GRK2:G q/11 Interaction: A Novel Surface on an RGS Homology

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GRK2:G q/11 Interaction: A Novel Surface on an RGS Homology Domain for

Binding G Subunits*

Rachel Sterne-Marr**1, John J. G. Tesmer2, Peter W. Day3, RoseAnn P. Stracquatanio1,3,

Jill-Ann E. Cilente1, Katharine E. O'Connor1, Alexey N. Pronin3,4, Jeffrey L. Benovic3, and

Philip B. Wedegaertner3

1 Biology Department, Siena College, 123 Morrell Science Center, 515 Loudon Rd.,

Loudonville, NY 12211, 2Department of Chemistry and Biochemistry, Institute for Cellular and

Molecular Biology, The University of Texas at Austin, Austin, TX 78712, 3Department of

Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, 233 S.

10th St., Philadelphia, PA 19107, 4Senomyx, Inc., 11099 North Torrey Pines Rd., Suite 160, La

Jolla, CA 92037

* This work was supported by National Science Foundation grant MCB9728179 and an

American Heart Association Southeastern Pennsylvania Affiliate Beginning Grant-in-Aid to

R.S.M., an American Heart Association Texas Affiliate Beginning Grant-in-Aid 0060118Y and a

Welch Foundation Chemical Research Grant F-1487 to J.J.G.T, a fellowship from American

Heart Association Pennsylvania-Delaware Affiliate to P.W.D., NIH grants GM44944 and

GM47417 to J.L.B., and NIH grants GM56444, GM628884 and a Pew Scholars Program in the

Biomedical Sciences grant to P.B.W.

**To whom correspondence should be addressed: Rachel Sterne-Marr, Siena College, Biology

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Department, 123 Morrell Science Center, 515 Loudon Road, Loudonville, NY 12211, Tel: (518)

783-2462, FAX: (518) 783-2986, Email: [email protected]

Running Title: Novel site on GRK2 RGS homology domain for binding G q/11

SUMMARY

G protein-coupled receptors (GPCRs) transduce cellular signals from hormones,

neurotransmitters, light and odorants by activating heterotrimeric guanine-nucleotide binding (G)

proteins. For many GPCRs, short-term regulation is initiated by agonist-dependent

phosphorylation by GPCR kinases (GRKs), such as GRK2, resulting in G protein/receptor

uncoupling. GRK2 also regulates signaling by binding G q/ll and inhibiting G q-stimulation of

the effector phospholipase C . The binding site for G q/ll resides within the amino terminal

domain of GRK2, which is homologous to the Regulator of G protein Signaling (RGS) family of

proteins. RGS proteins are negative regulators of G signaling. To map the G q/ll binding site

on GRK2, we carried out site-directed mutagenesis of the RGS homology (RH) domain of

GRK2 expressed as a GST fusion protein and identified eight residues, which when mutated,

alter binding to G q/ll. These mutations do not alter the ability of full-length GRK2 to

phosphorylate rhodopsin, an activity that also requires the amino terminal domain. Mutations

causing G q/ll binding defects impair recruitment to the plasma membrane by activated G q

when introduced into a GRK2(RH)-green fluorescent protein fusion, and regulation of G q-

stimulated phospholipase C activity when introduced into full-length GRK2. Two different

protein interaction sites have previously been identified on RH domains. The G binding sites


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on RGS4 and RGS9, called the “A“ site, is localized to the loops between helices 3 & 4, 5 &

6, and 7 & 8. The APC binding site of axin involves residues on helices 3, 4, and 5 (the

“B” site) of its RH domain. We demonstrate that the G q/ll binding site on the GRK2 RH

domain is distinct from the “A” and “B” sites and maps primarily to the C-terminus of its 5

helix. We suggest that this novel protein interaction site on an RH domain be designated the “C”

site. Additionally, GRK2 binds equally well to the G188S RGS-resistant mutant of G q which

suggests that the residues of G q that form the interface for binding GRK2 are distinct from

those used for binding the RH domain of RGS proteins.

INTRODUCTION

G protein-coupled receptors (GPCRs)1 are a large family of integral membrane proteins that

form seven transmembrane helices and couple to heterotrimeric guanine nucleotide (G) binding

proteins on their cytoplasmic surface. They transmit the signals from light and odorant receptors

as well as the signals initiated by numerous hormones and neurotransmitters. In their inactive

state, heterotrimeric G proteins are complexes of three polypeptide chains (G ). Upon

activation, GPCRs catalyze the exchange of GTP for GDP on the G subunit resulting in

dissociation of the GTP-bound G subunit from the G dimer [1]. G and G are then free to

regulate effectors such as adenylyl cyclase, phospholipase C (PLC ), cGMP phosphodiesterase

(PDE), ion channels, Rho family guanine-nucleotide exchange factors (RhoGEF), and activate

mitogen-activated protein kinase signal transduction pathways [2-5]. One common feature of

GPCR signaling is the rapid loss of cellular sensitivity even in the presence of a stimulus.

Insensitivity to the extracellular stimulus reflects intracellular events: receptor/G protein

uncoupling, G protein inactivation, and receptor sequestration (and receptor degradation), which


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together act to regulate the duration and/or magnitude of the signaling event [6]. One mode of

receptor desensitization is initiated by phosphorylation of the activated receptor by a kinase of

the G protein-coupled receptor kinase (GRK) family [7]. Phosphorylation then promotes binding

of the GPCR to a family of proteins called arrestins [8]. This occludes G interaction with

receptor and, in some non-visual cells, leads to sequestration of the receptor away from the

plasma membrane into endocytic vesicles[8-11].

GRKs are found in metazoans and, in mammals, the GRK family has seven members [7, 12].

GRKs are serine/threonine kinases with a tripartite modular structure. A central ~350 amino acid

kinase domain is closely related by sequence identity to those of cAMP-dependent protein

kinases, protein kinase C and ribosomal S6 kinases [13]. At the carboxyl terminus of the catalytic

core [14] homology to cAMP-dependent protein kinase predicts a putative “nucleotide gate”

[15]. The catalytic domain is flanked by an amino-terminal domain of 178 residues and a

carboxyl-terminal domain that varies in structure among members of the family. Using distinct

mechanisms, the carboxyl-terminal domains of GRKs direct the membrane association of these

kinases [16, 17].

The amino-terminal domains of all GRK family members are homologous to the Regulator of G-

protein Signaling (RGS) family of proteins [18]. RGS proteins are a multifunctional family of

proteins of variable length which share a ~120 amino acid "RGS domain." In this paper, we refer

to this domain as the RGS homology (RH) domain. RGS proteins act as GTPase activating

proteins (GAPs) for the G i/o (including G t) and G q family of G subunits [19-21] and as

antagonists of G /effector interaction [19, 22]. In general, these proteins bind preferentially to


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the GDP-aluminum fluoride (GDP•AlF4-)>GTP S>>GDP-bound form of G [21]. The crystal

structures of the RGS4:G i1 complex and the RGS9:G t/i chimaera/PDE complex show that the

RGS proteins contact switch regions I, II, and III of G which are polypeptide loops that undergo

conformational changes in the transformation between the GDP-bound (inactive) and the GTP-

bound (active) and states of the G protein. In these examples, the binding of the RGS protein

appears to stabilize the three switch regions in a conformation that preferentially binds the

transition state for GTP hydrolysis [23, 24]. RH domains can be grouped into five subfamilies

based on their evolutionary relatedness: R4, R7, R12, RZ and RA (axin) families [21]. Members

of the R4, R7, R12, and RZ families are negative regulators of G protein signaling as described

above. The newly described G s-specific RGS, RGS-PX1 [25], likely defines another RGS

subfamily as this protein is similarly related to all 5 RGS subfamilies (~24% amino acid identity).

Other families of proteins have RH domains but their roles in regulating heterotrimeric G protein

signaling are either distinct from RGS proteins, not well characterized, or do not regulate

heterotrimeric G protein signaling. Axin plays a role in the wnt/embryonic development

signaling pathway [26] and shares ~30% amino acid identity with RGS proteins of other

subfamilies. This RH domain has never been demonstrated to bind or GAP a G subunit [27].

Instead the RH domain of axin binds the tumor suppressor protein, adenomatous polyposis coli

(APC) [28], a downstream target in the wnt signaling pathway. The APC binding site of axin is

distinct from the G binding site of RGS proteins. A family of guanine-nucleotide exchange

factors for the monomeric G protein Rho (RhoGEFs) also has RH domains that share <20%

identity to the RGS family. p115RhoGEF, PDZRhoGEF, and LARG (leukemia-associated

RhoGEF), bind and in some cases serve as GAPs for G 12, G 13, and G q, via their RH domains


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yet they are also downstream effectors of G 12 and G 13 [29-32]. D-AKAP2, Dual specificity A

Kinase Anchoring Protein 2, binds the regulatory subunit of cAMP-dependent protein kinase and

has 2 RH domains. However, no G protein interaction has been reported for this protein [33].

The RH domain of GRK2 is most closely related to that of axin (26% amino acid identity) and

RGS12 (24% amino acid identity) and binds to G q and G 11 in an AlF4--dependent fashion, but

not to G s, G i or G 12/13 [34-36]. While all GRKs have putative amino-terminal RH domains,

G interaction has only been observed for GRK2 and GRK3. Unlike other G q-binding RGS

proteins such as RGS2 [37], RGS3 [38], RGS4 [22], and RGS18 [39], the GRK2 RH domain

does not stimulate the GTPase activity of G q in a single turnover GAP assay and only weakly

stimulates GTPase when G q is reconstituted with M1 muscarinic receptor and assayed in an

agonist-induced steady-state GTPase assay [34]. Because the GRK2 RH domain inhibits G q-

stimulated PLC activity both in vivo and in vitro yet lacks significant GAP activity in vitro, it

has been postulated that GRK2 RGS acts by sequestration of G q. It is also possible that GRK2

is an effector of G q. In this scenario, activation of G q would recruit GRK2 to the site of an

activated receptor. To investigate the role of GRK2/G q interaction in the regulation of Gq

signaling, we have created mutations in the RH domain of GRK2 that result in altered binding to

G q/11. Surprisingly, we found that the surface of GRK2 used to bind G q/11 is distinct from the

interaction site utilized by other RGS proteins to bind G subunits and from the site used by axin

to bind APC.


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EXPERIMENTAL PROCEDURES

Materials

Human embryonic kidney cells (HEK293) and African Green monkey kidney cells (COS-1) were

from the American Tissue Culture Collection. G q/11-specific polyclonal antibodies were

generously provided by Dr. D. Manning or purchased from Santa Cruz Antibodies, and EE-

specific monoclonal antibody was provided by Dr. H. Bourne. RGS2-GFP [40] and GRK2(45-

178)-GFP were expressed from the plasmid pEGFP (Clontech, Palo Alto, CA) and were

generously provided by Dr. S. Heximer and Dr. R. Penn, respectively. [3H]myo-inositol was from

Amersham, Dowex AG1-X8 resin was from Biorad and scintillation fluid was from Packard.

Molecular biologicals were from Roche unless otherwise indicated, immunoblotting detection

reagents were from Pierce, and all other biochemicals were from Sigma or Fisher.

Preparation and Mutagenesis of pGEX-GRK2(45-178) Constructs. Nucleotides encoding

residues 45-178 of bovine GRK2 cDNA were amplified by the polymerase chain reaction using

primers which incorporated BamHI and EcoRI restriction sites at the 5’ and 3’ ends of the coding

region, respectively. The resulting PCR fragment was subcloned into BamHI and EcoRI sites of

the glutathione-S-transferase fusion protein vector, pGEX-2T (Pharmacia) to generate pGEX-

GRK2(45-178). Sequential PCR [41] was used to produce the E78K, V83A, and D160K

derivatives of pGEX-GRK2(45-178) and QuikChange Mutagenesis (Stratagene) was used to

generate all other mutations. The GRK2 portion of each construct was sequenced to verify that

only the intended mutation had occurred.

Purification of GST-GRK2 Fusion Proteins. GST-GRK2 fusion proteins were expressed and


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purified by modifications of the procedures of Smith and Johnson [42] and Frangioni and Neel

[43]. Briefly, 40 ml cultures in Luria Broth containing 5mg/ml carbenicillin were grown at 37°C

to an optical density of 0.5, fusion protein expression was induced by the addition of IPTG to 0.5

mM, and incubation was continued for 3 h at 25°C. Cells were pelleted, washed in STE (20mM

Tris/150 mM NaCl/1mM EDTA, pH 8) and frozen at –70°C. Pellets were resuspended on ice in

STE containing 100 µg/ml lysozyme and incubated on ice for 15 min before the addition of -

mercaptoethanol ( ME) to 10 mM, PMSF to 100 µM, leupeptin to 1 µg/ml, benzamidine to 20

µg/ml, and sarkosyl to 1.5%. Lysates were sonicated in 10 s bursts followed by 15 s rest periods

to reduce viscosity. Insoluble protein was removed by centrifugation at 12,000 rpm for 10 min at

4°C and Triton X-100 was added to a final concentration of 2%. The lysate was adjusted to 25

mg/ml protein as determined by Bradford assay using gamma globulin as a standard (Biorad) and

fusion proteins were bound to glutathione-agarose beads (3 ml packed beads/mg protein) by

mixing for 1 h at 4°C. The beads were washed once with STE/1.5% sarkosyl/2% Triton X-100,

three times with STE, and stored in STE/ 25% glycerol/ 10 mM ME at –20°C. To determine the

amount of GRK2 associated with glutathione agarose beads, fusion proteins were eluted in 50

mM Tris-HCl pH8/10 mM glutathione/10 mM ME at room temperature for 1 h. Bradford

assays were then carried out on the eluates.

GST-GRK2 Pull-Down Assays with Bovine Brain G q/11. Bovine brain extract was used as a

source of G q/11 for in vitro binding assays. For 1 ml binding assays, 8 µg fusion protein was

incubated overnight at 4°C with 200 µg brain extract protein, prepared as described by Carman et

al. [34], in 20 mM Tris-HCl pH 8/2 mM MgSO4/6 mM ME/100 mM NaCl/0.05% C12E10/5%

glycerol/100 µM GDP in the absence or presence of 30 µM aluminum chloride and 5 mM sodium


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fluoride (AlF4-). Glutathione-agarose beads were washed 4 times with the buffer described above

(in the absence or presence of AlF4-

as appropriate), proteins were eluted with SDS-PAGE

sample buffer, and separated on 12% polyacrylamide gels. The proteins were transferred to

nitrocellulose, probed with anti-G q/11-specific polyclonal antibodies, incubated with peroxidase-

conjugated secondary antibody, and G q/11 was visualized by chemiluminescence using

SuperSignal West Pico (Pierce).

Rhodopsin Phosphorylation

COS-1 cells were grown at 37°C to 50-90% confluence on 10 cm dishes in DMEM

supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.

The cells were transfected with 10 µg total DNA (pcDNA3 alone, pcDNA3-GRK2 or pcDNA3-

mutant GRK2) using Fugene-6 (Roche) following the manufacturer’s recommendations. The

cells were harvested after 48 h, washed twice in ice cold PBS, and lysed in 1 ml buffer (20mM

HEPES, pH 7.2, 150 mM NaCl, 10 mM EDTA, 0.02% Triton-X100, 0.5 mM PMSF, 20 µg/mL

leupeptin, and 100 µg/mL benzamidine) by Polytron homogenization (two 15 sec bursts at 2500

rpm). Lysates were centrifuged for 10 min at 40,000 x g to remove particulate matter and

supernatants were then assayed.

To test for GRK activity, lysates containing WT or mutant GRK2 protein were assayed for their

ability to phosphorylate light-activated rhodopsin. Two microliters of COS-1 cell lysate were

incubated with 20 mM Tris-HCl pH 7.5, 2 mM EDTA, 5 mM MgCl2, 100 µM ATP, 1 uCi

[ 32P] ATP and 3.5 µM rhodopsin for 10 min at 30°C in room light. Reactions were quenched


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by addition of SDS sample buffer followed by 30 min incubation at room temperature.

Rhodopsin was separated by electrophoresis on a 10% SDS polyacrylamide gel, and gels were

fixed in 0.7 M trichloroacetic acid, 0.14 M 5’sulfosalicylic acid for 10 min to remove

unincorporated radionucleotide, washed twice in 50% ethanol, 16% acetic acid for 10 min, dried

and then subjected to autoradiography. Rhodopsin bands were excised and counted in a liquid

scintillation counter. Repeated measures ANOVA was used to test the statistical significance.

Homology Modeling. A homology model of the RH domain of GRK2 (residues 42-178) was

based on the structure of the RH domain of axin (PDB code 1EMU), which is the closest

homolog based on a BLAST [44] search of the protein data bank (26% identity within residues

64-174 of GRK2). The GRK2 model was built manually using the program O [45] by choosing

appropriate and reasonable rotamers for non-identical residues [46]. In regions that appeared to

have higher sequence identity with other RH domains of known structure, the GRK2 model was

adjusted locally according to those models. The 5- 6 loop of the GRK2 RH domain has no

obvious sequence homology to RH domains of known structure and was ultimately modeled

based on the axin structure because the fit of the side chains appeared to be reasonable and

because the 5- 6 loops of GRK2 and axin are identical in length (the loop is one amino acid

shorter in the RGS family of proteins). The overall model was refined in O to idealize its

stereochemistry.

In Vivo Inositol Phosphate Determination

GRK2 Mutants. To measure in vivo synthesized inositol phosphate (IP), 3.3 x 105 COS-1 cells

were plated on 6 cm dishes in DMEM (Mediatech, Herndon, VA) containing penicillin (100


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units/ml), streptomycin (100 ug/ml), and 10% fetal bovine serum. After 24 h, cells were

transfected using Fugene-6 (Roche) with 1 µg total DNA at a ratio of 3:1 pcDNA3-HA-G q-

R183C:pcDNA3-GRK2 (or mutant derivatives of GRK2). Following a 24 h incubation,

transfected cells were replated (~7 x 104 cell/well) in triplicate on 24-well plates and incubated in

complete DMEM. The media was removed and cells were labeled with [3H]myo-inositol

(Amersham) for 13-18 h in DMEM, w/o sodium pyruvate, w/high glucose, w/L-glutamine,

w/pyridoxine hydrochloride. In early experiments, labeling was carried out in inositol-free

DMEM (GibcoBRL), while later experiments utilized complete DMEM. Cells were washed in

the same media lacking radiolabel but containing 5 mM LiCl for 1 h at 37°C. The media was

removed and cells were lysed with 0.75 ml 20 mM formic acid for 30 min at 4°C before 0.1 ml

3% ammonium hydroxide was added. Inositol was separated from IP by sequential elution from

1 ml Dowex AG1-X8 (100-200 mesh) columns. The inositol fraction was eluted with 0.18%

ammonium hydroxide, while IPs were eluted with 4 M ammonium formate/0.2 M formic acid.

The inositol and IP fractions were mixed with Ultima Gold and Ultima Flo AF scintillation fluid

(Packard), respectively, and subjected to scintillation counting. To compare experiments with

differing levels of [3H]myo-inositol incorporation, IP production was determined as a fraction,

IP/(IP + inositol), and plotted relative to the control G q-R183C-stimulated IP production.

Statistical significance was assessed using repeated measures ANOVA with a Dunnett's post-test.

G q-G188S Mutant. IP accumulation experiments shown in Figure 7 were carried out as

described above except that HEK293 cells were utilized. In addition, 250 ng of plasmids

encoding G q-R183C or G q-R183C/G188S were transfected with increasing amounts of

pcDNA3-GRK2, pcDNA3-RGS2 and pB6-GAIP plasmids as indicated in the figure. pcDNA3


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vector was used as carrier DNA such that 1 µg of DNA was transfected in each well of the 6-

well plate. A unpaired t-test was used to assess statistical significance.

Confocal Microscopy. HEK293 cells were transfected in 6-well plates with the indicated

amounts of expression plasmids for GRK2(45-178)-GFP, RGS2-GFP, and/or G q using

FuGENE-6 reagent (Roche). After 24 h, transfected cells were replated onto glass coverslips and

grown for an additional 24 h before fixing in 3.7 % formaldehyde for 20 min. Cells were washed

with phosphate-buffered saline and then incubated in blocking buffer (50 mM Tris, pH 7.5, 150

mM NaCl, 1% Triton X-100, and 2.5% nonfat milk). Coverslips were then incubated in

blocking buffer containing a 1:100 dilution of anti-G q polyclonal antibody (Santa Cruz) for 1 h.

Following washes with blocking buffer, cells were incubated in a 1:100 dilution of Alexa Fluor

594-conjugated goat anti-rabbit secondary antibody (Molecular Probes) for 30 min. The

coverslips were washed and mounted on glass slides with Prolong Antifade reagent.

Representative images were recorded by confocal microscopy at the Kimmel Cancer Center

Bioimaging Facility using a Bio-Rad MRC-600 laser scanning confocal microscope running

CoMos 7.0a software and interfaced to a Zeiss Axiovert 100 microscope with Zeiss Plan-Apo

63x 1.40 NA oil immersion objective. Dual-labeled samples were analyzed using simultaneous

excitation at 488 and 568 nm. Images of “x-y” sections through the middle of a cell were

recorded.

RESULTS

Identification of GRK2 RGS Domain Mutants that are Defective in Binding to G q/11.

For a growing list of Gq-coupled receptors, it has been reported that desensitization can occur in a


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GRK2-dependent but phosphorylation-independent fashion. For example, hormone-mediated

PLC activation via the metabotropic glutamate receptor (mGluR1a) [47], the parathyroid

hormone receptor [48], the thromboxane A2 receptor [34], the endothelin receptor [49] and the

angiotensin II-1A receptor [50], is inhibited by overexpression of kinase-deficient GRK2-K220R.

The parathyroid receptor and mGluR1a interact with full-length GRK2 [47, 48] and the RH

domain of GRK2 co-immunoprecipitates with mGluR1a [51]. For the mGluR1a [51], the

endothelin receptor [49], and the thromboxane A2 receptor [34], overexpression of the RH

domain of GRK2 inhibits Gq-stimulated phosphoinositide hydrolysis. Thus, phosphorylation-

independent regulation of these receptors may be due either to GRK2/receptor interaction or to

GRK2/G q complex formation (or both). As a first step to determine the extent to which G q

binding by GRK2 regulates Gq-coupled receptor signaling, we used site-directed mutagenesis to

create GRK2 mutants which are defective in G q binding.

The three dimensional structures of several RH domains have been determined by X-ray

crystallography (RGS4, RGS9, axin, PDZRhoGEF and p115RhoGEF) [23, 24, 52-54], and

solution NMR (GAIP, RGS4) [55, 56]. Together these studies show that these RH domains share

a very similar fold (two four-helix bundles, see Fig. 1B, 1C). The X-ray structures of two of

these proteins, RGS4 and RGS9, were determined both alone and in complex with their G

ligands. The structures demonstrate that the RGS protein contacts all three switch regions of the

G subunit and that the RH domain does not undergo a large conformational change in tertiary

structure upon binding the G subunit. Crystal structures in combination with mutational

analyses have identified amino acids in the R4 family that contact the G subunit [23, 24, 57, 58].

For RGS4 and RGS9, G contact sites are primarily localized to the loops between helices, 3


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& 4, 5 & 6, and 7 & 8 (see Figure 1A and 1B for details). This has been designated the

“A” site [59]. G i residues important for RGS4 interaction are also conserved in other G

subunits including G q. Furthermore, mutation of an RGS4 residue in the 3/ 4 loop critical to

the RGS4:G i1 interaction (E87K) prevents RGS4:G q interaction [60]. Therefore, it is presumed

that the RGS4:G q interface mimics the RGS4:G i1 interaction.

The first residues of GRK2 targeted for mutagenesis were those that are identical or similar to

residues in RGS4 known to contact G i1 (GRK2 residues D160 and K164 corresponding to

RGS4 residues D163 and R167, respectively) (Figure 1A). A construct that encodes a GST

fusion protein bearing the RH domain of GRK2 (amino acids 45-178) was used as a template for

mutagenesis. Purified wildtype (WT) and mutant GST fusion proteins on glutathione-agarose

beads were incubated with bovine brain lysates in the presence of GDP or GDP•AlF4-. Beads

were pelleted and the binding of G q/11 was assessed by immunoblotting with G q/11-specific

antibody. WT, D160K, and K164A bound similarly to G q/11 in an AlF4--dependent fashion

(data not shown). Further mutagenesis of residues in the putative loops between 3 and 4 and

between 7 and 8 (H75A/L76A, E77A, E78K, K80A, V83A, N156A) was carried out without

identifying any residues important for G q/11 binding. Furthermore, double mutants such as

E78K/D160K, V83A/D160K, D160K/K164A retained the ability to bind G q/11 in the presence

of AlF4- (data not shown). Thus, unlike RGS4 and RGS9 binding to G i and G t, respectively,

residues in the 3/ 4 and 7/ 8 loops did not appear to be critical for G q/11 binding by GRK2.


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Binding of axin to its RH domain ligand, an helix from APC, utilizes residues within the 3,

4, and 5 helices [52]. This region, which has been designated the ”B” site [59], was targeted

in the next round of mutagenesis. While mutations in 4 (E84A, E87A, K90A) and a double

mutant in 5 (V103A/C104A) had no effect on binding, 5 substitutions R106A and D110A

resulted in diminished binding to G q/11 (Figure 2).

We then compiled a structural alignment to compare the RH domain of GRKs to RH domains

whose structures have been solved (Figure 1A) and used the known three-dimensional structures

of RH domains that had the highest sequence identity to the GRK2 RH domain (axin and GAIP)

to model residues 42-175. The model was then used to predict residues that might be close to

R106 and D110 in three-dimensional space. Substitutions F109I, M114A, and E116A exhibited

the greatest diminution in the binding to G q/11 in the presence of GDP•AlF4-, while L117A and

V137A had a lesser effect (Figure 2). E107A, Q133A and K139A, while spatially near to

R106A and D110A, had no observable effect on binding G q/11. Interestingly, K115A showed

no reproducible decrease in GDP•AlF4--dependent binding, but a dramatic increase in the ability

to bind GDP-bound G q/11. Mutations with the greatest effect map to the 5 helix and the

beginning of the 5/ 6 loop (and to the 6/ 7 loop to a much lesser extent) and form a

continuous surface (assuming that the axin-based model is a good representation of the GRK2 RH

domain).

GRK Mutants with G q-Binding Defects are not Impaired in In Vitro Receptor

Phosphorylation


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All amino acids selected for mutagenesis were predicted by our homology model to be exposed at

the surface of the RH domain. However, it is possible that some of these residues are in fact

buried and substitution to alanine might cause alteration in the tertiary structure. To test whether

single amino acid mutations in the RH domain altered another function of the N-terminal domain,

we looked at the ability of G q-binding mutants to phosphorylate activated rhodopsin. The N-

terminal domain of GRKs is thought to be involved in the activation of the kinase domain by

agonist-stimulated receptors. An antibody directed at residues 17-34 of GRK1 blocks rhodopsin,

but not peptide, phosphorylation [61]. Likewise, E7A-GRK1 and E5A-GRK2 mutants are

defective in rhodopsin, but not peptide, phosphorylation [62]. To test the integrity of the N-

terminal domain of G q-binding mutants, the R106A, D110A, M114A, K115A, and V137A

mutations were introduced into an expression vector encoding full length GRK2 (pcDNA3-

GRK2). Lysates from COS-1 cells transfected with WT or mutant GRK2 were assayed in a

rhodopsin phosphorylation assay. We did not observe any statistically significant defects in the

ability of G q-binding mutants of GRK2 to phosphorylate light-activated rhodopsin (Figure 3).

GRK Mutants with G q-Binding Defects are not Recruited to Plasma Membrane by

Activated G q.

A constitutively active mutant of G q, G q-Q209L, is able to promote plasma membrane

recruitment of a GFP fusion protein containing the RH domain of GRK2, GRK2(45-178)-GFP2.

The ability to induce plasma membrane localization of GRK2(45-178)-GFP is specific to G q

since other activated G subunits fail to recruit GRK2(45-178)-GFP to plasma membranes2. We

thus used this assay to examine interactions between G q-Q209L and a G q binding-defective


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mutant of GRK2 in cultured cells (Figure 4). When expressed in HEK293 cells, the GFP-tagged

RH domain of GRK2 is localized in the nucleus and throughout the cytoplasm (Figure 4A).

Likewise, GRK2(45-178)-GFP bearing the D110A mutation is diffusely localized throughout the

nucleus and cytoplasm when expressed alone (Figure 4D). Co-expression of G q-Q209L and

GRK2(45-178)-GFP results in their co-localization at cellular plasma membranes (Figure 4B and

4C). However, D110A-GRK2(45-178)-GFP remains in the nucleus and cytoplasm when co-

expressed with G q-Q209L (Figure 4E and 4F). This defect in G q-Q209L-induced plasma

membrane recruitment of D110A-GRK2(45-178)-GFP parallels the failure of this mutant to bind

G q/11 in the in vitro binding assay. When R106A-GRK2(45-178)-GFP was co-expressed with

G q-Q209L in similar experiments, this mutant demonstrated consistent but weak plasma

membrane localization (data not shown).

G q Binding Mutants of GRK2 are Defective in the Regulation of G q-R183C-stimulated

PLC Activity In Vivo

To determine whether the binding defects are manifested in full-length GRK2, we tested the

ability of GRK2 mutants to regulate G q-R183C activation of PLC in vivo. COS-1 cells were

transfected with G q-R183C alone, or co-transfected with G q-R183C and WT or mutant

GRK2, and inositol phosphate production was determined. WT GRK2 reduced G q-R183C-

stimulated inositol phosphate production by ~48% (P<0.01, Figure 5). Likewise, the K115A

mutant, which binds both the GDP- and GDP•AlF4--complexed forms of G q, was equally

effective (53% reduction) as WT GRK2 at preventing PLC activation (P<0.01). In contrast, the


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R106A, D110A, and M114A derivatives had no statistically significant effect on G q-R183C-

stimulated PLC activity. The V137A mutant, which exhibited only a modest binding defect in

the in vitro binding assay, only inhibited PLC activation by 27% (P<0.05). In general, the

ability of full length WT or mutant GRK2 to inhibit G q-R183C-stimulated inositol phosphate

production in vivo reflects the propensity of the analogous GST-GRK2(45-178) fusion protein to

bind brain G q/11 in vitro.

GRK2 Binds RGS-resistant G q-G188S Mutant

Because the RH domain of GRK2 preferentially binds to the transition state of G q/11, at least

one of the three switch regions is expected to be involved in their interaction. However, because

G q binds a surface of the GRK2 RH domain that is distinct from RGS4:G interface, the G q

contribution to the GRK2/G q interface is likely to be distinct as well. To test this hypothesis,

we evaluated the ability of the RGS resistant mutant, G q-G188S, to interact with GRK2. An

RGS-resistant G mutant was first described for the G subunit of the S. cerevisiae trimeric G

protein, Gpa1p [63]. gpa1sst mutants are supersensitive to the mating pheromone, -factor, due

to a failure to bind the RGS protein, Sst2p. The binding defect is due to a G�S substitution in

switch I of the G subunit. G�S substitutions in mammalian G subunits G i, G o, and G q

also result in resistance to RGS proteins of the R4 and R7 subfamilies [63, 64].

We first looked at the ability of the RGS-resistant mutant of G q to recruit GRK2-GFP to the

plasma membrane. HEK293 cells were transiently transfected with either G q, G q-R183C, or


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G q-R183C/G188S and either GRK2(45-178)-GFP or RGS2-GFP. A previous report

demonstrated that co-expression of an activated mutant of G q promoted the plasma membrane

localization of RGS2-GFP, while expression of RGS2-GFP alone resulted in nuclear and

cytoplasmic localization, with very weak plasma membrane localization [40]. When co-

transfected with G q, GRK2-GFP was found throughout the cell, and RGS2-GFP displayed

prominent nuclear staining with some cytoplasmic and faint plasma membrane localization

(Figure 6). In contrast, when transfected with the R183C GTPase mutant of G q, GFP-GRK2

was partially recruited to the plasma membrane (Figure 6), as also observed with expression of

G q-Q209L (Figure 4B), and GFP-RGS2 was strongly recruited to the plasma membrane

(Figure 6). However, GFP-RGS2 and GFP-GRK2 differed when assayed for plasma membrane

recruitment upon co-expression with the RGS-resistant mutant of G q. When co-expressed with

G q-R183C/G188S, GRK2-GFP was recruited to the plasma membrane to the same extent as

when co-expressed with G q-R183C. In contrast, RGS2-GFP was only faintly detected at the

plasma membrane, and instead, was localized to the nucleus and cytoplasm (Figure 6). Thus, the

G188S mutation of G q prevents RGS2 but not GRK2 binding.

We next looked at the ability of GRK2 to attenuate signaling by the RGS-resistant mutant.

HEK293 cells were co-transfected with G q-R183C or G q-R183C/G188S and increasing

amounts of RGS2, GAIP, or GRK2 (full-length) cDNAs, and inositol phosphate production was

measured. GRK2, RGS2 and GAIP each inhibited the G q-stimulated PLC activity in a dose-

dependent fashion with the inhibition being 89%, 71%, and 54%, respectively, at the highest


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level of DNA transfected (Figure 7). Immunoblotting suggested that G q expression was

similar in all experiments and that increasing the level of GRK2, RGS2 and GAIP cDNA

increased the level of expression (data not shown). While GAIP inhibited G q-R183C-

stimulated PLC activity by 54%, it was much less effective at inhibiting G q-R183C/G188S-

stimulated IP production (19% decrease, P<0.05). Likewise, RGS2 showed diminished ability to

regulate IP production stimulated by the RGS-resistant mutant (31% decrease) relative to its

ability to decrease the G q-R183C-stimulated PLC activity (71%, P<0.05). In stark contrast,

GRK2 inhibited PLC activity stimulated by both G q-R183C (89% decrease) and G q-

R183C/G188S (95% decrease) to a similar extent. Thus, unlike RGS proteins RGS2 and GAIP,

GRK2 can tolerate substitution of Ser for the conserved Gly in the switch I region.

DISCUSSION

Through extensive mutational analysis, we have identified eight residues in the GRK2 RH

domain (R106, F109, D110, M114, K115, E116, L117, and V137) which, when mutated, alter

binding to G q/11. The binding defects attributed to these mutations are not likely to be due to

misfolding of the RH domain since we were unable to detect any differences between the ability

of WT and GRK2 mutants to carry out receptor phosphorylation, a function that requires an intact

N-terminal domain. With the exception of K115A, all of the mutants exhibit diminished binding

to GDP•AlF4--bound G q/11. Mutations with the most dramatic effects map to the C-terminal

half of the 5 helix and the N-terminal region of the 5/ 6 loop (Figure 1A, 1D). The GRK2

binding surface is distinct from both the binding interface used by RGS4 and RGS9 to bind G


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subunits ( 3/ 4, 5/ 6, and 7/ 8 loops) and the surface used by axin to bind APC helical

peptide ( helices 3, 4, and 5). Comparison of the G binding site on GRK2 with the G contact

sites on RGS4/RGS9 suggests that these sites do not overlap (Figure 1B-D). Although RGS4 and

RGS9 also utilize the 5/ 6 loop, the GRK2 contact sites are in the amino-terminus of that loop

while the RGS4/RGS9 contact sites are concentrated toward the carboxyl-terminus. Therefore,

our experiments identify a novel G binding site on the RH domain. Consistent with the

nomenclature of Zhong and Neubig [59] we propose that this surface be termed the “C” site.

G q binding-defective mutants not only fail to bind G q/11 in an in vitro pull-down assay, but

they are also defective in cell-based assays. First, unlike the WT GRK2 RH domain fusion

protein GRK2(45-178)-GFP which co-localizes with activated G q at the plasma membrane, the

G q binding-defective mutant D110A-GRK2(45-178)-GFP fails to be recruited to the plasma

membrane and instead remains in the nucleus and cytoplasm. Likewise, whereas full-length WT

GRK2 inhibits G q-R183C-stimulated PLC activity by ~50%, G q binding-defective mutants

GRK2-R106, GRK2-D110A and GRK2-M114A have no affect on PLC activity. Thus, the

failure to bind to G q in vitro has the expected in vivo ramifications: these GRK2 mutants fail to

bind to activated G q at the plasma membrane and fail to regulate the activity of this G subunit.

One mutant, K115A, has a particularly interesting phenotype. WT GRK2 prefers binding to the

transition state over the GDP-bound form of G q (Figure 1, [34]). While undiminished in its

capacity to bind G q/11-GDP•AlF4-, the mutant has a greatly enhanced ability to bind G q/11-

GDP. One hypothesis is that binding by the K115A mutant traps G q/11-GDP in a G q/11-


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GDP•AlF4--like conformation. Another idea is that K115 may normally play a negative role in

preventing GRK2 from binding the GDP-bound form of G q/11. In this scenario, the K�A

substitution would relieve that inhibition. An alternative hypothesis is that the K115A mutation

allows recognition of some feature of the GDP-bound state that is not accessible to the WT

protein.

Switch regions I, II and III constitute most of the buried surfaces on G subunits in the

RGS4:G i1 and RGS9:G t interfaces [23, 24]. Since the RH domain interface in the GRK2:G q

interaction is novel, we propose that some aspect of G q interaction surface is distinct from the

surfaces utilized by G i1 and G t in their RGS4 and RGS9 interactions. In support of this

hypothesis, we found that the RGS-resistant G188S mutant of G q is not refractory to the GRK2

RH domain. The G q-R183C/G188S mutant recruits GFP-GRK2(45-178) to the plasma

membrane and full length GRK2 effectively blocks G q-R183C/G188S-stimulated PLC

activity. The analogous residue in G i1, G183 sits in close proximity to RGS4 E83 in the

RGS4:G i1 co-crystal and is surrounded by other switch I residues which make hydrogen bonds

and ionic contacts with RGS4. Substitution of the G183 with almost any residue would be

predicted to alter the tertiary structure and decrease complementarity to the RGS4 interface.

Because G q-R183C/G188S does not inhibit GRK2 interaction, we speculate either that G188 is

not located in the GRK2 interface or that minor changes in the G q and/or GRK2 tertiary

structure can accommodate the G q G�S substitution within the interface.


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Of the eight residues whose alterations cause G q binding defects, six are strictly conserved in

GRK3 but not in other members of the GRK family (Figure 1). This clearly explains why GRK2

and GRK3, but not GRK1, GRK4, GRK5 nor GRK6, bind G q. Surprisingly, four residues that

make important contributions to G q binding by GRK2 are conserved in other RGS proteins. For

example, the R106 position is a basic residue in RGS2, RGS3, RGS4, RGS5, RGS8, RGS13,

RGS16, RGS17, RGS18, RGS19, and RGS20. F109, a residue that is exposed to the solvent in

RGS4, is F or Y in most RGS proteins. D110 is an asparagine or glutamate in RGS4, RGS5,

RGS8, RGS16, RGS17, RGS18, GAIP, and RGS20. Finally, E116 is glutamate, aspartate, or

glutamine in RGS2, RGS3, RGS4, RGS5, RGS8, RGS16, and RGS18. Thus, four of the

presumed GRK2 contact residues are each conserved in RGS2, RGS4, RGS5, RGS8, RGS16, and

RGS18. RGS proteins are modular and the family members are sometimes categorized based on

presence or absence of protein interaction domains in the full-length polypeptide [20]. RGS2,

RGS4, RGS5, RGS8, RGS16, and RGS18 are all members of the “small” RGS subfamily and

contain only short sequences outside the RGS domain. Because RGS2, RGS4, and RGS16 can

bind G q and because the GRK2:G q interface is contiguous with the RGS4:G i1 and RGS9:G t

interface, perhaps residues in the 5 helix and N-terminal portion of the 5/ 6 loop may play a

role in binding of G q by other RGS family members.

Residues in the RGS 5 helix may be conserved because they bind other regulatory ligands.

Indeed, RGS4 binds phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) but a double mutant

K112E/K113E lacks this capability [65]. In our alignment (Figure 1A), RGS4 K112 corresponds

to GRK2 R106; therefore, a G q contact site in GRK2 corresponds to a PIP3 contact site in


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RGS4. PIP3 inhibits the GAP activity toward G i1 of RGS1, RGS10, and GAIP, but not that of

RGS16. Furthermore, many RGS proteins have putative calmodulin (CaM) binding sites that

map to a region spanning the C terminus of 4 helix and the N-terminal half of the 5 helix.

CaM binds to RGS1, RGS2, RGS4, RGS10, RGS16 and GAIP in a calcium-dependent fashion,

but does not alter the GAP activity of RGS4 [65]. However, CaM does reverse the inhibitory

effect of PIP3 on GAP activity. K112 and K113 of RGS4 represent the C terminus of the

consensus CaM binding site, yet the K112E/K113E double mutant does not alter CaM binding to

RGS4. Because of the proximity of the putative PIP3- and CaM-binding sites to the "C" site of

the GRK2 RH domain, it would be interesting to test whether PIP3 (or other

phosphatidylinositides) or CaM affect binding to G q by RGS family proteins. Incidentally,

GRK2 binds to phosphatidyl inositides and phosphatidylserine [66-70] and to CaM with low

affinity [71], but none of the these ligand-binding sites are located within its RH domain.

It has recently been shown that LARG, in addition to binding G 12 and G 13, can also bind G q,

a characteristic that distinguishes it from its close relatives, p115RhoGEF and PDZRhoGEF [32].

The RH domain of RhoGEF family members shares only a small number of residues implicated

in G interaction by RGS4 and RGS9. Likewise the 5, 5/ 6 loop region of LARG does not

bear similarity to the G q binding region of GRK2 (see alignment in Figure 1A). Thus, the G q

interaction site on this RH domain-containing protein appears distinct from the "C" site.

In summary, extensive mutational analysis of GRK2 shows that G q/11 binding to the RH domain

occurs at a novel G binding site which we have called a “C” site. This, in combination with the


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inability of the switch I mutant G q-G188S to affect GRK2 RH domain association, suggests that

GRK2 binding occurs on a distinct surface of G q/11 as well. Interestingly, several mutations in

the 5 helix which inhibit G q/11-binding do not impair rhodopsin phosphorylation, suggesting

that the region of the GRK2 N-terminal domain necessary for receptor phosphorylation is distinct

from the "C" site of the RH domain. Further studies are necessary to map residues of the GRK2

N-terminus that are necessary for receptor phosphorylation and to define features of G q that are

required for GRK2 interaction.

ACKNOWLEDGMENTS

We thank Drs. Dave Manning and Henry Bourne for providing G q/11-specific and EE

antibodies, respectively, and Drs. Scott Heximer, Ray Penn, and Chris Carman for providing

RGS2-GFP, GRK2-GFP, and GST-GRK2(RH)-K164A constructs. R.S.M. thanks Drs. Richard

Neubig for helpful suggestions, Dr. Ray Penn for statistical advice, and Siena undergraduates

Erin Twiss, Carlos Gonzalez, Billy Robinson, Jay Kubik and Mike Ragusa for their contributions

to this work. R.S.M. also thanks Dr. Ken Helm for sharing equipment and reagents, Mrs. Betsey

Harvey for her support, and Drs. Jim Angstadt, Tom Coohill, and Doug Fraser for

encouragement. Finally, we thank the reviewer for helpful comments.

FOOTNOTES

1The abbreviations are: GPCR, G protein-coupled receptor; PLC , phospholipase C ; PDE,

cGMP phosphodiesterase; GRK, GPCR kinase; RGS, regulator of G protein signaling; GAP,

GTPase activating protein; RH, RGS homology; AlF4-, aluminum fluoride; GFP, green

fluorescent protein; WT, wildtype; GST, glutathione-S-transferase


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2P. W. Day and P. B. Wedegaertner, manuscript in preparation

FIGURE LEGENDS

Figure 1. Protein bindingsurfaces of RH domains. A) Structural alignment of the RH

domain of GRKs with RGS, axin, and p115RhoGEF family members. An initial multiple

sequence alignment of the RH domains was generated by ClustalW [72]. Subsequently, RH

domains of known structure were superimposed within the program O [73] and were used to

verify and adjust the sequence alignment. Sequences with a known crystal or NMR structure are

labeled with an asterisk. Boundaries of alpha helices within the domains are depicted as blue

boxes above the alignment. In p115RhoGEF and presumably LARG, the lengths of helices 7

and 8 differ from other RGS proteins and their locations are indicated by the orange helices

below the alignment. Residues that were mutated (usually to alanine, see text) in GRK2 but do

not alter binding to G q-GDP•AlF4- have backgrounds colored gold, while those that decrease

binding to G q-GDP•AlF4- or increase binding to G q-GDP are colored white on top of a

magenta background (this work, see text). Residues of RGS4 and RGS9 that contact the switch

regions of G i1 and the G i/t chimaera, respectively, are yellow [23] [24], while contacts of

RGS9 with the alpha helical domain of the G i/t chimaera are light blue [24]. Axin contacts with

the APC peptide are green [52]. Residues in RGS2 that, when converted to their equivalents in


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RGS4, enhance GAP activity toward G i [74] are indigo. Mutations in p115RhoGEF that

decrease G 13 GAP activity are hot pink [53]. Dashed boxes indicate regions that are

structurally heterogeneous among the known structures. All sequences are human unless

otherwise indicated. Accession numbers for the sequences used in the alignment are Q15835

(GRK1), P21146 (GRK2), P26818 (GRK3), P32298 (GRK4), P34947 (GRK5), P43250 (GRK6),

NP_631948 (GRK7), P41220 (RGS2), P49799 (RGS4), O46469 (RGS9), NP_005864

(RGS19/GAIP), AAC51624 (axin), BAA20834 (PDZRhoGEF), NP_004697 (P115RhoGEF),

and NP_056128 (LARG). B) Structure of the RH domain from RGS4. Residues that contact

G i are drawn with yellow carbons atoms. The nine -helices of the canonical RH domain are

labelled 1- 9. [23] Nitrogen atoms are colored blue and oxygen atoms red. C) Structure of

axin bound to the APC peptide [52]. Residues that contact the APC peptide are drawn with

green carbon atoms. The APC peptide is drawn as a black coil. D) Homology model of the RH

domain of GRK2. The model was built using the structures of axin and GAIP as a guide (see

methods). The gold and magenta color scheme described above applies to parts B, C, and D of

this figure).

Figure 2. Identification of eight GRK2 RH domain mutants with altered binding to G q/11.

Upper Panel. Glutathione-agarose beads bearing GST fusion proteins, either WT (GST-

GRK2[45-178]) or GST-GRK2(45-178) substituted at one of eight single amino acid positions,

were incubated with bovine brain extract in the presence (+) or absence (-) of aluminum fluoride

(AlF4-). Bound G q/11 was visualized by immunoblotting.


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Lower Panel. GST fusion proteins used in the GST pull-down assay above were separated by

SDS-PAGE and visualized by Coomassie staining.

Figure 3. G q/11 binding-defective mutants phosphorylate rhodopsin.

Upper Panel. Lysates were prepared from COS-1 cells transfected with full length WT or

mutant GRK2 constructs and equal volumes were used in a kinase assay with light-activated

rhodopsin as a substrate. An autoradiograph representative of three separate experiments is

shown.

Middle Panel. Levels of WT or mutant GRK2 present in each lysate were compared by

immunoblotting of equal volumes of lysate.

Lower Panel. The kinase activity in WT and mutant GRK2 lysates was quantified and means ±

SEM for the three separate experiments are displayed.

Figure 4. G q-Q209L induces plasma membrane recruitment of GRK(45-178)-GFP but not

D110A-GRK2(45-178)-GFP. HEK293 cells were transfected with 0.02 µg of either WT

GRK2(45-178)-GFP (A-C), D110A-GRK2(45-178)-GFP (D-F), or along with 1 µg pcDNA3 (A

and D) or 1 µg pcDNA3 containing EE epitope-tagged G q-Q209L (B, C, E, F). Subcellular

localization was determined by confocal microscopy. GRK2(45-178)-GFP (A, B) and D110A-

GRK2(45-178)-GFP (D, E) were visualized by GFP fluorescence, while G q-Q209L (C and F)

was visualized using an anti- q polyclonal antibody followed by an Alexa 594 conjugated anti-

rabbit antibody. Representative micrographs are shown. More than one hundred cells were

examined in at least five separate experiments. Bar, 10 µm.


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Figure 5. Inhibition of G q-R183C-stimulated Inositol Phosphate Accumulation by WT but

not Mutant GRK2. COS-1 cells, transfected with G q-R183C alone or in combination with

full-length GRK2 or mutant derivative, were labeled with [3H]myo-inositol for 18 hours. Inositol

was separated from inositol phosphates as described in “Experimental Procedures” and

quantified by scintillation counting. Shown are means ± SEM for 4-10 experiments performed

in triplicate. Differences between G q-R183C alone and G q-R183C + GRK2-WT, GRK2-

K115A, or GRK2-V137A were statistically significant (P<0.05). Differences between G q-

R183C alone and G q-R183C + GRK2-R106A, GRK2-D110A or GRK2-M114A, were not

statistically significant.

Figure 6. G q-R183C/G188S recruits GRK2 RH domain, but not RGS2, to the plasma

membrane. HEK293 cells were co-transfected with 0.25 µg pcDNA3 containing HA-tagged

versions of either G q, G q-R183C, or G q-R183C/G188S, and either 0.025 µg GRK2(45-178)-

GFP (left panel) or 0.25 µg RGS2-GFP (right panel) as indicated. Either 0.725 µg or 0.5 µg

pcDNA3 was included to adjust total transfected DNA to 1 µg. Cells on coverslips were fixed in

formaldehyde and mounted on glass slides as described in “Experimental Procedures.” The

localization of GFP-tagged proteins was visualized by confocal microscopy. Only cells

expressing plasma membrane localized G q, G q-R183C, or G q-R183C/G188S (not shown), as

determined by using an anti- q polyclonal antibody followed by an Alexa 594 conjugated anti-

rabbit antibody, were chosen to identify subcellular localization of the GFP-tagged proteins.


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Representative micrographs are shown. More than one hundred cells were examined in at least

five experiments. Bar, 10 µm.

Figure 7. G q-R183C/G188S-stimulated Inositol Phosphate Production Is Sensitive to

GRK2 but Resistant to RGS2 and GAIP. HEK293 cells were transfected with G q-R183C

(A) or G q-R183C/G188S (B) alone or with increasing amounts of full-length GRK2 (�), GAIP

(�) or RGS2 (�). Cells were labeled with [3H]myo-inositol and inositol was separated from

inositol phosphates as described in “Experimental Procedures” and quantified by scintillation

counting. The experiment shown is representative of three independent experiments and

displayed as the average of triplicates ± SD.

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WedegaertnerE. Cilente, Katharine E. O'Connor, Alexey N. Pronin, Jeffrey L. Benovic and Philip B.

Rachel Sterne-Marr, John J. G. Tesmer, Peter W. Day, RoseAnn P. Stracquatanio, Jill-Ann subunitsαG

q/11 interaction: A novel surface on an RGS homology domain for bindingαGRK2:G

published online November 8, 2002J. Biol. Chem.

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GRK2:G q/11 Interaction: A Novel Surface on an RGS Homology