JBC Biosymposia, Inc.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M412996200 on January 14, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11052-11058, March 25, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/12/11052    most recent
M412996200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Pao, C. S.
Right arrow Articles by Benovic, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pao, C. S.
Right arrow Articles by Benovic, J. L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Structure/Function Analysis of {alpha}2A-Adrenergic Receptor Interaction with G Protein-coupledReceptor Kinase 2*

Christina S. Pao and Jeffrey L. Benovic{ddagger}

From the Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Received for publication, November 17, 2004 , and in revised form, January 10, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
G protein-coupled receptors (GPCRs) mediate the ability of a diverse array of extracellular stimuli to control intracellular signaling. Many GPCRs are phosphorylated by G protein-coupled receptor kinases (GRKs), a process that mediates agonist-specific desensitization in many cells. Although GRK binding to activated GPCRs results in kinase activation and receptor phosphorylation, relatively little is known about the mechanism of GRK/GPCR interaction or how this interaction results in kinase activation. Here, we used the {alpha}2A-adrenergic receptor ({alpha}2AAR) as a model to study GRK/receptor interaction because GRK2 phosphorylation of four adjacent serines within the large third intracellular loop of this receptor is known to mediate desensitization. Various domains of the {alpha}2AAR were expressed as glutathione S-transferase fusion proteins and tested for the ability to bind purified GRK2. The second and third intracellular loops of the {alpha}2AAR directly interacted with GRK2, whereas the first intracellular loop and C-terminal domain did not. Truncation mutagenesis identified three discrete regions within the third loop that contributed to GRK2 binding, the membrane proximal N- and C-terminal regions as well as a central region adjacent to the phosphorylation sites. Site-directed mutagenesis revealed a critical role for specific basic residues within these regions in mediating GRK2 interaction with the {alpha}2AAR. Mutation of these residues within the holo-{alpha}2AAR diminished GRK2-promoted phosphorylation of the receptor as well as the ability of the kinase to be activated by receptor binding. These studies provide new insight into the mechanism of interaction and activation of GRK2 by GPCRs and suggest that GRK2 binding is critical not only for receptor phosphorylation but also for full activity of the kinase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The superfamily of G protein-coupled receptors (GPCRs)1 forms a large class of cell surface molecules that respond to a diverse collection of stimuli including light, chemoattractants, hormones, and neurotransmitters (1, 2). GPCRs are bound to heterotrimeric G proteins, which contain GDP in the nucleotide-binding pocket of the {alpha}-subunit. Upon agonist stimulation, a conformational change in the receptor promotes the exchange of GDP for GTP and leads to subsequent dissociation of the {alpha}-GTP and {beta}{gamma} subunits from the receptor. These subunits then transduce and amplify the signal to specific downstream effector molecules that are important for cell growth and metabolism (3). Regulation of GPCR function is achieved through three principal mechanisms: desensitization, defined as the waning of receptor responsiveness to agonist; endocytosis, a process by which receptors are removed from the cell surface; and down-regulation, in which total receptor levels are decreased through degradation (4, 5). GPCR regulation is mediated largely by G protein-coupled receptor kinases (GRKs), a family of enzymes that specifically phosphorylate activated GPCRs on serine/threonine residues (4, 6, 7). Receptor phosphorylation promotes high affinity binding of arrestins, which function to uncouple receptor/G protein interaction and promote receptor endocytosis. However, GRKs have also been implicated in phosphorylation-independent desensitization (8–10), whereas additional receptors appear to bind arrestins in a phosphorylation-independent manner (11, 12). In addition, many GPCRs are also regulated by second messenger-dependent kinases such as cAMP-dependent protein kinase and protein kinase C (6).

The seven members of the GRK family can be classified into three subgroups: (i) GRK1 (rhodopsin kinase) and GRK7; (ii) GRK2 ({beta}ARK1) and GRK3 ({beta}ARK2); and (iii) GRK4, GRK5, and GRK6. GRK2 is the most extensively studied member. GRK2 is ubiquitously expressed and phosphorylates a wide variety of GPCRs including {alpha}2-adrenergic, {beta}-adrenergic, opioid, muscarinic, adenosine, and substance P (4, 7, 13). Although the actual mechanism by which GRK2 specifically phosphorylates an activated receptor is not known, it has been proposed that GRKs interact with receptor domains that become accessible during receptor activation. For example, previous studies have demonstrated that peptides derived from the first and third intracellular loops of the {beta}2-adrenergic receptor ({beta}2AR) inhibit GRK2 phosphorylation of receptor substrates, suggesting that there are multiple sites of interaction on the receptor for GRK2 (14). In addition, GRKs also appear to be activated upon binding to an agonist-occupied receptor. GRK1 is activated by binding to rhodopsin, and this activation is attenuated when the third intracellular loop of the receptor is proteolyzed (15). Analogous studies have demonstrated that GRK2 is activated by binding to rhodopsin or the {beta}2AR (16). Several intracellular loop peptides from the M2 muscarinic cholinergic receptor as well as mastoparan, a peptide that mimics activated receptors, have also been shown to activate GRK1 and GRK2 (17). Taken together, these studies reveal that GRK2 interacts with multiple domains on the intracellular surface of the receptor and suggest that agonist binding promotes a conformational change that enables the receptor to directly interact with and activate GRK2. However, the specific interaction between GRK2 and receptor remains poorly characterized both in terms of binding determinants and mechanism of action.

Three {alpha}2-adrenergic receptor ({alpha}2AR) subtypes have been cloned: {alpha}2AAR, {alpha}2BAR, and {alpha}2CAR (18–20). Activation of these receptors by epinephrine and norepinephrine in the central nervous system results in the suppression of pain perception, sedation, and lowering of blood pressure (21). These receptors are expressed in the central and peripheral nervous system and modulate a variety of pathways through coupling with Gi resulting in the inhibition of adenylyl cyclase, suppression of voltage-gated Ca2+ channels, and activation of receptor operated K+ channels (20, 22). {alpha}2ARs also activate Ras (23), the mitogen-activated protein kinase cascade (24), and phospholipase D (25). {alpha}2AAR and {alpha}2BAR are phosphorylated by GRK2 in an agonist-dependent manner, and in particular the phosphorylation of the {alpha}2AAR occurs at four adjacent serines in the third intracellular loop (26, 27). Interestingly, previous studies have revealed that the {alpha}2AAR third intracellular loop interacts with a number of proteins including arrestins (28, 29), heterotrimeric G proteins (30), spinophilin (31), and 14-3-3 (32). In fact, spinophilin has been suggested to antagonize GRK2-promoted phosphorylation of the {alpha}2AAR (33).

Much work has focused on the interactions between GPCRs and G proteins; however, far less is known in regard to the interaction of GPCRs and GRKs. How do GRKs recognize activated receptors and what is the molecular mechanism involved in GRK activation? To begin to address these questions, we used the {alpha}2AAR as a model to identify regions that interact with GRK2. We generated glutathione S-transferase (GST) fusion proteins of various regions of the {alpha}2AAR and used them in direct binding assays with purified GRK2. Through these studies we have defined four specific regions that bind to GRK2. Truncation and site-directed mutagenesis revealed the importance of several basic residues in {alpha}2AAR/GRK2 interaction. Furthermore, disruption of GRK2 binding led to decreased activation of the kinase and attenuated phosphorylation of the receptor. These studies provide important insight into the mechanism of interaction and activation of GRK2 by the {alpha}2AAR and suggest that GRK2 binding is not only critical for receptor phosphorylation but also for full activity of the kinase.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Plasmid Construction—Full-length FLAG-tagged {alpha}2AAR was cloned into pcDNA3 as described previously (34). The first (residues 61–71), second (residues 130–149), and third (residues 218–375) intracellular loops and the C terminus (residues 431–451) of the {alpha}2AAR were amplified by PCR using the full-length human {alpha}2AAR cDNA as a template. PCR products were digested with EcoRI and SalI, purified, and inserted in-frame into EcoRI/SalI-digested pGEX5X-1. The DNA sequence was verified by dideoxy chain sequence analysis. Truncation mutants of the third intracellular loop were prepared as described above and cloned into pGEX5X-1: residues 218–292, 218–245, 218–232, 224–291, 292–324, 300–324, 324–349, 349–375, 358–375, and 361–375. Point mutations of {alpha}2AAR were generated through oligonucleotide-directed PCR mutagenesis in constructs 218–245 (R218A, R225A, P242A, P243A), 300–324 (K320A, R322A) and 358–375 (K358A, R361A, K370A, R368A, K371A). The mutant R225A/K320A/K358A was generated in the complete third loop by nested PCR. The N terminus of the third loop was made using a forward mutagenic primer containing the R225A mutation and reverse mutagenic primer with the K320A mutation using the full-length {alpha}2AAR as the template. The C terminus of the third loop was generated with a forward mutagenic primer with K320A and reverse mutagenic primer with K358A using the full-length {alpha}2AAR as the template. The PCR products of the N and C-terminal reactions (100 ng each) were then utilized as template using the forward R225A primer and K358A reverse mutagenic primers for amplification. The PCR product was digested with EcoRI and SalI and subsequently cloned into pGEX5X-1. The QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene) was used according to the manufacturer's recommendations to generate the mutant R225A/K320A/K358A in the holo-{alpha}2AAR. Briefly, PCR amplification was performed using pcDNA3-{alpha}2AAR as the template, the three mutagenic primers (12.5 pmol each) R225A (5'-tac cag atc gcc aag gcg cgc acc cgc gtg -3'), K320A (5'-cgc ggt ccc cgg ggc aaa ggc gcg gcc cga gcg agc-3'), and K358A (5'-gag cgc gtc ggg gct gcc gcg gcg tcg cgc tgg cgc-3'), and the following cycling parameters: 1 cycle at 95 °C for 1 min, 30 cycles of 95 °C 1 min, 55 °C 1 min, and 65 °C 11 min. PCR product was digested with DpnI for 1–2 h at 37 °C and transformed into XL-10 Gold-competent cells. DNA sequencing was used to confirm the mutant.

GRK2 Expression and Purification—GRK2 was overexpressed and purified from Sf9 insect cells as detailed previously (35). Briefly, cells were harvested by low speed centrifugation 48 h after infection. The pellet was homogenized in 20 mM Hepes, pH 7.2, 250 mM NaCl, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 0.2 mg/ml benzamidine, and 0.02% Triton X-100 and centrifuged at high speed. The supernatant was diluted and applied onto an SP-Sepharose column, washed, and eluted with a 50–300 mM NaCl gradient. Peak fractions were pooled, loaded onto a heparin-Sepharose CL-6B column, and eluted with a 100–600 mM NaCl gradient. Peak fractions were pooled, aliquoted, and stored at –80 °C. Purity was determined by SDS-PAGE and Coomassie Blue staining.

GST-{alpha}2AAR Fusion Protein Binding with Purified GRK2—GST fusion constructs were expressed and purified as described previously (29). 5–10 µg of GST or GST-{alpha}2AAR fusion proteins immobilized on glutathione-agarose beads were incubated with 25–500 nM purified GRK2 in 100 µl of binding buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 2 mM MgCl2, and 0.02% Triton X-100) at 4 °C for 1 h. Beads were centrifuged (1000 x g) for 1 min and washed three times with binding buffer. Bound GRK2 was eluted with SDS sample buffer, electrophoresed on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and detected by immunoblotting using a GRK2/3 monoclonal antibody (Upstate Biotechnology) at 1:10,000 dilution. Blots were scanned and quantitated by densitometry.

Cell Culture and Transfection—COS-1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate at 37 °C in a humidified atmosphere containing 5% CO2. COS-1 cells grown to 50–75% confluence in 15-cm dishes were transfected with either wild-type or mutant (R225A/K320A/K358A) FLAG-tagged pcDNA3-{alpha}2AAR using FuGENE 6 according to the manufacturer's protocol. Briefly, 72 µl of FuGENE 6 was incubated with 2 ml of Dulbecco's modified Eagle's medium for 5 min at room temperature; 12 µg of pcDNA3-{alpha}2AAR DNA was then added and incubated for 20 min, and this mixture was added to the COS-1 cells and incubated for 48 h at 37 °C.

Preparation of Urea-treated Membranes—COS-1 cells were harvested 48 h after transfection by washing twice with PBS and then were scraped into 6 ml of ice cold buffer A (20 mM Hepes, pH 7.5, 1 mM MgCl2, 2 mM EDTA, 3 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin) per 15-cm dish. The cells were centrifuged at 200 x g for 10 min, and the pellet was homogenized using a polytron and then centrifuged at 100,000 x g for 30 min. The pellet was resuspended in buffer A and centrifuged again at 100,000 x g. The final pellet was resuspended in 6 ml of ice-cold buffer A containing 5 M urea and sonicated on ice until the pellet was resuspended (~3 x 15 s). The sample was centrifuged at 100,000 x g for 30 min, and the pellet was washed six times with buffer A to remove urea. The final pellet was resuspended in buffer A by Dounce homogenization, assayed for protein and ligand binding, and then snap-frozen in liquid nitrogen and stored at –80 °C. Crude membranes were prepared using this membrane-stripping procedure to enable us to evaluate agonist-dependent phosphorylation of {alpha}2AAR by GRK2. Pretreatment of membranes with urea effectively strips away peripheral membrane proteins and enriches the {alpha}2AAR in membranes.

Radioligand Binding Assays—{alpha}2AAR expression in urea-treated COS-1 membranes was determined by radioligand binding using the {alpha}2AR selective antagonist [3H]yohimbine. Briefly, 5–10 µg of membrane protein was incubated with 20 nM [3H]yohimbine in the absence or presence of 100 µM yohimbine in 75 mM Tris-HCl, pH 7.5, 12 mM MgCl2, and 2 mM EDTA for 1 h at room temperature. Receptor-bound ligand was separated from free radioligand by vacuum filtration over GF/C filters and washing with ice-cold 20 mM Tris-HCl, pH 7.5, 2 mM EDTA buffer. Filters were mixed with scintillation fluid and counted.

Phosphorylation of {alpha}2AAR—Urea-treated membranes containing 20 nM {alpha}2AAR (~100 pmol of {alpha}2AAR/mg of protein) was incubated with 5–10 nM GRK2 at 30 °C for 15–30 min in the presence or absence of epinephrine (100 µM) in 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM MgCl2, 0.2 mM ATP, 1–2 µCi of [{gamma}32P]ATP in a final volume of 20 µl. Reactions were stopped by the addition of 5 µl of SDS sample buffer and incubation at room temperature for 30 min. Samples were then electrophoresed on a 10% SDS-polyacrylamide gel, the gel was dried, and 32P-labeled receptors were visualized by autoradiography and quantified by excising the bands and scintillation counting.

To assess phosphorylation of various GST-{alpha}2AAR fusion proteins, the proteins were purified on glutathione beads, eluted with 20 mM glutathione, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, and dialyzed overnight in 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 50 mM NaCl. The purified fusions (2 µM final concentration) were incubated for 30 min with 10 nM GRK2, 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM MgCl2, 0.2 mM ATP, and 1–2 µCi of [{gamma}32P]ATP in a final volume of 20 µl. Reactions were stopped by the addition of 5 µl of SDS sample buffer and subjected to SDS-PAGE. 32P-Labeled fusion proteins were visualized by autoradiography and quantified by excising the bands and scintillation counting.

Activation of GRK2 by {alpha}2AAR—The peptide RRRASAAASAA was synthesized using t-butoxycarbonyl chemistry with an Applied Biosystems 430A peptide synthesizer. The peptide was purified by high pressure liquid chromatography on a Dynamax C-18 reverse phase column using 0–20% acetonitrile gradient in 0.1% trifluoroacetic acid. A stock solution of the purified synthetic peptide was prepared, and the pH was adjusted to 7.4 by the addition of Tris base. Activation assays (20 µl) were carried out by incubating 1 mM synthetic peptide with 50 nM GRK2 and either purified GST-{alpha}2AAR fusion protein (2 µM) or urea-treated membranes (10–25 nM {alpha}2AAR) for 0–60 min at 30 °C. The incubations were performed in the presence or absence of 100 µM epinephrine in a buffer containing 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM MgCl2, 0.2 mM ATP, and 1–2 µCi of [{gamma}32P]ATP. Reactions were quenched by the addition of 15% trichloroacetic acid and then centrifuged for 10 min at 14,000 x g. Supernatants (10 µl) were spotted onto P81 paper and washed six times with 75 mM phosphoric acid. GRK2 activity was defined as phosphate incorporation in the peptide.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
GRK2 Primarily Binds to the Third Intracellular Loop of the {alpha}2AAR—The {alpha}2AAR contains relatively small first (11 amino acids) and second (20 amino acids) intracellular loops, a large third intracellular loop (158 amino acids), and a short C terminus (21 amino acids) (Fig. 1A). Phosphorylation of four adjacent serine residues (residues 296–299) within the third loop of the {alpha}2AAR by GRK2 is known to mediate rapid homologous desensitization (27). Although the acidotropic environment of these serines appears important for GRK2-promoted phosphorylation (36), relatively little is known about the mechanism by which GRK2 targets the activated {alpha}2AAR for phosphorylation. In an effort to identify the regions in the {alpha}2AAR that mediate GRK2 interaction, the first (I1, residues 61–71), second (I2, residues 130–149), and third (I3, residues 218–375) intracellular loops and the C terminus (CT, residues 431–451) of the {alpha}2AAR were expressed as GST fusion proteins, purified on glutathione-agarose, and assessed for their ability to bind purified GRK2 (Fig. 1B). GRK2 binds to the GST-I2 and GST-I3 loops, but not to the I1 loop or CT fusion proteins. To further characterize GRK2 binding to the I2 and I3 loops, we used various concentrations of purified GRK2 in a binding assay (Fig. 1C). At all concentrations, GRK2 interacted more effectively with the I3 loop than with the I2 loop. Analysis of these data revealed a Kd of 0.08 ± 0.02 µM for GRK2 binding to the I3 loop, whereas binding to the I2 loop did not approach saturation and thus could not be adequately fit. The Kd for binding of GRK2 to the I3 loop of the {alpha}2AAR is comparable with the Km for GRK2 phosphorylation of the {beta}2-adrenergic receptor (14). Since these results suggest that the {alpha}2AAR I3 loop provides the primary binding domain for GRK2, additional studies focused on this region of the receptor.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 1.
Analysis of GRK2 binding to the{alpha}2AAR. A, schematic diagram of the human {alpha}2AAR. Shown are the putative membrane topology and amino acid sequences of the first, second, and third intracellular loops and carboxyl tail of the {alpha}2AAR. The I3 loop contains four adjacent serine residues, denoted as S in the diagram, that are known to be specific phosphorylation sites for GRK2 (27). B, upper panel, direct binding of GRK2 to GST-{alpha}2AAR fusion proteins. The first, second, and third intracellular loops as well as the carboxyl tail (C-tail) of the {alpha}2AAR were expressed as GST fusion proteins in Escherichia coli and purified on glutathione-agarose beads. One µM purified GST or GST fusion protein was incubated with 62 nM purified GRK2 as described under "Experimental Procedures." Bound proteins were detected by SDS-PAGE and immunoblotting using a monoclonal GRK2/3 antibody. Data are representative of three independent experiments. Lower panel, a representative Ponceau S stain of the immunoblot to show comparable loading of the GST fusion proteins. C, various concentrations of GRK2 (25–500 nM) were incubated with purified GST-I3 loop and GST-I2 loop proteins and processed as described under "Experimental Procedures." A representative immunoblot is shown on the left, and the results from a densitometric analysis of three experiments is shown on the right.

 
Identification of GRK2 Binding Domains within the I3 Loop of the {alpha}2AAR—To further define the specific GRK2 binding domains within the {alpha}2AAR I3 loop, several GST-I3 loop truncation mutants were expressed, purified, and tested for GRK2 binding. This analysis identified three discrete regions within the I3 loop that bind GRK2 (Fig. 2B). The first region was located within the N terminus of the I3 loop (residues 218–245) and was shown to lose binding when truncated further (residues 218–232) (Fig. 2A). This implicates a role for residues 233–245 in GRK2 interaction. However, since a construct containing this latter region (residues 224–291) did not bind GRK2 there may be two subregions within the 218–245 construct that mediate GRK2 interaction. The second GRK2 binding region was positioned in the central part of the I3 loop (residues 300–324) and was adjacent to the known phosphorylation sites on the {alpha}2AAR (serines 296–299). The third binding domain was located in the C-terminal portion of the I3 loop (residues 358–375). Interestingly, truncation of three additional residues from this construct (to residues 361–375) completely abolished GRK2 binding. The membrane-proximal regions of the I3 loop seem to play a prominent role in agonist-dependent protein interactions and also to mediate interaction with arrestin (29) and G protein (30). Taken together, these studies reveal that there are at least three distinct regions within the I3 loop of the {alpha}2AAR that are involved in binding GRK2.



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 2.
GRK2 binds three distinct regions in the {alpha}2AAR I3 loop. A, GST-I3 loop fusion proteins were incubated with 62 nM purified GRK2 as described under "Experimental Procedures." Bound proteins were detected by SDS-PAGE and immunoblotting using a monoclonal GRK2/3 antibody. Shown is a representative of 3–4 experiments performed. B, depiction of the GST-I3 loop {alpha}2AAR constructs used in binding assays with purified GRK2. Comparative binding is represented relative to full-length I3 loop binding as follows: ++++, 80–100%; +++, 60–80%; ++, 30–60%; +, 10–30%; –, <10%.

 
To identify specific residues within these three regions that are critical for GRK2 binding, we engineered a series of alanine point mutations within each of the three binding regions (Fig. 3 and Table I). Several mutations within the GST-{alpha}2AAR-(218–245) fusion protein significantly reduced GRK2 binding including R218A, R225A, and R226A, whereas mutation of Lys-224, Pro-242, and Pro-243 had minimal effect (Table I). The R225A mutant was particularly effective in attenuating GRK2 binding (Fig. 3B). Mutation of the acidic residues (Asp-292, Glu-294, Glu-295) important for GRK2 phosphorylation of the {alpha}2AAR had no effect on binding to GST-{alpha}2AAR-(292–324). Mutation of Lys-318 was also without effect, whereas mutation of Lys-320 or Arg-322 completely disrupted binding (Table I). In the C-terminal binding region, mutation of Lys-358 almost completely attenuated binding, whereas mutation of additional basic residues (Arg-361, Arg-368, Lys-370, Arg-371) had minimal effect on GRK2 binding (Table I). It is also worth noting that the putative BXBB G protein binding and activation motif in the {alpha}2AAR (Arg-368, Lys-370, Arg-371) does not appear to play a role in GRK2 binding. Taken together, these studies demonstrate that selective point mutations within the three binding regions effectively disrupt GRK2 interaction.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 3.
Identification of specific residues critical for GRK2 binding in the {alpha}2AAR I3 loop. A, the amino acid sequences of the three regions in the {alpha}2AAR I3 loop that bind GRK2. The bold-faced amino acids were individually mutated to alanine. The BXBB motif in construct 358–375 represents the activation motif for G proteins. B, GST-I3 loop fusion proteins were incubated with 62 nM purified GRK2 as described under "Experimental Procedures," Bound proteins were detected by SDS-PAGE and immunoblotting using a monoclonal GRK2/3 antibody. Data are representative of three independent experiments.

 


View this table:
[in this window]
[in a new window]
  		TABLE I
Characterization of GRK2 binding to 13 loop constructs

Various mutants were generated as described under "Experimental Procedures." Binding assays with purified GRK2 were performed as detailed under "Experimental Procedures." Shown in bold are the three discrete GRK2 binding regions, and below each region are different single-residue mutations. Densitometric scanning was used to quantify the blots. The data were normalized using each of the three wild-type binding domains as 100%. Data shown are the mean of 3-5 experiments.

 
GRK2 Phosphorylation of Wild-type and Mutant {alpha}2AAR I3 Loops—To evaluate whether the residues found to disrupt GRK2 interaction with the various I3 loop constructs also disrupted phosphorylation of the I3 loop, we generated a GST-mutant I3 loop (R225A, K320A, K358A) through nested PCR and used this construct in a phosphorylation assay with purified GRK2. The mutant I3 loop was severely impaired in its ability to be phosphorylated by GRK2 compared with the wild-type I3 loop (Fig. 4), demonstrating a correlation between binding and phosphorylation. The three point mutations (R225A, K320A, K358A) were next introduced into the holo-{alpha}2AAR, and the receptor was then tested as a substrate for GRK2. COS-1 cells were transfected with either wild-type or mutant {alpha}2AAR, and membranes were prepared and treated with 5 M urea, a procedure previously used to study GRK phosphorylation of muscarinic receptors (37). The mutant {alpha}2AAR (118 pmol of receptor/mg of protein) expression was comparable with that of wild-type {alpha}2AAR (123 pmol of receptor/mg of protein), whereas control membrane preparations did not express any {alpha}2AAR. The wild-type {alpha}2AAR was phosphorylated by GRK2 in an agonist-dependent manner, whereas the phosphorylation of the mutant {alpha}2AAR was significantly attenuated (Fig. 5A). In addition, a time course experiment further demonstrated that the wild-type {alpha}2AAR was more effectively phosphorylated than the mutant receptor (Fig. 5B). At 60 min, the stoichiometries of phosphorylation were 3.1 ± 0.3 mol/mol for wild-type {alpha}2AAR and 1.3 ± 0.2 mol/mol for the mutant {alpha}2AAR. Thus, the I3 loop mutations that attenuate GRK2 binding are also effective in disrupting the phosphorylation of the receptor by GRK2.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 4.
GRK2 phosphorylation of wild-type and mutant GST-{alpha}2AAR I3 loops. A, the mutant GST-{alpha}2AAR I3 loop has the mutations R225A, K320A, and K358A, generated through nested PCR. Phosphorylation reactions were performed in a total volume of 20 µl containing either 2 µM purified wild-type (WT) or mutant GST-{alpha}2AAR I3 loop, 10 nM GRK2, 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM MgCl2, 0.2 mM ATP, and ~1–2 µCi of [{gamma}-32P]ATP. The reactions were incubated at 30 °C for the times indicated and analyzed by SDS-PAGE and autoradiography. B, after autoradiography, phosphorylated bands were excised and counted, and phosphate incorporation was determined. The mean ± S.D. from three experiments are shown.

 



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 5.
Agonist-dependent phosphorylation of wild-type and mutant {alpha}2AAR. A, urea-treated membranes containing wild-type or mutant (R225A/K320A/K358A) {alpha}2AAR were prepared as described under "Experimental Procedures." Phosphorylation reactions were performed with or without 100 µM epinephrine (Epi) in a total volume of 20 µl and contained urea-treated membranes (0.4 pmol of {alpha}2AAR), 5 nM purified GRK2, 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM MgCl2, 0.2 mM ATP, and ~1–2 µCi of [{gamma}32P]ATP. Control membranes (Ctrl) did not contain any {alpha}2AAR. The reactions were incubated at 30 °C for 30 min and analyzed by SDS-PAGE and autoradiography. The experiment was repeated three times with similar results. B, time course using urea-treated membranes containing wild-type (WT) and mutant {alpha}2AAR. Phosphorylation reactions were performed as described in A. The reactions were incubated for the times indicated and analyzed by SDS-PAGE and autoradiography. The experiment was repeated 3–4 times with similar results.

 
Activation of GRK2 by Wild-type and Mutant {alpha}2AAR—GRK2 utilizes a number of co-factors to mediate GPCR phosphorylation including acidic phospholipids and G protein {beta}{gamma} subunits (35, 38–43). In addition, GRK2 can be stimulated by binding to the agonist-occupied form of the {beta}2AR (16). The mechanism of this activation is not known. To test whether the {alpha}2AAR can also activate GRK2, we assayed the ability of GRK2 to phosphorylate the relatively poor peptide substrate RRRASAAASAA (16) in the presence or absence of various {alpha}2AAR preparations. GRK2 phosphorylation of the peptide in the presence of control membranes that did not express {alpha}2AAR was low (Fig. 6A). The urea-treated membrane preparation containing the {alpha}2AAR increased peptide phosphorylation ~3-fold above the control, suggesting that the receptor is able to bind and induce some activation of GRK2. The addition of agonist to the {alpha}2AAR promoted a further 3–4-fold increase in peptide phosphorylation above the phosphorylation of the receptor sample without epinephrine, demonstrating that the agonist-occupied {alpha}2AAR is able to effectively activate GRK2 (Fig. 6A). We next compared the ability of the wild-type and mutant {alpha}2AARs to activate GRK2. Similar to Fig. 6A, the wild-type {alpha}2AAR exhibited an ability to partially activate GRK2 in the absence of agonist, whereas the agonist-occupied receptor effectively activated GRK2 (Fig. 6B). In contrast, the ability of the mutant {alpha}2AAR to activate GRK2 was ~4-fold lower than the wild-type {alpha}2AAR (Fig. 6B). In an effort to further correlate GRK2 binding with activation, we also tested whether any of our GST-{alpha}2AAR third loop constructs activated GRK2. The GST-I3 loop, the three smaller regions within the I3 loop that bind to GRK2, and the various mutants were used in a GRK2 activation assay (Fig. 6C). Given that only small regions of the holoreceptor were used in the activation assay, we did not expect full activation of the kinase. Nevertheless, the I3 loop, as well as the three smaller regions within the I3 loop, was able to enhance peptide phosphorylation ~2 fold. In contrast, the various mutant constructs did not activate GRK2. Although we still do not fully understand the mechanism involved in GRK2 activation, these studies are the first to demonstrate that small receptor domains are able to activate GRK2 and that activation is directly correlated with binding.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 6.
Activation of GRK2 by wild-type and mutant {alpha}2AAR. A, GRK2 activation by wild-type {alpha}2AAR. The synthetic peptide RRRASAAASAA (1 mM) was incubated with 50 nM GRK2 and urea-treated COS-1 membranes (either control membranes (Ctrl) or membranes containing 0.4 pmol {alpha}2AAR) in the presence or absence of 100 µM epinephrine (Epi) in a total volume of 20 µl in 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM MgCl2, 0.2 mM ATP, and ~1–2 µCi of [{gamma}32P]ATP. Reactions were incubated at 30 °C for the times indicated, quenched by the addition of 15% trichloroacetic acid, and centrifuged for 10 min at 14,000 x g. Supernatants (10 µl) were spotted on P81 paper and washed six times with 75 mM phosphoric acid, and peptide phosphorylation was then assessed by scintillation. Data points are the mean of three experiments. B, activation of GRK2 by wild-type and mutant {alpha}2AAR. Activation assays were performed as described above except that the incubations used urea-treated membranes containing 0.4 pmol of wild-type or mutant (R225A/K320A/K358A) {alpha}2AAR and were quenched after 30 min. Data are the mean of three experiments performed in triplicate. C, activation of GRK2 by GST-I3 loop constructs. The wild-type and mutant (R225A/K320A/K358A) I3 loop and the wild type and mutant 218–245, 300–324, and 358–375 GST fusions were assayed for their ability to activate GRK2. Assays contained 1 mM RRRASAAASAA, 0.2 µM GRK2, 2 µM GST or GST-{alpha}2AAR, 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 5 mM MgCl2, 0.2 mM ATP, and ~1–2 µCi of [{gamma}32P]ATP. Reactions were incubated at 30 °C for 30 min, quenched with 15% trichloroacetic acid, centrifuged, spotted on P81 paper, washed with phosphoric acid, and counted. Data are the mean of three experiments performed in triplicate.

 
Conclusions—We have characterized four regions within the {alpha}2AAR that interact with GRK2, the I2 loop as well as three specific sites within the I3 loop. One site is located adjacent to the four serines that are phosphorylated by GRK2 (phosphorylation site), and the other sites are located at the membrane proximal regions of the I3 loop. Even though acidic residues adjacent to the serines are important for phosphorylation, these residues did not play a significant role in GRK2 binding. In addition, we identified several basic residues within these three binding domains that are critical for GRK2 interaction with the {alpha}2AAR, including Arg-225, Lys-320, and Lys-358. Mutation of these three residues effectively attenuated the ability of the {alpha}2AAR to both activate and serve as a substrate for GRK2. However, there are likely additional regions of the {alpha}2AAR involved in GRK2 binding, including the I2 loop (Fig. 1).

Recently, the x-ray crystal structure of the inactive holo-GRK2 bound to G{beta}{gamma} has been solved (44). Based on the docking analysis of GRK2 with rhodopsin, the catalytic domain of GRK2 is predicted to have many contacts with the receptor with one important site being the structurally disordered nucleotide gate. Because we determined that several basic residues in the {alpha}2AAR are important for GRK2 binding, we hypothesize that acidic residues on the kinase might contribute to receptor binding. Interestingly, the 20-amino acid nucleotide gate in GRK2 is particularly rich in acidic residues, including Glu-476, Asp-481, Asp-484, Asp-489, Glu-490, Glu-491, and Glu-492, and thus might provide potential ionic interactions with basic residues in the {alpha}2AAR. It is intriguing that the nucleotide gates within several other members of the GRK family contain an adjacent serine and threonine in place of the Asp-489, Glu-490, Glu-491, and Glu-492 found in GRK2 and GRK3. In GRK5, autophosphorylation of these residues (Ser-484 and Thr-485) enhances receptor phosphorylation 15–20-fold (45). It is also worth noting that receptor phosphorylation by GRK2 is very sensitive to inhibition by salt (46), further suggesting the importance of ionic interactions in GRK2/substrate binding. An additional region that has been implicated in GRK2 interaction with GPCRs is a proline-rich motif that is just N-terminal to the nucleotide gate (47). Mutagenesis of this proline-rich motif in GRK2 disrupted its ability to bind to light activated rhodopsin, suggesting that this region contributes to recognition of activated receptor. Further studies with GRK2 mutants should provide additional insight on the mechanism of GRK/receptor interaction.

In summary, we have shown that multiple regions of the {alpha}2AAR play a key role in substrate recognition. Mutations of Arg-225, Lys-320, and Lys-358 in the I3 loop disrupted GRK2 binding and resulted in decreased activation of GRK2 and decreased receptor phosphorylation. These results suggest that ionic interactions will likely play an important role in mediating GRK/GPCR interaction. Our findings also demonstrate that small receptor domains are able to activate GRK2 and that activation is directly correlated with binding. The membrane-proximal regions on GPCRs have been implicated in binding a number of proteins including G proteins, arrestins, and now GRKs. These regions are likely exposed upon agonist binding and are then able to interact with target proteins. This will likely serve as a general mechanism for mediating the agonist-dependent targeting of GPCRs by GRKs.


   FOOTNOTES
 
* This study was supported by National Institutes of Health Grant GM44944. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. E-mail: benovic{at}mail.jci.tju.edu.

1 The abbreviations used are: GPCR, G protein-coupled receptor; AR, adrenergic receptor; GRK, G protein-coupled receptor kinase; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
We thank members of the Benovic laboratory for continuous encouragement.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Marinissen, M. J., and Gutkind, J. S. (2001) Trends Pharmacol. Sci. 22, 368–376[CrossRef][Medline] [Order article via Infotrieve]
  2. Pierce, K. L., Premont, R. T., and Lefkowitz, R. J. (2002) Nat. Rev. Mol. Cell. Biol. 3, 639–650[CrossRef][Medline] [Order article via Infotrieve]
  3. Neer, E. J. (1995) Cell 80, 249–257[CrossRef][Medline] [Order article via Infotrieve]
  4. Krupnick, J. G., and Benovic, J. L. (1998) Annu. Rev. Pharmacol. Toxicol. 38, 289–319[CrossRef][Medline] [Order article via Infotrieve]
  5. Tsao, P. I., and von Zastrow, M. (2001) Pharmacol. Ther. 89, 139–147[CrossRef][Medline] [Order article via Infotrieve]
  6. Ferguson, S. S. (2001) Pharmacol. Rev. 53, 1–24[Abstract/Free Full Text]
  7. Kohout, T. A., and Lefkowitz, R. J. (2003) Mol. Pharmacol. 63, 9–18[Free Full Text]
  8. Pao, C. S., and Benovic, J. L. (2002) Science's STKE http://stke.sciencemag.org/cgi/content/full/sigtrans;2002/153/pe42
  9. Dicker, F., Quitterer, U., Winstel, R., Honold, K., and Lohse, M. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5476–5481[Abstract/Free Full Text]
  10. Freedman, N. J., Ament, A. S., Oppermann, M., Stoffel, R. H., Exum, S. T., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 17734–17743[Abstract/Free Full Text]
  11. Jala, V. R., Shao, W. H., and Haribabu, B. (2005) J. Biol. Chem. 280, 4880–4887[Abstract/Free Full Text]
  12. Min, L., Galet, C., and Ascoli, M. (2002) J. Biol. Chem. 277, 702–710[Abstract/Free Full Text]
  13. Carman, C. V., and Benovic, J. L. (1998) Curr. Opin. Neurobiol. 8, 335–344[CrossRef][Medline] [Order article via Infotrieve]
  14. Benovic, J. L., Onorato, J., Lohse, M. J., Dohlman, H. G., Staniszewski, C., Caron, M. G., and Lefkowitz, R. J. (1990) Br. J. Clin. Pharmacol. 30, Suppl. 1, 3S–12S
  15. Palczewski, K., Buczylko, J., Kaplan, M. W., Polans, A. S., and Crabb, J. W. (1991) J. Biol. Chem. 266, 12949–12955[Abstract/Free Full Text]
  16. Chen, C. Y., Dion, S. B., Kim, C. M., and Benovic, J. L. (1993) J. Biol. Chem. 268, 7825–7831[Abstract/Free Full Text]
  17. Haga, K., Kameyama, K., and Haga, T. (1994) J. Biol. Chem. 269, 12594–12599[Abstract/Free Full Text]
  18. Lomasney, J. W., Lorenz, W., Allen, L. F., King, K., Regan, J. W., Yang-Feng, T. L., Caron, M. G., and Lefkowitz, R. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5094–5098[Abstract/Free Full Text]
  19. Kobilka, B. K., Matsui, H., Kobilka, T. S., Yang-Feng, T. L., Francke, U., Caron, M. G., Lefkowitz, R. J., and Regan, J. W. (1987) Science 238, 650–656[Abstract/Free Full Text]
  20. Regan, J. W., Kobilka, T. S., Yang-Feng, T. L., Caron, M. G., Lefkowitz, R. J., and Kobilka, B. K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6301–6305[Abstract/Free Full Text]
  21. Ruffolo, R. R., Jr., Nichols, A. J., Stadel, J. M., and Hieble, J. P. (1993) Annu. Rev. Pharmacol. Toxicol. 33, 243–279[Medline] [Order article via Infotrieve]
  22. Limbird, L. E. (1988) FASEB J. 2, 2686–2695[Abstract]
  23. Koch, W. J., Hawes, B. E., Allen, L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12706–12710[Abstract/Free Full Text]
  24. Della Rocca, G. J., van Biesen, T., Daaka, Y., Luttrell, D. K., Luttrell, L. M., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 19125–19132[Abstract/Free Full Text]
  25. Jinsi, A., Paradise, J., and Deth, R. C. (1996) Eur. J. Pharmacol. 302, 183–190[CrossRef][Medline] [Order article via Infotrieve]
  26. Liggett, S. B., Ostrowski, J., Chesnut, L. C., Kurose, H., Raymond, J. R., Caron, M. G., and Lefkowitz, R. J. (1992) J. Biol. Chem. 267, 4740–4746[Abstract/Free Full Text]
  27. Eason, M. G., Moreira, S. P., and Liggett, S. B. (1995) J. Biol. Chem. 270, 4681–4688[Abstract/Free Full Text]
  28. Wu, G., Krupnick, J. G., Benovic, J. L., and Lanier, S. M. (1997) J. Biol. Chem. 272, 17836–17842[Abstract/Free Full Text]
  29. DeGraff, J. L., Gurevich, V. V., and Benovic, J. L. (2002) J. Biol. Chem. 277, 43247–43252[Abstract/Free Full Text]
  30. Wade, S. M., Dalman, H. M., Yang, S. Z., and Neubig, R. R. (1994) Mol. Pharmacol. 45, 1191–1197[Abstract]
  31. Richman, J. G., Brady, A. E., Wang, Q., Hensel, J. L., Colbran, R. J., and Limbird, L. E. (2001) J. Biol. Chem. 276, 15003–15008[Abstract/Free Full Text]
  32. Prezeau, L., Richman, J. G., Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 13462–13469[Abstract/Free Full Text]
  33. Wang, Q., Zhao, J., Brady, A. E., Feng, J., Allen, P. B., Lefkowitz, R. J., Greengard, P., and Limbird, L. E. (2004) Science 304, 1940–1944[Abstract/Free Full Text]
  34. DeGraff, J. L., Gagnon, A. W., Benovic, J. L., and Orsini, M. J. (1999) J. Biol. Chem. 274, 11253–11259[Abstract/Free Full Text]
  35. Kim, C. M., Dion, S. B., Onorato, J. J., and Benovic, J. L. (1993) Receptor 3, 39–55[Medline] [Order article via Infotrieve]
  36. Onorato, J. J., Palczewski, K., Regan, J. W., Caron, M. G., Lefkowitz, R. J., and Benovic, J. L. (1991) Biochemistry 30, 5118–5125[CrossRef][Medline] [Order article via Infotrieve]
  37. Debburman, S. K., Kunapuli, P., Benovic, J. L., and Hosey, M. M. (1995) Mol. Pharmacol. 47, 224–233[Abstract]
  38. Onorato, J. J., Gillis, M. E., Liu, Y., Benovic, J. L., and Ruoho, A. E. (1995) J. Biol. Chem. 270, 21346–21353[Abstract/Free Full Text]
  39. Carman, C. V., Barak, L. S., Chen, C., Liu-Chen, L. Y., Onorato, J. J., Kennedy, S. P., Caron, M. G., and Benovic, J. L. (2000) J. Biol. Chem. 275, 10443–10452[Abstract/Free Full Text]
  40. DebBurman, S. K., Ptasienski, J., Boetticher, E., Lomasney, J. W., Benovic, J. L., and Hosey, M. M. (1995) J. Biol. Chem. 270, 5742–5747[Abstract/Free Full Text]
  41. Wu, G., Bogatkevich, G. S., Mukhin, Y. V., Benovic, J. L., Hildebrandt, J. D., and Lanier, S. M. (2000) J. Biol. Chem. 275, 9026–9034[Abstract/Free Full Text]
  42. Pitcher, J. A., Fredericks, Z. L., Stone, W. C., Premont, R. T., Stoffel, R. H., Koch, W. J., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 24907–24913[Abstract/Free Full Text]
  43. Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264–1267[Abstract/Free Full Text]
  44. Lodowski, D. T., Pitcher, J. A., Capel, W. D., Lefkowitz, R. J., and Tesmer, J. J. (2003) Science 300, 1256–1262[Abstract/Free Full Text]
  45. Kunapuli, P., Gurevich, V. V., and Benovic, J. L. (1994) J. Biol. Chem. 269, 10209–10212[Abstract/Free Full Text]
  46. Benovic, J. L., Mayor, F., Jr., Staniszewski, C., Lefkowitz, R. J., and Caron, M. G. (1987) J. Biol. Chem. 262, 9026–9032[Abstract/Free Full Text]
  47. Gan, X., Ma, Z., Deng, N., Wang, J., Ding, J., and Li, L. (2004) J. Biol. Chem. 279, 49741–49746[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
S. U. Miedlich and A. B. Abou-Samra
Eliminating phosphorylation sites of the parathyroid hormone receptor type 1 differentially affects stimulation of phospholipase C and receptor internalization
Am J Physiol Endocrinol Metab, September 1, 2008; 295(3): E665 - E671.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
T. Kohno and Y. Igarashi
Attenuation of cell motility observed with high doses of sphingosine 1-phosphate or phosphorylated FTY720 involves RGS2 through its interactions with the receptor S1P.
Genes Cells, July 1, 2008; 13(7): 747 - 757.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Parameswaran, C. S. Pao, K. S. Leonhard, D. S. Kang, M. Kratz, S. C. Ley, and J. L. Benovic
Arrestin-2 and G Protein-coupled Receptor Kinase 5 Interact with NF{kappa}B1 p105 and Negatively Regulate Lipopolysaccharide-stimulated ERK1/2 Activation in Macrophages
J. Biol. Chem., November 10, 2006; 281(45): 34159 - 34170.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
N. D. Khoa, M. Postow, J. Danielsson, and B. N. Cronstein
Tumor Necrosis Factor-{alpha} Prevents Desensitization of G{alpha}s-Coupled Receptors by Regulating GRK2 Association with the Plasma Membrane
Mol. Pharmacol., April 1, 2006; 69(4): 1311 - 1319.
[Abstract] [Full Text] [PDF]

Home page
 Protein Eng Des SelHome page
D. Japrung, S. Chusacultanachai, J. Yuvaniyama, P. Wilairat, and Y. Yuthavong
A simple dual selection for functionally active mutants of Plasmodium falciparum dihydrofolate reductase with improved solubility
Protein Eng. Des. Sel., October 1, 2005; 18(10): 457 - 464.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. K. Dhami, A. V. Babwah, R. Sterne-Marr, and S. S. G. Ferguson
Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 1 Signaling Requires G Protein-coupled Receptor Kinase 2 Binding to the Second Intracellular Loop
J. Biol. Chem., July 1, 2005; 280(26): 24420 - 24427.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow<