specificity.

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Many hormones and neurotransmitters bind and activate heptahelical transmembrane

receptors which in turn catalyze exchange of bound GDP for GTP on G protein α subunits. GTP

binding induces dissociation of Gα from Gβγ subunits and enables these activated proteins to

signal downstream effectors. RGS1 proteins (regulators of G protein signaling) help terminate

signaling by greatly enhancing the weak intrinsic GTPase activity of Gα proteins, thus driving

reassembly of the inactive, GDP-bound Gαβγ heterotrimer (1). All RGS proteins contain a ~120

amino acid region known as the RGS domain that contacts Gα subunits and functions as the

GTPase activation domain (2). To date more than 20 mammalian RGS proteins and about 20

mammalian Gα proteins have been identified (1).

Although RGS proteins have been extensively studied in vitro and by overexpression in

cultured cells, their in vivo Gα targets and physiological functions remain largely unclear. For

example, the RGS proteins RGS4 and GAIP act similarly on both Gαi and Gαo in vitro (3), but

show strong and opposing selectivity between these targets when assayed in cultured chick

sensory neurons (4). However, when overexpressed in HEK293 cells, both RGS proteins acted

similarly to block Gαi signaling (5). Another dilemma is illustrated by the fact that the RGS2

protein shows different preferences for Gαi versus Gαq depending on the specific in vitro assay

system used (6, 7). An additional complication arises from the fact that many studies have been

carried out on small RGS domain-containing fragments of RGS proteins. RGS proteins typically

contain additional conserved regions outside the RGS domain that appear to affect their functions

and target specificities (1).

Genetic studies have the potential to conclusively demonstrate the true physiological

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functions and Gα target specificities of RGS proteins. To date only a few RGS proteins have

been genetically characterized. A mouse knockout of RGS9 shows that it functions in the retina

to limit the duration of visual signaling (8). RGS9 is the only abundant RGS protein in rod outer

segments (9), which also contain only one Gα protein, the visual G protein Gαt. Thus the

selectivity of the RGS-Gα interaction in this case appears to be a relatively simple issue. A more

complex, and likely more typical case, is revealed by genetic studies of the C. elegans RGS

proteins EGL-10 and EAT-16. Although both are widely expressed in the nervous system, they

select different Gα targets and thus have opposite effects on C. elegans behavior (Fig. 1C).

Genetic experiments have shown that EGL-10 inhibits signaling by the C. elegans Gαo homolog

GOA-1, which in turn inhibits C. elegans egg-laying behavior (10). EAT-16, on the other hand,

inhibits signaling by the C. elegans Gαq homolog EGL-30, which in turn activates egg-laying

behavior (11). EGL-10 and EAT-16 constitute the clearest example of RGS proteins that have

been shown through rigorous genetic experiments to have distinct Gα target specificities.

EGL-10 and EAT-16 are members of a subfamily of RGS proteins that includes RGS9

as well as the mammalian RGS6, RGS7, and RGS11 proteins. Just N-terminal to their RGS

domains, these proteins contain a ~60 amino acid G gamma-like (GGL) domain (Fig. 1A and

1B) that mediates binding to a divergent Gβ subunit, Gβ5 (12). The exact role of Gβ5 in signaling is

unclear, but genetic and molecular studies in C. elegans show that EGL-10 and EAT-16 require

association with the C. elegans Gβ5 ortholog, GPB-2, for their stability and function (13). All

GGL-containing RGS proteins also contain a conserved N-terminal region of ~220 amino acids

(Fig. 1A) that has been termed the RGS-N domain (9). The significance of this region is

indicated by its extraordinary conservation (e.g. 69% identity comparing EGL-10 and RGS7,

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Fig. 1B).

The function of the RGS-N region is not clearly understood. It contains an 80-100

amino acid subregion known as the DEP domain, named for its weak sequence similarity (10-

19% identity) comparing the Dishevelled, EGL-10, and pleckstrin proteins (14). Two studies

analyzed the stimulation of Gαt GTPase activity by N-terminal truncation mutants of RGS9

deleted for the RGS-N region. One study saw little effect of the RGS-N region (15). The other

(16), using a different method of kinetic analysis, found that the RGS-N region contributed to

the ability of RGS9 to act preferentially on Gαt when Gαt is complexed with the gamma subunit

of its effector, phosphodiesterase. Recent studies of RGS9 have also shown that its RGS-N

region binds an anchoring protein that tethers it to the membrane (17, 18).

In this study, we show that the RGS-N regions of EGL-10 and EAT-16 contribute to the

functions and distinct Gα target specificities of these RGS proteins in vivo. Using EGL-

10/EAT-16 chimeras, we found that the manner in which the RGS-N and GGL/RGS regions are

linked influences whether Gαo or Gαq is selected for inhibition. Most intriguingly, we found

that the RGS-N region can direct Gα inhibition by the GGL/RGS region even when these

regions are expressed as separate polypeptides. This observation can be explained by our finding

that the RGS-N region directly or indirectly binds to the GGL/RGS region and its associated Gβ

subunit GPB-2, and the complex thus formed appears to be the functional unit that acts on Gα

targets in vivo.

EXPERIMENTAL PROCEDURES

Plasmids for Neural Expression of RGS proteins--A 2.2 kb PstI/PflMI fragment of the

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rgs-1 promoter (19) was inserted into pPD49.26 (20) to construct pGP3, a vector that directs

expression of inserted open reading frames in all neurons of C. elegans. cDNA fragments coding

full-length EGL-10 or EAT-16 were inserted into pGP3 to generate plasmids pGP4 and

pMK340, respectively. Conserved protein subregions were identified in alignments of EGL-10

and EAT-16 with each other and their human homologs (Fig. 1B), and cDNA fragments coding

some of these regions were inserted into pGP3. For expression of C-terminal fragments of

EGL-10 or EAT-16, an artificial AUG codon was added to initiate translation. The extents of

the EGL-10 subregions, the names designating them, and the corresponding expression plasmids

were: residues 1-223, EGL-N, pGP39; residues 224-334, EGL-L, (not expressed

individually); and residues 335-555, EGL-C, pGP40. The subregions, designations, and

plasmids for EAT-16 were: residues 1-201, EAT-N, pGP51; residues 202-211, EAT-L, (not

expressed individually); and residues 212-473, EAT-C, pGP52. An alternative EGL-N

fragment, consisting of residues 1-243 of EGL-10, was also tested: it behaved in all respects

like the smaller 223 residue EGL-N fragment (data not shown). The FL-N construct pGP91 was

made by adding sequences encoding DYKDDDDKDYKDDDDK (containing two FLAG tags)

immediately after the initiating AUG codon of the EGL-N construct pGP39. Similarly, the HA-

C construct pGP92 was made by adding sequences encoding GYPYDVPDYAGYPYDVPDYA

(containing two HA tags) after the AUG codon of the EGL-C construct pGP40.

Derivatives of pGP3 were also constructed to express EGL-10/EAT-16 chimeras

composed of the N, L, and C subregions described above. The chimeras used and the

corresponding expression plasmids were: EGL-N/EGL-L/EAT-C, pGP55; EGL-N/EAT-

L/EAT-C, pGP58; EGL-N/EAT-L/EGL-C, pGP59.

Transgenic animals--Test plasmids were coinjected with a marker plasmid into the

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gonad of C. elegans to generate extrachromosomal transgene arrays (20). Animals from

transgenic lines were recognized among the F2 progeny because they showed the phenotype

induced by the marker plasmid. Rescue of egl-10 was tested by injection into animals of

genotype egl-10(md176); lin-15(n765). The lin-15 rescuing plasmid pL15EK (11) was

coinjected at 50 ng/µl as the marker. Rescue of eat-16 was tested by injection into animals of

genotype eat-16(ad702), and the marker plasmid pRF4 (20), which induces a dominant Rol

phenotype, was coinjected at 80 ng/µl as the marker. To test for dominant negative effects,

injections were into lin-15(n765) animals carrying no RGS mutations, and pL15EK was used as

the marker plasmid. Each RGS expression plasmid tested was injected at 80 ng/µl, and pGP3

(empty vector) DNA was included in certain injections so that the total expression plasmid DNA

concentration was identical for every injection.

Extrachromosomal transgenes expressing FLAG and HA epitope-tagged EGL-10

fragments were chromosomally integrated by irradiating transgenic animals with γ-rays. The

resulting strains were outcrossed to egl-10(md176); lin-15(n765) animals at least two times to

produce clean genetic backgrounds. The integrated FL-N and HA-C transgenes shown in this

work have the allele designations vsIs20 and vsIs18, respectively. vsIs18 male animals were

mated to vsIs20 hermaphrodites and the resulting cross-progeny were allowed to self-fertilize to

produce animals homozygous for both vsIs20 and vsIs18. The genotypes of all these strains were

verified by PCR amplification of the transgenes.

Behavioral Assays--Unlaid eggs were counted by dissolving adult animals in bleach and

counting the bleach-resistant fertilized eggs under a microscope (10). All assays were on

animals selected as late L4 larvae and aged at 20° C for 30 hours to produce precisely-staged

adults. For each extrachromosomal transgene analyzed, at least 50 animals were assayed (e10

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animals from at least five independent transgenic lines). For integrated transgenes, e30 animals

were assayed. In certain cases we also carried out a second egg-laying assay in which the

developmental stages of freshly laid eggs were determined (10). In each case, this verified that

the transgenes affected egg-laying behavior, not egg production, indicating that the transgenes

generated normal EGL-10 and EAT-16 activities (data not shown).

C. elegans Protein Extracts--Worm strains carrying the egl-10(md176) null mutation as

well as the integrated transgene(s) vsIs18 and/or vsIs20 were grown in liquid culture at 20º C as

mixed-stage populations. Worms were purified by flotation on 30% sucrose and transferred to

lysis buffer (50 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 5 mM MgCl2, 150 mM NaCl,

protease inhibitors, and in certain experiments, 1% Triton X-100). Lysis was by three passages

through a French Press followed by 60 seconds of sonication with a microtip probe (Fisher

Scientific Model 550 Sonic Dismembrator). Debris and unlysed worms were removed by

centrifugation at 2,000 RPM in a clinical centrifuge. The resulting “total lysates” were flash-

frozen in liquid nitrogen and stored at –80º C. Protein concentrations were determined by

Bradford analysis. When required, total lysates were fractionated into soluble and pellet fractions

by centrifugation at 100,000Xg for 30 minutes. To assess the levels of FLAG- and HA-tagged

proteins in total lysates (Fig. 6), 50 and 160 µg total protein, respectively, were fractionated by

SDS-PAGE. We further analyzed total lysates by sucrose density gradient centrifugation.

Sucrose gradients were formed by successively overlaying a 49% (w/v) sucrose cushion with

equal volumes of 20% sucrose and total lysate. Centrifugation was carried out in a TLS-55

swinging-bucket rotor at 55,000 RPM for 2 hours at 4º C. Twelve equal volume fractions were

collected with fraction 1 at the top and fraction 12 (including any pellet fraction) at the bottom.

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The results shown in Fig. 6 and Fig. 7 are representative of those obtained in multiple trials

carried out with independently prepared lysates.

Western Blotting-- Proteins were fractionated by SDS-PAGE and then transferred to

nitrocellulose filters. The primary antibodies used to probe Western blots were: mouse anti-

FLAG (M2; Sigma); rat anti-HA High Affinity (3F10; Roche Molecular Biochemicals); mouse

anti-beta tubulin (E7; developed by Michael Klymkowsky and obtained from the Developmental

Studies Hybridoma Bank maintained by The University of Iowa, Department of Biological

Sciences); rabbit anti-GPB-2 (13); and rabbit anti-GOA-1 (13). The secondary antibodies were

horseradish peroxidase-coupled goat anti-mouse (Bio-Rad), goat anti-rabbit (Bio-Rad) or goat

anti-rat (Pierce). Protein bands were visualized using chemiluminescence detection reagents

(Pierce) and Kodak BioMax MR film. The proteins studied had mobilities on SDS-PAGE

analysis approximately as follows: HA-C, 28 kDa; FL-N, 32 kDa; beta tubulin, 55 kDa; GPB-

2 isoforms, 42 and 44 kDa; and GOA-1, 40 kDa.

Immunoprecipitation--Soluble fractions of extracts made in lysis buffer with 1% Triton

X-100 were incubated with anti-FLAG M2 antibodies coupled to agarose beads (Sigma),

tumbling for 2 hours at 4º C. The beads were washed three times in lysis buffer containing 1%

Triton X-100 and pelleted by centrifugation. Proteins bound to the pelleted beads were eluted in

100 mM glycine, pH 3.5 for 5 minutes with gentle shaking at room temperature and neutralized

with 10% elution volume of 0.5 M Tris, pH 7.4, 1.5 M NaCl. The results shown in Fig. 8 are

representative of those obtained in >3 trials.

RESULTS

EGL-10 and EAT-16 Have Opposite Effects Even When Expressed from the Same

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Promoter--EGL-10 and EAT-16 have precisely opposite mutant phenotypes because EGL-10

specifically inhibits Gαo and EAT-16 specifically inhibits Gαq. In principle, these RGS proteins

might achieve such specificity by being expressed in different cells that also express different Gα

proteins. Alternatively, the RGS and Gα proteins might all be found in the same cells, but each

RGS protein could have the ability to selectively act on only one of the Gα targets available to it.

This latter possibility is supported by the observation that both RGS and both Gα proteins are

expressed in all the neurons of C. elegans, although each RGS and Gα protein is also

additionally expressed in muscle cells that may differ for each protein (10, 11, 21-23).

To distinguish between these alternative models, we constructed transgenes in which the

same promoter was used to direct expression of either the EGL-10 or the EAT-16 cDNA. We

used the rgs-1 promoter, which is active in all neurons of C. elegans but in no other cells (19).

The constructs were transformed into egl-10 or eat-16 null mutants and tested for their ability to

rescue the opposite defects in egg-laying behavior seen in the two mutants.

egl-10 mutants are lethargic in their egg-laying behavior and thus accumulate excess

unlaid eggs compared to control animals (compare Fig. 2 A and B). Transgenic expression of

EGL-10 rescued the egl-10 egg-laying defect (Fig. 2C). In contrast, expression of EAT-16

from the same heterologous promoter did not affect the egl-10 mutant (Fig. 2D). We quantitated

our results by counting the number of unlaid eggs retained inside animals carrying the different

transgenes (Fig. 2E). The egl-10 transgene rescued the egl-10 defect fully, and even resulted in

slightly hyperactive egg laying, seen as a decrease in the accumulation of unlaid eggs relative to

the wild-type control (Fig. 2E, compare bars 1 and 3). Overexpressing EGL-10 has previously

been shown to cause a gain-of-function effect that results in hyperactive egg laying (10). The

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EAT-16 protein had no detectable EGL-10 activity, since expression of EAT-16 did not reduce

the accumulation of unlaid eggs (Fig. 2E, bar 4).

eat-16 mutants are hyperactive in their egg-laying behavior (11). Whereas wild-type

animals retain their fertilized eggs for two or more hours before laying them, eat-16 mutants lay

their eggs very soon after fertilization, resulting in a decreased accumulation of unlaid eggs (Fig.

2F, compare bars 1 and 2). Transgenic expression of EAT-16 resulted in substantial rescue of

the eat-16 egg-laying defect (Fig. 2F, bar 4), whereas expression of EGL-10 using the same

promoter resulted in no detectable rescue of the eat-16 defect (Fig. 2F, bar 3). The rescue

resulting from EAT-16 expression may have been incomplete either due to insufficient levels of

expression from the heterologous promoter, or because EAT-16 expression may be required

outside of the nervous system to achieve full rescue.

These experiments demonstrate that, even when expressed from the same heterologous

promoter, EGL-10 and EAT-16 have opposite effects on egg laying. Neural expression of either

RGS protein can fully (EGL-10) or substantially (EAT-16) rescue loss of endogenous

expression of the same RGS protein, but cannot rescue loss of the other RGS protein at all. We

conclude that EGL-10 and EAT-16 have distinct effects on egg laying due to distinct properties

of these RGS proteins themselves, and that any small differences in their endogenous expression

patterns do not account for their different functions.

The EGL-10 N- and C-Terminal Fragments Need Not Be Covalently Attached for

EGL-10 Function--We tested the ability of subregions of EGL-10 and EAT-16 to function in

vivo. The fact that the linker between the RGS-N region and the C-terminal GGL/RGS region is

variable in length and sequence among different RGS proteins (Fig. 1B) suggested that a precise

attachment between these two regions may not be required, and encouraged us to try expressing

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the two regions as separate polypeptides in transgenic animals to test for their function. The same

neural-specific promoter used above for expressing full-length RGS proteins was also used to

express the protein subfragments. In addition, we tested coexpression of these fragments by

cotransforming expression constructs for each. The expression constructs were transformed into

egl-10 or eat-16 null mutants and tested for their ability to rescue the egg-laying defects of these

mutants. The constructs were also transformed into animals with no RGS mutations to test for

dominant-negative effects on egg laying.

Experiments in which protein fragments are expressed suffer from the problem that such

fragments may not be properly folded or stable, making negative results difficult to interpret. We

set a stringent criterion for dealing with this issue: we do not present or interpret results from

constructs that give only negative results. However, if a construct gives strong rescuing activity

in one experiment, we do interpret negative results it may give in other experiments because the

positive result demonstrates that the construct successfully expresses an active protein fragment.

Figure 3A shows the effects of EGL-10 fragment expression in an egl-10 null mutant

background. We refer to the N- and C-terminal fragments of EGL-10 (indicated in Fig. 1A) as

EGL-N and EGL-C, respectively. Expression of either EGL-N or EGL-C only weakly rescued

the egl-10 egg-laying defect (Fig. 3A, bars 3 and 4). Surprisingly, coexpression of both

fragments gave full rescue of the egl-10 mutant (Fig. 3A, bar 5), equivalent to the strong rescue

previously seen by expressing full-length EGL-10 (Fig. 2E, bar 3). This result demonstrates that

EGL-N and EGL-C act together to inhibit Gαo signaling, and that EGL-N need not be

covalently attached to EGL-C for full EGL-10 function.

We tested the EGL-10 fragments for EAT-16 activity by expressing them in the eat-16

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null mutant, but found that neither fragment nor the combination of both showed much activity

(Fig. 3B). We also tested the EGL-10 fragments in a background carrying no RGS mutations to

test the fragments for dominant-negative effects (Fig. 3C). Interference with endogenous EGL-

10 function would be seen as an increased accumulation of unlaid eggs. Expression of EGL-N

alone (Fig 3C, bar 2) gave a result similar to the control (bar 1). Expression of either EGL-C

alone or coexpression of EGL-C and EGL-N also did not show dominant-negative effects.

Rather, expression of these fragments resulted in small decreases in the accumulation of unlaid

eggs ( Fig. 3C, bars 3 and 4) showing that these constructs gave positive EGL-10 activity, just as

they did in the egl-10 mutant background (Fig 3A, bars 4 and 5).

We also tested constructs expressing N- and C-terminal fragments of EAT-16 (termed

EAT-N and EAT-C, respectively) in egl-10, eat-16, and wild-type RGS backgrounds. Neither

fragment nor the combination of both showed strong effects in any background tested (data not

shown). According to our criterion for interpretability outlined above, we therefore do not

interpret these experiments. Although we know EAT-C is active (see below), EAT-N may

simply not be folded or stable.

The RGS-N Region of EGL-10 Can Direct the GGL/RGS Region of EAT-16 to Have

Full EGL-10 Activity--To identify the regions of EGL-10 and EAT-16 responsible for their

distinct Gα target specificities, we generated transgenes to express EGL-10/EAT-16 chimeras,

transformed them into egl-10 and eat-16 null mutants, and tested for their ability to rescue the

egg-laying defects of these mutants. In these experiments we hoped to identify discrete protein

subregion(s) that determine EGL-10 activity versus EAT-16 activity.

Our first strategy for generating chimeras was based on the observation that the N- and

C-terminal fragments of EGL-10, when coexpressed, give full EGL-10 function. We

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coexpressed combinations of N- and C-terminal fragments from EGL-10 and EAT-16 to see if

these combinations, which we term "noncovalent chimeras", could give EGL-10 or EAT-16

function.

We found that coexpression of the EGL-N fragment with the C-terminal GGL/RGS

fragment of EAT-16 (EAT-C) gave full EGL-10 function. As shown in Fig. 4A, expression of

either fragment alone in an egl-10 null background gave little rescue of the egl-10 egg-laying

defect (bars 3 and 4). When coexpressed, however, the EGL-N and EAT-C fragments fully

rescued the egl-10 mutant (bar 5). Indeed, the number of unlaid eggs in the animals

coexpressing the fragments was below that of the wild-type control (bar 1), indicating that

animals coexpressing EGL-N and EAT-C have excess EGL-10 activity and thus are slightly

hyperactive for egg laying. Expression of EGL-N, EAT-C, or both gave no rescue of the eat-16

egg-laying defect (Fig. 4B, bars 3-5), showing that these fragments have no detectable eat-16

activity.

We also coexpressed the EAT-N fragment with the C-terminal fragment of EGL-10, but

found this noncovalent chimera was unable to rescue either egl-10 or eat-16 mutants (data not

shown). As discussed above, we have no evidence that the EAT-N fragment was successfully

expressed and folded in these experiments and thus do not interpret these negative results.

Our main finding from the use of noncovalent chimeras was that expression of the EGL-

N and EAT-C fragments resulted in full EGL-10 activity and gave results essentially identical

to those seen when EGL-N and EGL-C were coexpressed (Fig. 3). In the context of these

experiments, the EGL-C and EAT-C fragments are thus equivalent and interchangeable.

Whereas the GGL/RGS region of EAT-16 would normally act to inhibit Gαq signaling, when

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this region of EAT-16 is coexpressed with the RGS-N region of EGL-10, it is instead

apparently directed to inhibit Gαo signaling.

The Relationship between the RGS-N and GGL/RGS Regions Directs Gα Target

Specificity--To refine our understanding of Gα target specification, we generated transgenes

that express full-length chimeric RGS proteins as single polypeptides. We term these "covalent

chimeras" to distinguish them from the noncovalent chimeras described above. In designing

these chimeras, we divided EGL-10 and EAT-16 into three subregions: 1) the N-terminal

RGS-N domain; 2) the linker region between the RGS-N domain and the GGL domain; 3) a C-

terminal region consisting of the GGL and RGS domains and residues C-terminal to them (see

Fig. 1A). We generated the complete set of six chimeras in which each of the three subregions

was derived from either EGL-10 or EAT-16 in every possible combination. For example, the

"EGL-N/EAT-L/ EAT-C" chimera contains the RGS-N domain of EGL-10, followed by the

linker from EAT-16, and finally the C-terminal GGL/RGS region from EAT-16. The chimeras

were expressed using the same neural-specific promoter employed in the experiments described

above. As before, we present data only from chimeras that gave strong rescuing activity in either

the egl-10 or eat-16 backgrounds. Purely negative results were obtained from the three chimeras

containing the EAT-N region, and these results were considered uninterpretable.

A key factor determining whether a chimera had EGL-10 activity or EAT-16 activity

was the manner in which the RGS-N and C-terminal regions were linked. For example, the

EGL-N/EGL-L/EAT-C chimera gave strong EGL-10 activity (Fig. 4A, bar 6) and little EAT-

16 activity (Fig. 4B, bar 6). In this chimera, the EGL-N region apparently directs the EAT-C

region to have EGL-10 activity, just as occurred when these two regions were coexpressed as

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separate polypeptides (Fig. 4 A and B, bars 5). However, swapping from the EGL-10 to the

EAT-16 linker caused a switch from EGL-10 activity to EAT-16 activity. This is seen in the

EGL-N/EAT-L/EAT-C chimera, which has strong eat-16 rescuing activity (Fig. 4B, bar 7) but

little egl-10 rescuing activity (Fig. 4A, bar 7).

The linker region is not the sole determinant of Gα target specificity. If this were true, the

EGL-N/EAT-L/EGL-C chimera would be expected to show EAT-16 activity rather than EGL-

10 activity. Instead, this chimera shows strong EGL-10 activity (Fig. 4A, bar 8) and little EAT-

16 activity (Fig. 4B, bar 8). This chimera demonstrates that the C-terminal GGL/RGS region

also contributes to Gα target specificity, since the EGL-10 activity of this chimera can be

converted to EAT-16 activity by swapping the C-terminal region to that of EAT-16 (Fig. 4 A

and B, compare bars 7 and 8).

In summary, analysis of EGL-10 and EAT-16 transgenes shows that the C-terminal

GGL/RGS regions of these proteins are not sufficient for in vivo function, but require a N-

terminal conserved region to gain full function in vivo. Surprisingly, the two protein regions

need not be covalently linked to function together. However, if they are covalently linked, the

manner in which they are attached can determine which Gα protein is selected as a target.

Functional Epitope-Tagged EGL-10 N- and C-Terminal Fragments for Biochemical

Analysis of Their Interactions--Perhaps the most intriguing result from our transgenic

experiments is our finding that neither the EGL-N nor EGL-C fragments are able to rescue egl-

10 mutant animals, but that coexpression of these fragments as separate polypeptides does give

full EGL-10 function (Fig. 3A). For the remainder of this work, we present a biochemical

analysis of these two protein fragments and their interactions in extracts of transgenic animals.

We modified our original EGL-10 N- and C-terminal fragments to include FLAG and

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HA epitope tags, respectively. We refer to these modified fragments as FL-N and HA-C. We

generated transgenes expressing these fragments in C. elegans and chromosomally integrated the

transgenes to produce stable transgenic lines that could be grown in biochemical quantities. An

additional benefit of the integrated transgenes is that we could genetically cross the two strains

carrying the individual FL-N and HA-C transgenes to generate a strain carrying both

transgenes. Thus proteins expressed from the exact same FL-N or HA-C transgenes could be

compared when expressed alone or in combination.

Before analyzing the tagged proteins biochemically, we checked their function in vivo by

testing their ability to rescue egl-10 mutant animals. We found that the tagged fragments (Fig. 5)

behaved similarly to their untagged counterparts (Fig. 3A). We note one subtle but interesting

difference: while the untagged N transgene appeared to have a small amount of egl-10 rescuing

activity (Fig. 3A, bar 3), the FL-N transgene (perhaps due to a lower expression level) had no

such detectable activity (Fig. 5, bar 3). Nevertheless, the FL-N transgene, when combined with a

partially rescuing C-terminal transgene, could give rise to full egl-10 rescuing activity (Fig. 5,

bar 5). These results suggest that the cooperation between N- and C-terminal fragments may be

synergistic rather than merely additive.

FL-N and HA-C Proteins Show Increased Abundance When Coexpressed--We carried

out Western analyses of total worm extracts from strains carrying the same FL-N and HA-C

transgenes separately or together. Our results showed that the FL-N and HA-C proteins were

both increased in abundance when coexpressed with each other (Fig. 6). Blots carrying different

loadings of the samples to achieve equivalent signals revealed about a two-fold enhancement in

the levels of FL-N and HA-C when coexpressed (data not shown). Since the HA-C protein by

itself showed partial rescue of the egl-10 mutant (Fig. 5, bar 4), the increase in HA-C protein

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upon coexpression with FL-N could, at least in part, explain the full rescue observed when these

proteins were coexpressed (Fig. 5, bar 5). One way the FL-N and HA-C proteins might increase

each others’ abundance is by forming a complex and thus stabilizing each other. In order to test

this hypothesis by coimmunoprecipitation, we first searched for conditions that could solubilize

the FL-N and HA-C proteins for precipitation.

Coexpression of FL-N with HA-C Decreases the Solubility of HA-C, and Both Proteins

Can Be Solubilized with Triton X-100--We fractionated total worm extracts by 100,000Xg

centrifugation into soluble and pellet fractions in the presence or absence of detergent. We used

Western blots to determine the solubility of the FL-N and HA-C proteins when expressed

separately or together (Fig. 7A). In the absence of detergent, HA-C expressed alone was more

than 50% soluble. However, when coexpressed with FL-N, almost all of the HA-C protein

moved to the pellet fraction. This insoluble HA-C could be substantially solubilized by addition

of 1% Triton X-100 (compare top two panels in Fig. 7A). In contrast to HA-C, FL-N, whether

expressed alone or in the presence of HA-C, was almost entirely in the pellet fraction. Addition

of 1% Triton X-100 also resulted in solubilization of a significant amount of FL-N (compare

bottom two panels in Fig. 7A).

To distinguish whether the insolubility of FL-N and HA-C observed in the absence of

detergent was due to membrane association or due to the formation of particulate/aggregate

structures, we carried out sucrose density gradient centrifugation on total lysates containing these

proteins. Our results revealed that the bulk of the insoluble FL-N and HA-C floated in fractions

8 and 9 of these gradients, which comprised the 20%/49% sucrose interface where membrane-

associated proteins typically reside (top two panels in Fig. 7B). No FL-N or HA-C were

detected in fraction 12, which included the pellet where any aggregates should be found. Western

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analysis of the same sucrose gradients showed that fractions 8 and 9, in addition to containing

FL-N and HA-C, also contained endogenous membrane-associated GOA-1, the Gα target of

EGL-10 (lower panel in Fig. 7B). FL-N and HA-C floated rather than pelleted in sucrose

gradients regardless of whether these two proteins were coexpressed (Fig. 7B), or expressed

individually (data not shown). FL-N and HA-C therefore appear to be membrane-associated,

and their insolubility is not due to aggregation.

Since the RGS domain found in the EGL-10 C terminus is believed to act directly on the

membrane-associated Gα protein GOA-1, the apparent increase in HA-C membrane

localization upon coexpression with FL-N could explain, at least in part, the increase from

partial to full egl-10 rescuing activity that occurs when HA-C is coexpressed with FL-N. The

effect of FL-N on the solubility of HA-C suggests that FL-N may form a complex with HA-C

and thereby affect not only its stability, but also its membrane localization. Using the 1% Triton

solubilization conditions shown in Fig. 7A, we have tested this hypothesis by

coimmunoprecipitation.

FL-N forms a complex with HA-C and its associated Gβ subunit--To determine if FL-

N and HA-C form a complex, we immunoprecipitated FL-N from Triton-solubilized extracts of

worms expressing both FL-N and HA-C and tested for coprecipitation of HA-C. As controls,

we used extracts of worms expressing only FL-N or HA-C, which should not show any

coprecipitated signal. We found that HA-C could be immunoprecipitated by the FLAG antibody

only in the presence of FL-N, demonstrating that HA-C forms a complex with FL-N in worm

extracts (Fig. 8). We expected that the Gβ subunit, GPB-2, should also be in this complex since

it had previously been shown to be an obligate subunit of EGL-10, presumably via an interaction

with the GGL domain (13). Indeed we found that GPB-2 coimmunoprecipitates with FL-N (Fig.

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8, lower panel). Interestingly, GPB-2 forms a complex with FL-N even in the absence of HA-

C, suggesting that both the RGS-N and GGL regions of EGL-10 may each have an independent

ability to bind the Gβ subunit.

DISCUSSION

An Experimental Approach Focused on the Physiologically Relevant Determinants of

RGS-Gα Specificity--The RGS domains of a number of RGS proteins have been found to

promiscuously activate the GTPase activities of many Gα proteins in vitro (1). These puzzling

results are at odds with the expectation that different RGS proteins might achieve distinct

functions in vivo at least in part by targeting different Gα proteins. The Gα target specificity of

RGS proteins might be increased in vivo by other cellular proteins or by non-RGS domain

regions of RGS proteins themselves. Several studies have compared purified full-length and

deleted versions of RGS proteins in in vitro GTPase activation assays to test for effects of

regions outside the RGS domain on Gα target specificity (12, 15, 16, 24). These studies might

not identify functions crucial to RGS protein activity in vivo, such as membrane localization or

interaction with cellular proteins other than Gα. An additional problem is that, in such studies,

RGS9-1 (the retinal-specific isoform of RGS9) was the only RGS protein tested that had a

known, physiologically relevant Gα target identified by genetic studies. RGS9-1 is atypical in

that it acts in rod outer segments where Gαt is the only Gα protein present at a significant level.

Thus RGS9-1 does not physiologically face the challenge of selecting a Gα target as do other

RGS proteins that are typically expressed in cells containing multiple Gα proteins. Another

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experimental approach used to analyze the basis of Gα target selection by RGS proteins involves

expressing full-length or deleted RGS proteins in cultured cells or Xenopus oocytes and using

assays that indirectly measure signaling by Gα targets (reviewed in Ref. 1). In these studies, the

physiologically relevant Gα targets of the RGS proteins used, again, are typically not known,

and the signaling readouts arranged for purposes of the experiments may have no relation to the

normal physiological functions of the RGS proteins studied.

In contrast, our studies analyze two RGS proteins, EGL-10 and EAT-16, that genetic

studies have shown to target two distinct Gα proteins, Gαo and Gαq, respectively. Previous

studies showed that these RGS and Gα proteins are all expressed in the same cells, suggesting

that the RGS proteins must actively select from at least two accessible Gα targets. We

demonstrated this more clearly by using transgenes to express EGL-10 and EAT-16 from the

same heterologous promoter and showing that they retained their proper Gα target specificities.

Using this same promoter, we have also expressed subregions or chimeras of EGL-10 and EAT-

16 and measured their functions in vivo using assays of egg-laying behavior. Importantly, this

readout of RGS function measures the normal physiological actions of EGL-10 and EAT-16 on

their genetically identified Gα targets. Our experimental approach also allows us to use extracts

of the transgenic animals to biochemically analyze the RGS proteins expressed. Thus, our

experimental system is designed to analyze RGS function and Gα target selectivity in a

physiological setting, and enables us to correlate in vivo and in vitro data.

The RGS-N Region Is Essential for Activity of EGL-10 and EAT-16, and Has a

Membrane Targeting Function--Studies of mammalian RGS proteins have shown that their

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GGL/RGS regions can function in vitro as efficient Gα GTPase activators even when the RGS-

N domain has been removed (15, 16). In contrast, our results show that the GGL/RGS regions of

either EGL-10 or EAT-16 have relatively little function in vivo unless attached to or

coexpressed with an RGS-N region. The differences between these results can be explained, at

least in part, by the fact that the in vitro assays were carried out in the absence of membranes,

while in vivo the RGS-N region functions to target RGS proteins to the membrane, the location

of their Gα targets.

By analyzing soluble and membrane fractions of C. elegans extracts, we found that the

RGS-N and GGL/RGS regions are each independently targeted to membrane fractions, although

less than 50% of the GGL/RGS region, when expressed alone, ended up in the membrane

fraction. When complexed with an RGS-N region, however, the GGL/RGS region was almost

entirely targeted to the membrane. Our results correlate with studies of RGS9, which also

identified membrane targeting functions in both the N- and C-termini of this protein. A portion

of the RGS-N region contains weak similarity to a region of Dishevelled (the "DEP domain")

that serves as a membrane anchor (25). In RGS9, the RGS-N domain binds the protein R9AP,

which anchors it to the rod outer segment membrane (18). Lishko et al. (17) showed that this

membrane association results in a ~70-fold increase in the activity of RGS9 on its Gα target. In

C. elegans there is no clear homolog of R9AP that could serve as a membrane anchor for EGL-10

and/or EAT-16, and the nature of the membrane attachment of RGS-N domains in C. elegans

remains to be elucidated. It is possible that there is a distant homolog of R9AP that does not

stand out as statistically significant in homology searches of the C. elegans genome, but may

serve as a membrane anchor for EGL-10 and/or EAT-16. It is also possible that such a

membrane anchor will be uncovered in the future by cloning additional genetically-identified

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genes that affect C. elegans egg-laying behavior.

The RGS-N Region Directs Gα Target Specificity--How are RGS proteins directed to a

specific Gα target? Our studies of EGL-10 and EAT-16, as well as recent work on the

mammalian members of the same RGS protein subfamily (RGS6, 7, 9, and 11), are beginning to

answer this question. Studies of mammalian RGS proteins have shown that the RGS domain is

sufficient on its own to act as an efficient Gα GTPase activator (26). Thus the RGS domain

might have been expected to fully specify the Gα target. However, addition of the GGL domain

and the Gβ5 subunit appeared to restrict the specificity of the RGS domain in vitro, making it

more selective for Gαo (12, 24, 27). RGS-Gβ5 complexes need not always be Gαo-selective in

vivo since EAT-16 is in a complex with the Gβ5-like protein GPB-2 but is a Gαq selective

regulator (11, 13). Thus, at least in the case of EGL-10 and EAT-16, which both individually

bind to GPB-2, specificity appears to be achieved by some means other than association with a

Gβ subunit.

Our in vivo experiments show that an interaction between the RGS-N region and the

GGL/RGS region specifies the Gα protein targets of EGL-10 and EAT-16. We were able to

direct the GGL/RGS regions of either EGL-10 or EAT-16 to act on either GOA-1 or EGL-30,

depending on how the GGL/RGS regions were attached to the RGS-N region of EGL-10. For

example, EAT-16 normally acts on the Gαq protein EGL-30, but the EAT-16 GGL/RGS

region can be made to act on the Gαo protein GOA-1 when coexpressed with the RGS-N

domain of EGL-10 (Fig. 4). Our results showing a role for the RGS-N region in recognition of

Gα targets are consistent with in vitro studies of RGS9. These studies showed that the RGS-N

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region of RGS9 helps allow it to act preferentially on a complex of Gαt with the gamma subunit

of phosphodiesterase versus on Gαt alone (15, 16). More recent work has shown that RGS9 can

form a stable complex with the GDP-bound form of Gαt using contacts other than those

previously characterized between the RGS domain of RGS9 and the switch regions of Gαt (18).

Our studies and the work on RGS9 together suggest that the RGS-N region may contact Gα

proteins and contribute to Gα target specificity. The RGS-N region is very large (~220 amino

acids). However, neither our work nor the studies of RGS9 have yet identified the features within

this region that allow it to specify Gα targets, nor have any RGS-N binding proteins been

identified other than the membrane anchor R9AP described above.

The RGS-N and GGL/RGS Regions Are Associated by Both a Noncovalent Interaction

as well as a Covalent Linker--A surprising aspect of our results is that the RGS-N and

GGL/RGS regions need not be covalently attached to function together. For example,

coexpression of these fragments of EGL-10 as separate polypeptides is sufficient to provide full

EGL-10 activity. Our results explain how this can occur, since we observed that the separately

expressed RGS-N and GGL/RGS fragments form a complex (Fig. 8), which appears to be the

functional unit. This identification of a noncovalent interaction between the RGS-N and

GGL/RGS regions is a novel observation. The binding interaction could be direct or indirect,

perhaps via the Gβ5-like GPB-2 subunit that is bound to the GGL region (13). The latter

possibility is suggested by our observation that the RGS-N fragment of EGL-10

coimmunoprecipitates with GPB-2 even in extracts lacking the GGL/RGS fragment of EGL-10.

Since GPB-2 binds to the GGL domains of both EGL-10 and EAT-16 (13), our observation

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that the RGS-N fragment of EGL-10 can function with the GGL/RGS fragments of either

protein (Fig. 3A and Fig. 4A) could be explained if the RGS-N fragment binds directly to GPB-

2.

In our experiments, we transgenically expressed RGS-N and GGL/RGS regions as

separate protein fragments and induced them to bind and function together. In endogenous RGS

proteins these regions are covalently attached by a linker, and the linker may simply allow the

two regions to more efficiently find each other and form a functional unit. Alternatively,

complexes between the RGS-N region of one molecule and the GGL/RGS region of another

might occur normally, so that higher order complexes containing one or more type of RGS

protein might exist.

Studies of the yeast RGS protein Sst2p have given results intriguingly analogous to those

presented here for EGL-10 and EAT-16. Coexpression of N- and C-terminal fragments of

Sst2p gave more function in vivo than expression of either fragment alone (28). Sst2p is only

distantly related to EGL-10: it has no recognizable GGL domain, although its N-terminus has

weak similarity to the DEP domain of the RGS-N region. Sst2p is endoproteolytically processed

so that it can naturally exist as separate N- and C-terminal fragments (28). There is currently no

evidence, however, that the N- and C-terminal fragments of Sst2p form a complex with each

other, or that EGL-10, EAT-16, or any of their mammalian counterparts (RGS6, 7, 9, and 11)

are proteolytically processed.

The noncovalent linkage between the RGS-N and GGL/RGS regions cannot exist merely

for the purpose of attaching the GGL/RGS region to the membrane via the RGS-N region. The

covalent linkage that also exists between these regions would be sufficient for this purpose. The

noncovalent association between the regions is likely to have additional functional consequences,

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including influencing Gα target specificity, as discussed above.

The covalent linkage between the RGS-N and GGL/RGS regions is not absolutely

required for RGS protein function, as shown by our functional coexpression of these regions as

separate polypeptides. However, the nature of the covalent linker can influence Gα target

specificity as demonstrated in our experiments with EGL-10/EAT-16 chimeras. The linker

regions of EGL-10 and EAT-16 are very different. The EGL-10 linker is ~110 residues long,

shows no conservation with the linker of the human EGL-10 ortholog RGS7, and is very rich in

proline, serine, alanine, and glycine (Fig. 1B). This linker may not fold into an ordered structure,

and thus may provide a long and flexible attachment between the RGS-N and GGL/RGS

regions. The ten residue EAT-16 linker, in contrast, would provide a short, rigid attachment that

might alter the geometry of the complex between the RGS-N and GGL/RGS regions. We note

that coexpression of RGS-N and GGL/RGS regions using the long EGL-10 linker gave results

similar to coexpressing these regions as separate polypeptides: in every case where activity was

seen, it was EGL-10 activity, not EAT-16 activity. These cases include full-length EGL-10,

coexpression of EGL-N with either EGL-C or EAT-C, and the EGL-N/EGL-L/EAT-C

chimera. In contrast, when the short EAT-16 linker was used, any activity observed was usually

EAT-16 activity, not EGL-10 activity. These cases include full-length EAT-16, and the EGL-

N/EAT-L/EAT-C chimera. We speculate that the geometry of the complex allowed by a

flexible linker, or no linker at all, between the RGS-N and GGL/RGS regions promotes EGL-10

activity (targeting Gαo), while a rigid attachment between these regions promotes EAT-16

activity (targeting Gαq).

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Acknowledgements--This paper is dedicated to the memory of our late colleague Paul

Sigler who provided inspiration and insight during the early stages of this work.

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Footnotes:

* This work was supported by a National Science Foundation predoctoral fellowship to G.A.P.,

by a Leukemia and Lymphoma Society Scholar award to M.R.K., and by grants from the

National Institutes of Health and the Robert Leet & Clara Guthrie Patterson Trust.

‡ To whom correspondence should be addressed: Dept. of Molecular Biophysics and

Biochemistry, Yale University School of Medicine, SHM C-E30, 333 Cedar Street, New Haven,

CT 06520-8024. Tel.: 203-737-5808; Fax: 203-785-6404; E-mail: [email protected].

1The abbreviations used are: RGS, regulator of G protein signaling; GGL, G gamma-like; DEP,

Dishevelled/EGL-10/pleckstrin homology; RGS-N, conserved N-terminal region found in

GGL-containing RGS proteins; EAT–(N, L, or C), N-terminal, linker, or C-terminal region of

EAT-16, respectively; EGL–(N, L, or C), N-terminal, linker, or C-terminal region of EGL-10,

respectively; FL-N, FLAG epitope-tagged EGL-10 N-terminal fragment; HA-C, HA epitope-

tagged EGL-10 C-terminal fragment.

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FIGURE LEGENDS

FIG. 1. RGS proteins with opposing effects on C. elegans egg laying share several conserved

regions. A, schematic representation of the 555 amino acid EGL-10 protein. An N-terminal

conserved region of unknown function (RGS-N; gray box) contains a subregion known as the

DEP domain. A poorly conserved linker region (white box) is followed by a G gamma-like

domain (GGL, hatched box) and an RGS domain (black box). In this work we analyze N- and

C-terminal fragments of EGL-10 indicated by bars under the schematic. B, alignment of the

EGL-10, human RGS7 (hRGS7), and EAT-16 RGS proteins. Gray, hatched, and black shaded

bars (top) overlie sequences corresponding to the gray, hatched, and black shaded regions in the

EGL-10 schematic in A. Amino acids identical in two or three of the sequences are shaded

black. C, schematic representation of the opposing G protein signaling pathways that control

egg-laying behavior in C. elegans. Signaling through cell surface receptors activates the Gαο

and Gαq proteins (known in C. elegans as GOA-1 and EGL-30, respectively). GOA-1 inhibits

egg laying whereas EGL-30 activates this behavior. The RGS proteins EGL-10 and EAT-16

each exist as obligate dimers with the Gβ5 ortholog, GPB-2. EGL-10 inhibits GOA-1 activity,

while EAT-16 inhibits EGL-30. Thus EGL-10 and EAT-16 have opposite effects on egg-

laying behavior.

FIG. 2. Effects of transgenic expression of EGL-10 and EAT-16, using a heterologous neural

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promoter, in egl-10 and eat-16 mutant animals. A, control adult hermaphrodite that carries an

empty vector transgene. B, egl-10 mutant carrying an empty vector transgene. C, egl-10 mutant

carrying a transgene expressing EGL-10 in all neurons using a heterologous promoter. D, egl-

10 mutant carrying a transgene expressing EAT-16 using the same promoter. Arrows in A-D

indicate individual unlaid fertilized eggs inside the adults. E, phenotypes shown in panels A-D

were quantitated by counting the number of unlaid eggs inside >50 animals for each genotype.

RGS mutant backgrounds and transgenes are indicated below the graph, and bars 1-4 correspond

to the genotypes shown in A-D, respectively. The egl-10 mutant phenotype, seen as an

accumulation of unlaid eggs, is rescued by expression of EGL-10 (bar 3), but not by expression

of EAT-16 (bar 4). Error bars indicate the 95% confidence interval of the mean. F, quantitation

of an experiment analogous to that shown in panel E, but examining rescue of eat-16 rather than

of egl-10. The eat-16 mutant phenotype, seen as a decrease in the number of unlaid eggs

retained in adults, is substantially rescued by expression of EAT-16 (bar 4), but not by

expression of EGL-10 (bar 3). Note the change of scale from panel E, and that the control (bar

1) is the same data as shown in panel E (bar 1). Control data from this figure are also replotted in

subsequent figures for purposes of comparison. The mutations used in this and subsequent

figures were egl-10(md176) and eat-16(ad702). Each is an apparent null mutation (10, 11).

FIG. 3. Effects of expressing EGL-10 protein fragments in egl-10, eat-16, and wild-type

animals. The fragments expressed were as indicated in Figure 1A: EGL-N, an N-terminal

fragment consisting of the RGS-N domain (indicated in figure as "N"); EGL-C, a C-terminal

fragment containing the GGL and RGS domains ("C"); or coexpression of both fragments

("N+C"). A, effects of EGL-10 protein fragment expression in the egl-10 null mutant

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background. Individual expression of EGL-N (bar 3) or EGL-C (bar 4) showed only partial

rescue of the egl-10 egg-laying defect, while coexpression of both fragments showed full rescue

(bar 5). B, expression of the same EGL-10 fragments as shown in panel A, but in an eat-16

mutant background. None of the EGL-10 fragments showed significant rescue of the eat-16

egg-laying defect. C, effects of EGL-10 protein fragment expression in a wild-type RGS

background. No significant dominant-negative effects were observed. The EGL-C transgene

(bar 3) and the coexpression of EGL-N and EGL-C (bar 4) both showed some positive egl-10

activity as evidenced by decreases in the number of unlaid eggs relative to the control (bar 1).

FIG. 4. Effects of expressing EGL-10/EAT-16 chimeric proteins in egl-10 and eat-16 mutant

animals. A, effects of chimera expression in the egl-10 null mutant background. B, effects of

chimera expression in the eat-16 mutant background. Egg-laying behavior was quantitated by

counting unlaid eggs. The three components of the RGS proteins were as indicated in Figure 1A:

an N-terminal subregion consisting of the RGS-N domain (denoted as "N"); a linker subregion

("L"); and a C-terminal subregion containing the GGL and RGS domains, ("C"). The label for

each subregion is printed under the graph on a gray or black background to indicate that it is

derived from EGL-10 or EAT-16, respectively. Bars 3-5 in panels A and B show the results of

expressing the EGL-10 N-terminal fragment and EAT-16 C-terminal fragment as separate

polypeptides in the absence of any linker region. Bars 6-8 show the results of expressing

individual chimeric polypeptides in which regions from the two proteins were covalently linked.

Coexpression of the EGL-10 N-terminal fragment and EAT-16 C-terminal fragment (bars 5)

gave strong EGL-10 activity, while the EGL-N/EAT-L/EAT-C chimeric protein (bars 7) gave

substantial EAT-16 activity. Other covalent chimeras (bars 6 and 8) showed partial EGL-10

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activity.

FIG. 5. Effects of chromosomally-integrated transgenes expressing epitope-tagged EGL-10

fragments in egl-10 mutant animals. The fragments expressed were: FL-N, a FLAG epitope-

tagged N-terminal fragment consisting of the RGS-N region; HA-C, an HA epitope-tagged C-

terminal fragment containing the GGL and RGS domains. The two transgenes expressing FL-N

or HA-C were chromosomally integrated to give stable expression, and the strain expressing

both FL-N and HA-C was generated by genetically crossing strains carrying the individual

transgenes (FL-N X HA-C). The results shown are similar to those in Figure 3A, indicating that

the epitope tags used do not interfere with the function of the EGL-10 fragments, and

reproducing the result that coexpression of both N- and C-terminal fragments is required for full

EGL-10 rescuing activity.

FIG. 6. Western blot analysis of EGL-10 protein fragment expression in transgenic animals.

Total worm lysates were separated by SDS-PAGE, transferred to nitrocellulose filters, and

immunoblotted with anti-HA, anti-FLAG, or anti-tubulin antibodies. Combining the HA-C and

FL-N transgenes by genetically crossing the strains carrying them resulted in increases in the

levels of both the HA-C and FL-N proteins. To control for loading, the same blots probed with

anti-FLAG and anti-HA antibodies were also probed with an anti-tubulin antibody.

FIG. 7. Solubility and effect of detergent on HA-C and FL-N expressed in transgenic animals.

A, total worm lysates (T), soluble fractions generated as supernatants from 100,000Xg

centrifugation of the same lysates (S), and the insoluble pellet fractions (P) were separated by

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SDS-PAGE, transferred to nitrocellulose filters, and immunoblotted with anti-HA and anti-

FLAG antibodies. This experiment was conducted either in the presence or absence of 1% Triton

X-100 in the lysis buffer. The HA-C protein was largely soluble when expressed alone, but

moved almost entirely to the pellet when coexpressed with the FL-N protein. In contrast, the

distribution of the FL-N protein remained unchanged in the presence or absence of the HA-C

protein, remaining predominantly in the pellet fraction. The use of 1% Triton resulted in

significant solubilization of both the HA-C and FL-N proteins. B, sucrose density gradient

fractionation of total lysates prepared in the absence of detergent from worms carrying integrated

transgenes expressing HA-C and FL-N. The bulk of these proteins floated in fractions 8 and 9,

as did endogenous membrane-associated GOA-1.

FIG. 8. Coimmunoprecipitation of the HA-C and FL-N proteins. Triton X-100 solubilized

protein extracts from worm strains carrying integrated transgene(s) expressing HA-C and/or

FL-N were subjected to immunoprecipitation (IP) with anti-FLAG antibodies. The pellets,

along with control extracts representing 5% of the material used for the IPs, were fractionated by

SDS-PAGE, transferred to nitrocellulose filters, and immunoblotted with anti-HA, anti-FLAG,

or anti-GPB-2 antibodies. Anti-FLAG antibody coprecipitates HA-C only in the presence of

FL-N, indicating that the HA-C and FL-N proteins form a complex.

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Georgia A. Patikoglou and Michael R. Koelleq signalingαo versus Gαinhibition of G

An N-terminal region of C. elegans RGS proteins EGL-10 and EAT-16 directs

published online September 26, 2002J. Biol. Chem.

10.1074/jbc.M208186200Access the most updated version of this article at doi:

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An N-terminal region of C. elegans RGS proteins EGL-10 and ...functions and Gα target specificities...

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