1 Revised 3/700 The Regulator of G protein Signaling RGS4 ...

1

Revised 3/700

The Regulator of G protein Signaling RGS4 Selectively Enhances α2A-Adrenoceptor

Stimulation of the GTPase Activity of Go1α and Gi2α

Antonella Cavalli, Kirk M. Druey+ and Graeme Milligan

Molecular Pharmacology Group,

Division of Biochemistry and Molecular Biology,

Institute of Biomedical and Life Sciences,

University of Glasgow,

Glasgow G12 8QQ,

Scotland, U.K.

+Molecular Signal Transduction Section, Laboratory of Allergic Diseases

NIAID, National Institutes of Health

Bethesda, MD 20852, U.S.A.

JBC Papers in Press. Published on May 11, 2000 as Manuscript M910395199 by guest on M

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Correspondence to:

Graeme Milligan,

Davidson Building,

University of Glasgow,

Glasgow G12 8QQ

Scotland U.K.

Tel. 44 141 330 5557

FAX: 44 141 330 4620

e-mail: [email protected]

1Abbreviations

GAP: GTPase activating protein. GPCR: G protein-coupled receptor. RGS: Regulator of

G protein signaling.

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Agonist-stimulated high affinity GTPase activity of fusion proteins between the α2A-

adrenoceptor and the α subunits of forms of the G proteins Gi1, Gi2, Gi3 and Go1,

modified to render them insensitive to the action of pertussis toxin, was measured

following transient expression in COS-7 cells. Addition of a recombinant regulator

of G protein signaling protein, RGS4, did not significantly affect basal GTPase

activity nor agonist-stimulation of the fusion proteins containing Gi1α and Gi3α but

markedly enhanced agonist-stimulation of the proteins containing Gi2α and Go1α.

The effect of RGS4 on the α2A-adrenoceptor-Go1α fusion protein was concentration-

dependent with EC50 of 30 ± 3 nM and the potency of the receptor agonist UK14304

was reduced 3-fold by 100nM RGS4. Equivalent reconstitution with Asn88Ser RGS4

failed to enhance agonist function on the α2A-adrenoceptor-Go1α or α2A-

adrenoceptor-Gi2α fusion proteins. Enzyme kinetic analysis of the GTPase activity of

the α2A-adrenoceptor-Go1α and α2A-adrenoceptor-Gi2α fusion proteins demonstrated

that RGS4 both substantially increased GTPase Vmax and significantly increased Km

of the fusion proteins for GTP. The increase in Km for GTP was dependent upon

RGS4 amount and is consistent with previously proposed mechanisms of RGS

function. Agonist stimulated GTPase turnover number in the presence of 100nM

RGS4 was substantially higher for α2A-adrenoceptor-Go1α than for α2A-

adrenoceptor-Gi2α.

These studies demonstrate that although RGS4 has been described as a generic

stimulator of the GTPase activity of Gi-family G proteins, selectivity of this

interaction and quantitative variation in its function can be monitored in the

presence of receptor activation of the G proteins.


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Initiation of signal transduction cascades involving heterotrimeric G proteins requires that

the binding of an agonist ligand to a G protein-coupled receptor 1(GPCR) results in the

stabilization of conformations of the GPCR which increase the rate of dissociation of

bound GDP from the nucleotide binding pocket of the α subunit of a cognate G protein.

Binding of GTP is thus allowed. With GTP bound the G protein produces a series of

activating conformational changes and can thence regulate, directly or otherwise, the

activity of several enzymes which generate intracellular second messengers or the

probability of opening of a range of ion channels [1]. Effector regulation is terminated by

the intrinsic GTPase activity of the G protein α subunit. Although this cycle of events is

clear [1], it is only in recent years that explanations for the measured discrepancy in

GTPase activity rates of heterotrimeric G proteins, which appeared too slow to account

for biochemically and electrophysiologically measured functional end points, have begun

to become apparent. A number of effector enzymes have been shown to display GTPase

Activating Protein (GAP) activity towards their partner G proteins [2-4]. Moreover, a

relatively recently identified family of Regulators of G protein Signaling (RGS) proteins

[see 5-8 for reviews] play key roles in accelerating the GTP hydrolysis rates of at least the

Gi and Gq families of G proteins. RGS GAP activity is thought to facilitate attenuation of

functional output of G protein-coupled signaling systems [9-13]. Data from both

biochemical assays [14-15] and the crystal structure of the core catalytic domain of RGS4

bound to a transition-state model of Gi1α [16] has indicated this to be the key state for

these interactions.

Many initial studies on the interactions of G proteins and RGS family members simply

used “single turnover” studies to confirm RGS function [15, 17]. Such studies involve the

co-addition of a recombinant RGS and a G protein which has been preloaded with

γ[32P]GTP under ionic conditions which limit nucleotide hydrolysis. Release of this

constraint allows measurement of accelerated GTP hydrolysis in the presence of an active

RGS. Although highly useful, only a limited amount of kinetic information can be


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derived from such studies, and steady-state GTPase activity and its regulation by the

presence of GPCRs and appropriate agonist ligands can provide more detailed

information [4,11,18]. However, to date, such studies have been restricted to the

reconstitution of GPCR (e.g. the M1 and M2 muscarinic receptors), G protein and RGS

into phospholipid vesicles and have not been performed in native membrane systems

under conditions capable of monitoring GTPase turnover number.

We have recently generated a series of fusion proteins between the α2A-adrenoceptor and

the α subunits of the pertussis toxin-sensitive G proteins by the simple expedient of

fusing the N-terminus of the open reading frame of the G protein cDNA in frame with C-

terminal end of the GPCR cDNA from which the stop codon was removed [19-23].

These fusion proteins have a number of distinct properties ideal for quantitative

functional analysis [see 24-25 for reviews]. Firstly, they ensure a 1:1 stoichiometry of

expression of GPCR and G protein. This ratio establishes the validity of saturation ligand

binding studies using [3H]antagonist as a direct measurement of the level of expression

of the G protein as well as the GPCR, which is difficult to achieve with independently co-

expressed GPCRs and G proteins. Secondly, the fusion protein strategy ensures

equivalent proximity of the GPCR to each G protein linked to it. Most importantly, the

fusion proteins function as agonist-activated GTPases. Addition of agonist to membranes

expressing such a fusion protein results in stimulation of GTPase activity [20-21].

Although the two elements of such fusion proteins inherently cannot fully physically

separate upon agonist stimulation, a considerable range of studies have recently

questioned the previous assumption that such separation does indeed occur [26, see 27 for

review]. Furthermore, a role for RGS proteins in the stabilization of GPCR-G protein

interactions has also been suggested [4].

Many studies with closely related, isolated, G proteins have suggested that the effects of

RGS proteins are generally non-selective. However, very recent work has indicated that

the N-terminal region of RGS4 allows GPCR-selective regulation of signaling complexes


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which involve Gq [28-29]. In the current study we now demonstrate selective regulation

by RGS4 of Gi-family G proteins coupled to the same GPCR and that the effect of RGS4

includes both concentration dependent increase in rate of agonist-stimulated hydrolysis of

GTP and an increase in Km for this nucleotide.

MATERIALS AND METHODS

Materials

All materials for tissue culture were supplied by Life Technologies, Inc. (Paisley,

Strathclyde, Scotland, U.K.). [3H]RS-79948-197 (90 Ci/mmol) was purchased from

Amersham International (Amersham, Buckinghamshire, U.K.). γ[32P]GTP (30 Ci/mmol)

was obtained from DuPont-New England Nuclear (Boston, MA). Pertussis toxin (240

µg/ml) and all other basic chemicals were purchased from Sigma (Poole, Dorset, U.K.)

or Boehringer-Mannheim (Mannhein, Germany) and were of the highest purity available.

Reagents for molecular biological manipulation were obtained from Promega.

Construction of the α2A-adrenoceptor-G protein fusion proteins.

The porcine α2A-adrenoceptor [30] was obtained from Dr. L.E. Limbird, Vanderbilt

University, TN. Position Cys351 of rat Gi1α was mutated to Val by site directed

mutagenesis [31]. The amplicons generated using primers spanning the restriction sites

DraI and EcoRI ( respectively in Gi1α and in pcDNA3 ) were subcloned into the α2A-

adrenoceptor-Gi1α fusion construct [20] restricted with the same enzymes. An identical

strategy was applied to construct the pertussis toxin-resistant fusion protein α2A-

adrenoceptor- Val351Go1α. The mutagenic reverse primer was designed to include the

XhoI site to facilitate subcloning in pcDNA3, while the forward primer was designed to

anneal to the sequence spanning the ClaI site in Go1α. Equivalent strategies were used to

generate α2A-adrenoceptor-Ile352Gi2α and α2A-adrenoceptor-Ile351Gi3α.

Cell culture and transfection


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COS-7 cells were maintained in DMEM containing 10 % (v/v) newborn calf serum , 2

mM L-glutamine. Cells were seeded in 60 mm culture dishes and grown to 60-80 %

confluency (18-24 h) prior to transfection with pcDNA3 containing the relevant cDNA

species using lipofectamine reagent (Life Technologies, Inc.) [20]. For transfection, 2.5-

2.8 µg of DNA was mixed with 10 µl of lipofectamine in 0.2 ml of Opti-MEM (Life

Technologies, Inc.) and incubated at room temperature for 30 min prior to the addition of

1.8 ml of Opti-MEM. COS-7 cells were exposed to the DNA/lipofectamine mixture for 5

h. 2 ml of 20 % (v/v) newborn calf serum in DMEM was then added to the cells. Cells

were harvested 48 h after transfection. In all the experiments herein cells were treated for

the final 24 h prior to cell harvest with pertussis toxin (25 ng/ml).

Preparation of membranes

Plasma membrane-containing P2 particulate fractions were prepared from cell pastes that

had been stored at -80oC following harvest as described previously [32].

[3H]RS-79948-197 binding studies

Binding assays were initiated by the addition of 5 µg of protein to an assay buffer (10

mM Tris-HCl, 50 mM sucrose, 20 mM MgCl2, pH 7.5) containing [3H]RS-79948-

197 [19-21] (1 nM). Non-specific binding was determined in the presence of 100 µM

idazoxan. Reactions were incubated at 30oC for 45 min., and bound ligand was separated

from free by vacuum filtration through GF/C filters. The filters were washed with 3 x 5

ml of assay buffer, and bound ligand was estimated by liquid scintillation spectrometry.

High affinity GTPase assays

Were performed as described in [19-21]. Non-specific GTPase was assessed by parallel

assays containing 100 µM GTP. All experiments were performed at least three times on

membranes prepared from individual cell transfections.


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Production of recombinant RGS4

Hexahistidine-tagged wild type and Asn88Ser RGS4 were expressed in E. Coli and

purified as previously described [33].

Results

A fusion protein was constructed between the porcine α2A-adrenoceptor from which the

stop codon had been removed and a variant of Go1α in which Cys351, the site for pertussis

toxin-catalysed ADP-ribosylation, was converted to Val. This resulted in production of a

single open reading frame in which the N-terminus of the G protein was attached in

frame with the C-terminus of the GPCR. This construct was expressed transiently in

COS-7 cells and following extensive treatment with pertussis toxin (25ng/ml, 24h)

membranes were prepared. Addition of a maximally effective concentration of the α2-

adrenoceptor agonist UK14304 (100µM) caused a substantial increase in high affinity

GTPase activity measured using 0.5µM GTP as substrate. Parallel specific ligand binding

studies were performed in these membranes with the α2-adrenoceptor antagonist [3H]RS-

79948-197 [19-23, 34]. As the fusion protein strategy defines a 1:1 ratio of expression of

the α2-adrenoceptor and G protein, the stimulation of the GTPase activity by UK14304 at

this concentration of GTP was calculated to be 2.8 ± 0.4 mol GTP hydrolysed.mol fusion

protein-1. min-1 (mean ± S.E.M., n = 6). When equivalent studies were performed using a

fusion protein between the α2A-adrenoceptor and Val351Gi1α, UK14304 (100µM)

stimulation of the high affinity GTPase of this fusion protein was substantially greater at

8.5 ± 0.3 mol GTP hydrolysed.mol fusion protein-1.min-1 (mean ± S.E.M., n = 3). (Figure

1). Addition of purified, recombinant RGS4 [33] (30nM) to membranes expressing the

α2A-adrenoceptor-Val351Go1α fusion protein did not significantly alter basal high affinity

GTPase activity but produced a strong enhancement ( p< 0.005) of UK14304-stimulated


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activity (Figure 1), increasing the effect of agonist to 10.0 ± 0.2 mol GTP hydrolysed.mol

fusion protein-1.min-1. In contrast to this effect of RGS4, addition of the recombinant

protein to membranes expressing the α2A-adrenoceptor-Val351Gi1α fusion protein

produced neither an alteration in basal GTPase activity nor a significant enhancement

(p = 0.42) of the stimulation of GTPase activity produced by UK14304 (9.1 ± 0.3 mol

GTP hydrolysed.mol fusion protein-1.min-1) (Figure 1).

Addition of varying amounts of recombinant RGS4 to membranes expressing the α2A-

adrenoceptor-Val351Go1α fusion protein resulted in a concentration-dependent

enhancement of the effect of UK14304 with an EC50 for RGS4 of 30 ± 3 nM (Figure 2).

This effect of RGS4 was likely to be dependent on specific, high affinity G protein

binding since addition of equivalent amounts of an Asn88Ser RGS4 mutant was unable to

enhance the stimulation of GTPase produced by UK14304 (Figure 2). This mutation

produces an RGS4 protein which is unable to bind effectively to any form of Gi1α

(33,35). Although RGS4 enhanced the effect of UK14304 on maximal stimulation of the

GTPase activity of membranes expressing the α2A-adrenoceptor-Val351Go1α fusion

protein, it also increased the EC50 for the agonist by some 3 fold. In the absence of RGS4

this was 31 ± 5 nM whilst in the presence of added RGS4 it was 113 ± 23 nM (Figure 3).

The α2A-adrenoceptor-Val351Go1α fusion protein can be effectively considered as a

agonist-stimulated, single enzyme species. Basal and regulated GTPase activity in

membranes expressing this fusion protein were measured at a wide range of

concentrations of GTP to explore the kinetic basis of RGS4 action (Figure 4). Analysis of

this data by extrapolation to Vmax demonstrated that UK14304 increased the basal

GTPase rate, as previously demonstrated for a number of other GPCR-G protein fusion

proteins (20, 36-37), and reduced Km for GTP (basal = 0.36 ± 0.04µM, UK14304 = 0.21

± 0.03µM, means ± S.E.M. n = 5, p = 0.019). Addition of RGS4 (100 nM) along with

UK14304 substantially further increased the Vmax of the system. Now, however, a

marked increase (from 0.21 ± 0.03µM in the absence of RGS4 to 1.26 ± 0.16 µM in the


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presence of 100µM RGS4, means ± S.E.M. n = 5, p = < 0.001) in the estimated Km for

GTP was also observed (Figure 4). With knowledge of the levels of expression of the α2A-

adrenoceptor-Val351Go1α fusion protein from [3H]RS-79948-197 binding studies,

UK14304-stimulated GTPase turnover number at Vmax was calculated to increase from

6.3 ± 1.4 min-1 to 81.7 ± 16.0 min-1 with addition of 100nM RGS4 (means ± S.E.M. n =

5, p = 0.002) (Table 1).

When varying amounts (1-100nM) of RGS4 was added to membranes expressing the

α2A-adrenoceptor-Val351Go1α fusion protein and kinetic analysis of UK14304 (100µM)

stimulated GTPase activity was monitored Vmax was progressively increased as was the

measured Km for GTP (Figure 5).

To further explore selectivity of enhancement of agonist activation of G protein GTPase

activity by RGS4, fusion proteins were constructed between the α2A-adrenoceptor and

both pertussis toxin-resistant Ile351Gi2α and Ile351Gi3α. Following transient expression in

pertussis toxin-treated COS-7 cells, UK14304 (100µM) stimulation of the GTPase

activity of α2A-adrenoceptor-Ile351Gi2α at 0.5µM GTP (2.7 ± 0.5 mol GTP hydrolysed.mol

fusion protein-1.min-1) was further enhanced by addition of recombinant RGS4 (30nM) to

the membranes (6.3 ± 0.9 mol GTP hydrolysed.mol fusion protein-1.min-1 means ±

S.E.M. n = 4) (Figure 6). By contrast, although the GTPase activity of membranes

expressing the α2A-adrenoceptor-Ile351Gi3α fusion protein was effectively stimulated by

UK14304 (5.6 ± 0.9 mol GTP hydrolysed.mol fusion protein-1.min-1), no further

significant enhancement (p = 0.6) of the agonist effect was produced by addition of RGS4

(7.0 ± 0.6 mol GTP hydrolysed.mol fusion protein-1.min-1 means ± S.E.M. n = 3) (Figure

6). As with the α2A-adrenoceptor-Val351Go1α fusion protein, the effects of RGS4 on the

α2A-adrenoceptor-Ile351Gi2α was to increase both GTP turnover number and the Km for

GTP (Figure 7, Table 1). In the absence of RGS4, the UK14304 stimulated GTP turnover

number was 4.4 ± 0.3


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min-1 and this was increased to 23.8 ± 3.0 min-1 with addition of 100nM RGS4 (means ±

S.E.M. , n= 5, p = 0.001).

Discussion

The expression of more than 20 members of the mammalian family of RGS proteins [5-6]

suggests that they are likely to display marked selectivity of expression patterns and/or

function. Striking variation in their distribution patterns in the central nervous system has

been observed [38]. By contrast, early studies of purified G proteins and RGS proteins

indicated little selectivity in the capacity of individual RGS proteins to regulate the

GTPase activity of individual members of the Gi and Gq family G proteins. However,

emerging data suggest inherent selectivity of RGS proteins towards G proteins may

indeed exist in their native environment. For example, a recent study examining the

ability of a range of GPCRs to regulate Ca2+ signaling via Gq family G proteins in

pancreatic acinar cells showed both marked variation in potency of individual RGS

proteins to regulate function via a single GPCR and of the same RGS to regulate the

signaling output from multiple, related, GPCRs [29]. Although the basis of the

interactions between the highly conserved core domain of RGS proteins with Gi1α has

been elucidated at atomic level [16], recent studies have indicated that the N-terminal

region of RGS4 might confer selectivity towards particular GPCR-G protein tandems

[28]. Such studies thus provide a conceptual basis to further explore selectivity in

function of RGS proteins and suggest that direct interactions between RGS proteins and

GPCRs occur.

Previously, selectivity in regulation of individual Gi-family G proteins by the most

widely studied RGS, RGS4, has not been observed. Herein we demonstrate the selective

enhancement of the GTPase activity of Go1 and Gi2 by RGS4 when these G proteins are

activated by the α2A-adrenergic receptor. Furthermore, for the first time, the experiments

were performed in membrane preparations rather than in reconstituted lipid vesicles.


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These studies have utilised a series of fusion constructs between the α2A-adrenergic

receptor and the α subunit of each of Gi1, Gi2, Gi3 and Go1 in which the N-terminus of the

G protein was linked directly and in-frame with the C-terminus of the GPCR from which

the stop codon had been removed. We [19, 36-37] and others [39-42] have previously

made considerable use of this strategy as it allows detailed enzyme kinetics to be

performed on the G protein under conditions in which the ratio of expression of the

GPCR and each individual G protein is kept constant [see 24-25 for reviews]. Although

the two elements of such fusion proteins inherently cannot fully physically separate upon

agonist stimulation, a considerable range of studies have recently questioned the previous

inherent implication that this should indeed occur [26, see 27 for review].

All cell lines which are widely used for either transient or stable expression of GPCRs

express a range of endogenous pertussis toxin-sensitive G proteins. Therefore, to ensure

that agonist stimulation of high affinity GTPase activity following expression of the

fusion proteins measured guanine nucleotide exchange and hydrolysis by the G protein

linked to the GPCR we have used mutants of the Gi-family G proteins in which the Cys

residue which acts as the acceptor for pertussis toxin-catalysed ADP-ribosylation was

converted to either Val or Ile [31, 34]. Pertussis toxin-catalysed ADP-ribosylation

prevents effective interaction between modified Gi-family G proteins and GPCRs.

Therefore, following expression of fusion proteins containing a mutation at this position,

the cells were treated with pertussis toxin prior to membrane preparation and assay.

Stimulation of GTPase activity by agonist now represents only activation of the GPCR-

linked G protein as G proteins of the Gs ,Gq and G12 families, which are not sensitive to

pertussis toxin, produce too limited a signal to be detected with this assay design, even if

the receptor can interact productively with them.

Initial studies expressed the α2A-adrenoceptor-Val351Go1α fusion protein in pertussis

toxin-treated COS-7 cells. Parallel measures of the capacity of the agonist UK14304 to

stimulate high affinity GTPase activity (when using 0.5µM GTP as substrate) and the


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levels of expression of the fusion protein indicated that a maximally effective

concentration of UK14304 caused stimulation of hydrolysis of some 2-3 mol GTP.mol

fusion protein-1.min-1. Addition of recombinant RGS4 to these membranes increased the

effect of UK14304 some 3 fold. Such results, although quantitatively more detailed than

produced from previous studies, were essentially predictable from prior studies of RGS

function. In addition, from isolated agonist-induced activation of fusion protein GTPase

activity, we determined that the potency of UK14304 to stimulate the α2A-adrenoceptor-

Val351Go1α fusion protein was slightly increased by the presence of RGS4. Furthermore,

the lack of ability of the Asn88Ser form of RGS4 [33] at concentrations up to 1µM to

enhance the effect of UK14304 demonstrated the specific requirement for a high affinity

interaction between RGS4 and the α2A-adrenoceptor-Val351Go1α fusion protein.

When equivalent studies were performed using an α2A-adrenoceptor-Val351Gi1α fusion

protein, however, very different results were obtained. Firstly, UK14304 stimulation of

the GTPase activity of α2A-adrenoceptor-Val351Gi1α at 0.5µM GTP, in the absence of

added RGS4, was substantially greater than of α2A-adrenoceptor-Val351Go1α. More

importantly, however, addition of recombinant RGS4 now failed to increase significantly

the stimulation of GTPase activity produced by UK14304. These data provide the first

evidence that the capacity of a RGS protein to enhance the GTPase activity of a Gi-family

G protein might be dependent upon the GPCR which produces the primary stimulus of

GDP release and subsequent guanine nucleotide exchange and also suggest that a direct

contact between Gi-coupled GPCRs and RGS proteins might occur. Such a mechanism is

not without precedent: an alternatively spliced, larger form of the RGS protein RGS12

contains a PDZ motif which has been shown to bind certain GPCRs [43].

Analysis of UK14304 stimulation of the GTPase activity of α2A-adrenoceptor-Val351Go1α

and α2A-adrenoceptor-Val351Gi1α at varying concentrations of GTP indicated Km for the

substrate to be similar (Table1). The observed higher GTPase activity of α2A-

adrenoceptor-Val351Gi1α per mol, when measured at 0.5µM GTP compared to the α2A-


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adrenoceptor-Val351Go1α therefore cannot simply reflect a substantially lower Km for

substrate of this G protein. GDP release is well accepted to be the rate limiting element

of the guanine nucleotide exchange and hydrolysis which is promoted by receptor

activation. It is thus important to note that Go1α has a 5 times higher rate of basal GDP

release than does Gi1α [44]. This further indicates that the greater agonist stimulated GTP

turnover by α2A-adrenoceptor-Val351Gi1α must reflect more effective activation of this G

protein by the receptor.

Equivalent studies also demonstrated that the effect of RGS4 on the α2A-adrenoceptor-

Val351Go1α fusion protein was to substantially increase Vmax of the GTPase activity. Most

impressively, however, full kinetic analysis demonstrated that addition of RGS4 to

membranes expressing the α2A-adrenoceptor-Val351Go1α fusion protein resulted in a

marked increase in the Km for agonist stimulation of GTPase activity which was

dependent upon RGS4 levels. As such, assays performed at a single concentration of GTP

substantially underestimate the capacity of RGS4 to enhance agonist-mediated GTP

hydrolysis. Indeed, rather than the estimates of a 3-fold stimulation produced by different

preparations of recombinant RGS4 when the assays were performed at 0.5µM GTP,

extrapolation of the kinetic parameters to Vmax indicated the true stimulation to be some

15 fold.

If the role of RGS4 is to stabilize the transition state for GTP hydrolysis without altering

GDP release or GTP loading, as has previously been surmised [16] these are exactly the

kinetic characteristics expected.

k1 k2

E + S ES

k-1

An RGS4-induced increase in k2 without alteration in the other kinetic constants will

result in an increase in Km as:


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Km = k2 + k-1

k1

It was also the case that RGS4 increased both agonist-stimulated Vmax and the Km for

GTP of the α2A-adrenoceptor-Gi2α fusion protein (Table 1). However, the effects of RGS4

were such that with 100nM RGS4, a maximally effective concentration of agonist and

measurement of GTPase activity at Vmax , the enzyme turnover number of this fusion

protein was only 30% of that produced by the α2A-adrenoceptor-Go1α fusion protein under

equivalent conditions (Table 1). These result reinforce the quantitative differences in the

effects of RGS4 on receptor regulation of closely related G proteins and also indicate why

analysis at Vmax is vital to produce detailed insights.

It was also noticeable that the addition of recombinant RGS4 did not significantly

increase basal high affinity GTPase activity. This observation suggests “basal”, RGS4

resistant, GTPase in such assays, generally considered to represent a composite signal

which, at least in large part, represent the relatively high guanine nucleotide exchange of

endogenous Gi-family G proteins might actually be derived largely from non-G protein

GTPases, such as tubulin, which contaminate the membrane preparations used for these

studies. Alternatively, if high affinity interactions of RGS4 in native systems involve

interactions with both GPCR and G protein [28] then pertussis toxin-mediated

uncoupling of endogenously expressed G proteins and GPCRs may encourage selective

interactions of the added RGS4 with the expressed fusion protein.

` If` the`differencesìn`sensitivityòf` the`GPCR-G`protein` fusion` proteins``to`

`RGS4àre`not`due`toà` receptor` interaction,` an alternative explanation for its

selectivity might lie in primary structure differences òf the three switch regions of

closely related Giα proteins, particularly in switch 3. These regionsùndergo

conformational changes upon GDP-GTP exchange and are thought to be the


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principal`contact points with RGS proteins [16]. Interestingly, 2-hybrid approaches using

the RGS family member GAIP have shown strong interactions of this protein with Gi1,

Gi3 and Go1 but only weak interactions with Gi2 [44]. This difference in affinity of

interaction between Gi1 and Gi2 has been suggested to be due to the presence or absence

of a single Asp residue in the Switch 3 region in Gi1. In Gi2 the equivalent residue is an

Ala [45]. This may not represent the entire answer as Go1, which interacts well with

GAIP, has a Gly rather than Asp at the equivalent position. It should be noted, however,

that Gi1 and Gi3, which were not effectively regulated by RGS4 in this study, are the

most closely related in sequence of the Gi-family G proteins. It will be of interest to

ascertain if a similar pattern of selectivity for RGS4 is observed for activation of these G

proteins by different GPCRs or if different members of the RGS family will display such

selectivity.

Each of the GPCR, the G protein and the RGS4 are potential targets for post-translational

S-linked palmitoylation [46-47]. In the case of the α2A-adrenoceptor, acylation occurs at

Cys442 [48]. However, mutation of this residue to Ala does not intefere with the

effectiveness of coupling of this receptor either in co-expression studies [48] or following

construction of this mutant into an α2A-adrenoceptor-Gi1α fusion protein [19]. Gi1α is

palmitoylated at Cys3 [49] as are the other Gi-family α subunits. Although there is no

formal proof that this amino acid becomes palmitoylated in the fusion proteins used

herein the equivalent Cys residue does become palmitoylated, and can be regulated in an

agonist-dependent manner, in a β2-adrenoceptor-Gsα fusion protein [50]. This does not

appear directly relevant for the the present studies, however, as it has previously been

shown that an α2A-adrenoceptor-Gi1α fusion protein in which this Cys is substituted by

Ala allows as effective agonist stimulation of the G protein GTPase activity as in a wild

type fusion protein [19]. There has been considerable recent interest in observations that a

range of RGS family members including RGS4, RGS10 [51] and RGS16 [52] can be

palmitoylated. A palmitoylation-defective mutant of RGS16 is able to act as an RGS for


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Gi but following expression in HEK293 cells was impaired in its capacity to attenuate

both Gi and Gq mediated signal cascades [52]. In the case of RGS4, mutants designed to

limit palmitoylation had different effects on the GAP activity of the protein against Gi

dependent upon whether the assay was based on ‘single-turnover’ measurements of GTP

hydrolysis or in steady state, ligand-regulated, GTPase assays which are closer to the

assay system we have employed. In this system palmitoylation promoted GAP activity

[51]. The bacterially produced recombinant protein we have used herein will not be

palmitoylated and we may thus have underestimated the absolute GAP capacity of RGS4

in our assays. As such, this will be an important issue to be addressed in future studies.

However, there is no current evidence to suggest that the acylation status of an RGS

protein will differentially modulate its capacity for regulation of a series of closely

related G proteins which is the key observation in the current studies.

It has been well established in a range of systems that the α2A-adrenoceptor can

concomitantly interact with and activate each of the Gi-family G proteins [53-54]. As

such activation can produce regulation of effector systems as diverse as adenylyl cyclase

[53], ERK MAP kinases [55] , voltage-operated Ca2+ channels [56] and K+ channels

[56], then the ability of RGS4 to selectivity control the duration of action of these α2A-

adrenoceptor-activated G proteins is likely to allow distinct regulation of different end

points.

Acknowledgments

These studies were supported by the Medical Research Council (U.K.).

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Tables

Table 1.

Kinetic parameters of the regulation of the GTPase α2A-adrenoceptor-

Val351Go1α and α2A-adrenoceptor-Val351Gi1α fusion proteins by agonist and RGS4.

Construct GTP Km

(nM)

Turnover

number

(min-1)

Turnover

number

(min-1)

basal + UK14304 + UK14304

+ RGS4

+UK14304 + UK14304

+ RGS4

α2A-Go1α 361 ± 43 207 ± 31 1259 ± 162 6.3 ± 1.4 81.7 ± 16.0

α2A-Gi2α 241 ±31 248 ± 28 560 ± 24 4.4 ± 0.3 23.8 ± 3.0

Data are presented as means ± S.E.M., n = 5 for each construct. UK14304 was present at

100µM and RGS4 at 100nM.


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Figure Legends

Figure 1

Recombinant RGS4 enhances agonist stimulation of the GTPase activity of α2A-

adrenoceptor-Val351Go1α but not α2A-adrenoceptor-Val351Gi1α.

α2A-adrenoceptor-Val351Go1α (a2AGo C351V) or α2A-adrenoceptor-Val351Gi1α (a2AGi1

C351V) fusion proteins were expressed transiently in COS-7 cells. Pertussis toxin

(25ng/ml) was added 24h before cell harvest. Cell membranes were used to measure basal

(open bars) and UK14304 (100µM) (stippled and filled bars) stimulation of high affinity

GTPase activity in the absence (open and stippled bars) or presence (filled bars) of

recombinant RGS4 (30nM) using 0.5µM γ[32P]GTP as substrate. Parallel specific

[3H]RS-79948-197 binding studies measured levels of expression of the fusion proteins.

RGS4 did not significantly alter basal high affinity GTPase activity. Data represent

means ± S.E.M. from 6 (a2AGo C351V) or 3 (a2AGi1 C351V) independent experiments.

Figure 2

Wild type RGS4 but not Asn88Ser RGS4 promotes UK14034 stimulation of α2A-

adrenoceptor-Val351Go1α high affinity GTPase activity in a concentration-dependent

manner.

Pertussis toxin-treated COS-7 cell membranes expressing α2A-adrenoceptor-

Val351Go1α were produced as in Figure 1. The capacity of varying concentrations of either

wild type (squares) or Asn88Ser (circles) RGS4 to modulate basal GTPase activity (open

symbols) and to enhance the capacity of UK14304 (100µM) (filled symbols) to stimulate

fusion protein high affinity GTPase activity was then assessed using 0.5µM γ[32P]GTP as

substrate. Equivalent data were produced two further independent experiments.


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Figure 3

RGS4 decreases the potency of UK14304 to stimulate the GTPase activity of the of

α2A-adrenoceptor-Val351Go1α fusion protein.


Val351Go1α were produced as in Figure 1. The capacity of varying concentrations of

UK14304 to stimulate high affinity GTPase activity was then measured in the absence

(open symbols) or presence (filled symbols) of recombinant RGS4 (100nM) using 0.5µM

γ[32P]GTP.

Figure 4

RGS4 enhances Vmax and Km of UK14304 stimulated α2A-adrenoceptor-Val351Go1α

fusion protein GTPase activity.


Val351Go1α were produced as in Figure 1. Basal GTPase activity ( open symbols) and the

capacity of UK14304 (100µM) to regulate this activity in the absence (filled squares) or

presence (filled circles) of RGS4 (100nM) was assessed at a range of concentrations of

GTP. Data from a representative experiment of 5 performed is displayed as an Eadie-

Hofstee transformation. Results from the entire data set in shown in Table 1.

Figure 5.

Analysis of the effects of varying RGS4 levels on agonist stimulation of the GTPase

activity of an α2A-adrenoceptor-Val351Go1α fusion protein.

Varying concentrations of RGS4 (filled circles 100nM, open circles 30nM, filled

triangles 10nM, open triangles 3 nM, open diamonds 1 nM, filled squares, 0) were

added to pertussis toxin-treated COS-7 cell membranes expressing α2A-adrenoceptor-

Val351Go1α along with 100µM UK14304.


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25

5A. High affinity GTPase activity was then measured at a range of concentrations of GTP

as in Figure 4 and compared to basal GTPase activity (open squares).

5B. The Km for GTP at varying concentration of RGS4 is displayed.

Figure 6

Recombinant RGS4 enhances agonist stimulation of the GTPase activity of α2A-

adrenoceptor-Ile351Gi2α but not α2A-adrenoceptor-Ile351Gi3α.

α2A-adrenoceptor- Ile351Gi2α (a2AGi2 C351I) or α2A-adrenoceptor- Ile351Gi3α (a2AGi3

C351I) fusion proteins were expressed transiently in COS-7 cells. Pertussis toxin

(25ng/ml) was added 24h before cell harvest. Cell membranes were used to measure basal

(open bars) and UK14304 (100µM) (stippled and filled bars) stimulation of high affinity

GTPase activity in the absence (open and stippled bars) or presence (filled bars) of

recombinant RGS4 (30nM) using 0.5µM γ[32P]GTP as substrate. Parallel specific

[3H]RS-79948-197 binding studies measured levels of expression of the fusion proteins.

Data represent means ± S.E.M. from 4 (a2AGi2 C351I) or 3 (a2AGi3 C351I) independent

experiments.

Figure 7

RGS4 enhances Vmax and Km of UK14304 stimulated α2A-adrenoceptor-Ile352Gi2α

fusion protein GTPase activity.


Ile352Gi2α were produced. Basal GTPase activity ( open symbols) and the capacity of

UK14304 (100µM) to regulate this activity in the absence (filled squares) or presence

(filled circles) of RGS4 (100nM) was assessed at a range of concentrations of GTP. Data

from a representative experiment of 5 performed is displayed as an Eadie-Hofstee

transformation. Analysis of the entire data set is presented in Table 1.


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Antonella Cavalli, Kirk M. Druey and Graeme Milliganstimulation of the GTPase activity of Go1alpha and Gi2alpha

The regulator of G protein signaling RGS4 selectively enhances alpha2A adrenoceptor

published online May 11, 2000J. Biol. Chem.

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