©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of the N-Formyl Peptide Receptor Carboxyl Terminus by the G Protein-coupled Receptor Kinase, GRK2 (*)

(Received for publication, September 13, 1994; and in revised form, November 11, 1994)

Eric R. Prossnitz (1)(§) Chong M. Kim (2) Jeffrey L. Benovic (2) Richard D. Ye (1)

From the  (1)Department of Immunology, The Scripps Research Institute, La Jolla, California 92037 and the (2)Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Attenuation of receptor-mediated signal amplification in response to external stimuli, an essential step in the balance of cellular activation, may be mediated by receptor phosphorylation. We have recently shown that the carboxyl-terminal cytoplasmic domain of the N-formyl peptide receptor (FPR) interacts with G proteins and demonstrate here that this same region of the FPR is specifically phosphorylated by a neutrophil cytosolic kinase with properties similar to the G protein-coupled receptor kinase, GRK2. Both kinase activities show a lack of sensitivity toward protein kinase A, protein kinase C, and tyrosine kinase inhibitors but demonstrate almost identical sensitivity toward the kinase inhibitor heparin. Kinetic studies demonstrated that GRK2 has a K for the carboxyl-terminal domain of the FPR of approximately 1.5 µM and that denaturation of the substrate results in an almost complete loss of phosphorylation. Comparative studies reveal that GRK3 has approximately 50% of the activity of GRK2 toward the FPR carboxyl terminus, whereas GRK5 and GRK6 have no detectable activity. Site-directed mutagenesis of numerous regions of the FPR carboxyl terminus demonstrated that, whereas Glu/Asp and Asp are critical for phosphorylation, the carboxyl-terminal 10 amino acids are not required. Simultaneous substitution of Thr, Thr, Ser, and Thr resulted in an 50% reduction in phosphorylation, whereas simultaneous substitution of the upstream Ser, Thr, Thr, and Ser or merely the Ser and Thr residues resulted in an 80% reduction in phosphorylation. The introduction of negatively charged glutamate residues for Ser and Thr or Thr and Ser resulted in marked stimulation of phosphorylation. These results suggest a hierarchical mechanism in which phosphorylation of amino-terminal serine and threonine residues is required for the subsequent phosphorylation of carboxyl-terminal residues. These results provide the first direct evidence that an intracellular domain of a chemoattractant receptor is a high affinity substrate for GRK2 and further suggest a role for GRK2 or a closely related kinase in the attenuation of receptor-mediated activation of inflammatory cells.


INTRODUCTION

Neutrophils respond to a large number of structurally diverse ligands with functions such as chemotaxis, superoxide production, and degranulation. Ligands are recognized through their binding to cell surface receptors, which results in the stimulation of phospholipases and the mobilization of intracellular calcium(1) . Many of these chemotactic receptors have recently been cloned, including the receptors for N-formyl peptides(2) , complement component C(3) , platelet-activating factor(4) , IL-8 (5, 6) and homologous receptors for which the ligand has not yet been determined (7) . These receptors are all members of the class of receptors coupled to GTP-binding regulatory proteins (G proteins^1; for a review, see Refs. 8 and 9). The precise regulation of neutrophil responses is crucial due to the cytotoxic effects of superoxide and degradative enzymes on host tissues. Following an initial exposure to ligand, neutrophils rapidly but reversibly become unresponsive or desensitized to subsequent stimulation with either the same or unrelated chemoattractant(10, 11) . Multiple mechanisms may contribute to desensitization, including receptor phosphorylation, endocytosis, and the down-regulation of receptor expression(12) .

G protein-coupled receptor kinases (GRKs) are a rapidly growing family of protein kinases which phosphorylate their respective ligand-bound receptors on Ser/Thr-containing intracellular domains(13, 14, 15, 16) . Once phosphorylated, these receptors may bind accessory proteins, termed arrestins, which further uncouple the receptor from the G protein(17) . G proteincoupled receptor kinases were originally implicated in the light-stimulated phosphorylation of rhodopsin (18) and the agonist-dependent phosphorylation of the beta(2)-adrenergic receptor (19) and are presumed to be an essential component in the homologous desensitization of multiple receptors. GRK2 (also termed beta-adrenergic receptor kinase) has a relatively broad tissue distribution, but has been shown to be particularly abundant in peripheral blood leukocytes as well as myeloid and lymphoid cell lines (20) . Furthermore, platelet-activating factor was able to stimulate GRK2 translocation from the cytosol to the membrane of mononuclear cells(20) , where it may be anchored by the beta subunits of G proteins(21, 22) . This is proposed to be an initial step in the process of GRK2-dependent desensitization and to provide a mechanism for increasing the selectivity of GRK2 for its receptor substrates. Recently, the FPR has been demonstrated to become rapidly phosphorylated in a dose-dependent manner(23, 24) . Pretreatment of the cells with staurosporine, a protein kinase C inhibitor, failed to prevent the ligand-stimulated phosphorylation of the receptor, suggesting that a kinase other than protein kinase C was responsible for the observed phosphorylation.

In order to investigate whether ligand-dependent FPR phosphorylation and desensitization could be a result of phosphorylation of the Ser/Thr-rich carboxyl terminus of the FPR, we utilized a fusion protein containing the carboxyl-terminal 47 amino acids of the FPR, which we have previously shown interacts specifically with G proteins(25) . In this report, we provide the first direct evidence that 1) both serine and threonine residues within the carboxyl terminus of the FPR are phosphorylated by a neutrophil cytosolic kinase with properties similar to GRK2; 2) GRK2 has an affinity for the cytoplasmic tail of the FPR approaching that of the liganded beta-adrenergic receptor; 3) phosphorylation by GRK2 likely proceeds in a sequential manner with phosphorylation of residues between 334 and 339 being dependent on prior phosphorylation of residues between 328 and 329; and 4) introduction of negatively charged residues in the native receptor sequence stimulates subsequent phosphorylation of downstream residues.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes and T4 DNA ligase were from Life Technologies, Inc. [-P]ATP and alpha-S-dATP were from DuPont NEN. Oligonucleotides were obtained from Operon. Taq polymerase was purchased from Perkin Elmer-Cetus. Mutagenesis was carried out using the Amersham oligonucleotide-directed mutagenesis system. Sequencing was carried out using Sequenase version 2.0 (U. S. Biochemical Corp.). Reduced glutathione and glutathione-agarose beads were from Sigma. Staurosporine C, genistein, and H9 were obtained from Calbiochem. Bacterial cells were grown in Circlegrow medium (BIO 101).

Fusion Protein Synthesis and Purification

FPR cDNA (26) encoding residues 303 to 350, which comprise the three carboxyl-terminal-most amino acids from the seventh transmembrane region in addition to the entire cytoplasmic tail (see Fig. 1), was amplified by polymerase chain reaction using the following primers: 5`-TAGAATTCATGGGCCAGGACTTCCG and 5`-ACGAATTCTATTACTTTGACTGTAACTC (underlined sequences represent EcoRI restriction sites, bold sequences represent tandem stop codons). This fragment was isolated and cloned into the protein fusion vector pGEX-1T (Pharmacia Biotech) at the EcoRI restriction site. After transformation of the Escherichia coli strain DH5alpha/F` with recombinant pGEX-1T, an overnight culture of bacteria expressing the fusion protein was diluted 1:100 and grown at 37 °C to an A of 1.0. Subsequently, bacteria were induced with 1 mM isopropyl-1-thio-beta-D-galactopyranoside for 2-3 h at 37 °C, followed by centrifugation at 4000 times g. Bacterial pellets were resuspended in 50 ml of lysis buffer (10 mM NaP(i), 2.5 mM EDTA, 2.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, pH 7.0), followed by nitrogen cavitation (Parr Instruments) after equilibration at 550 p.s.i. for 15 min. After centrifugation of the cell lysate at 15,000 times g, the resulting supernatant was purified by binding to glutathione-agarose beads and elution with reduced glutathione when required. Fusion protein was dialyzed extensively against 1 mM NaP(i), pH 7.0, and, when isolated by this method, was judged to be >99% pure by SDS-polyacrylamide gel electrophoresis. Verification that the purified protein contained the complete cytoplasmic tail was obtained by immunoblotting with an anti-peptide antibody directed against the last 12 residues of the carboxyl terminus of the FPR(27, 28) .


Figure 1: Amino acid similarity between the carboxyl-terminal domains of three chemoattractant receptors. The amino acid sequences of the carboxyl-terminal domains of the FPR, C receptor, and IL8 receptor B are aligned so as to optimize similarity with the introduction of a minimal number of gaps. The following groups of amino acids were considered similar in overall properties: Ser and Thr; Asp and Glu; Lys, Arg, and His; Ala and Gly; Ala and Val; Leu, Ile, and Val.



Construction of Site-directed Mutants

The FPR gene was subcloned into the EcoRI site in the polylinker of M13mp18. Single-stranded M13 DNA, in conjunction with mutant oligonucleotides (containing the altered nucleotides necessary to generate the desired mutations), was used to introduce the desired mutations into the FPR gene. Plaques were amplified and the mutations were confirmed by dideoxy sequencing. Using the polymerase chain reaction primers described above, the sequences of the carboxyl-terminal 47 amino acids of the mutant FPR genes were amplified and cloned into pGEX-1T. The sequence was reconfirmed by dideoxy sequencing prior to expression and purification of the mutant fusion proteins.

Purification and Assay of G Protein-coupled Receptor Kinases

Bovine GRK2 was overexpressed and purified from infected Sf9 insect cells using the baculovirus expression system(29) . Briefly, Sf9 cells were harvested 48 h postinfection by low speed centrifugation. The cells were washed and subsequently homogenized in 20 mM HEPES, pH 7.2, 250 mM NaCl, 5 mM EDTA, 3 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine (50 ml of buffer per liter of cells). A high speed supernatant fraction was then diluted with buffer A (20 mM HEPES, pH 7.2, 5 mM EDTA, 0.02% Triton X-100), loaded onto an S-Sepharose column, and then eluted with a linear gradient from 50 to 300 mM NaCl in buffer A. Peak fractions were pooled, diluted, loaded onto a heparin-Sepharose column, and eluted with a linear gradient from 100 to 600 mM NaCl in buffer A. The specific activity of the GRK2 preparations used in these studies was 1 µmol/min/mg of protein using rhodopsin as the substrate. Other GRKs were similarly purified(29, 30, 31, 32) .

Phosphorylation was assayed as follows. Purified, eluted fusion protein, at the indicated concentration as determined with the BCA reagent (Pierce), was incubated with purified GRK in assay buffer (20 mM Tris-Cl, pH 7.2, 2 mM EDTA, 7 mM MgCl(2)) containing 0.1 mM [-P] ATP (1.5 fmol/cpm) in a final volume of 30 µl at 30 °C for the indicated time. Phosphorylation reactions with glutathione-agarose-bound fusion proteins were performed as follows. Fusion protein bound beads (containing 5-10 µg of protein as judged by Coomassie Blue staining of polyacrylamide gels) were incubated with either neutrophil cytosol extract (from 10^6 cell equivalents) or purified GRK (100 ng) in a final volume of 300 µl of assay buffer on ice for 60 min. The beads were centrifuged, washed three times with assay buffer, and brought to 50 µl with assay buffer. [-P]ATP (1-2 µCi) was added, and the sample was incubated at 30 °C for 20 min. All reactions were quenched with SDS sample buffer followed by electrophoresis on a 12.5% polyacrylamide gel. Gels were stained with Coomassie Blue and subsequently exposed to x-ray film for visualization of the P-labeled proteins. Relative determinations of protein and P content were performed with a Molecular Devices Densitometer and PhosphorImager, respectively. Absolute determinations of P were accomplished by cutting out the bands from the polyacrylamide gels and counting by liquid scintillation.

Phosphoamino acid content was determined as described(33) . Briefly, following transfer of the SDS gel to Immobilon P membrane (Millipore), the fusion protein band was excised and hydrolyzed in 6 M HCl for 1 h at 100 °C. The sample was dried and resuspended with 0.5 µg each of phosphoserine, phosphothreonine, and phosphotyrosine and spotted onto a cellulose thin layer plate (100 µm, EM Laboratories). Phosphoamino acids were separated by chromatography in 5:3 isobutyric acid, 0.5 M ammonium hydroxide. Unlabeled phosphoamino acid standards were visualized with ninhydrin, whereas P-labeled phosphoamino acids were visualized with a Molecular Devices PhosphorImager.


RESULTS

Chemoattractant receptors undergo a functional uncoupling from G proteins following exposure to agonist, likely contributing to the process of desensitization(10, 11) . The recent cloning of numerous chemoattractant receptors reveals that they possess strong structural homology, belonging to the class of seven transmembrane-spanning G protein-coupled receptors(8) . Furthermore, these receptors appear to belong to a subclass of G protein-coupled receptors of small size containing approximately 350 amino acids(34) . This compares to the well characterized adrenergic and muscarinic receptors whose larger size (from approximately 400 to almost 600 amino acids) is primarily due to larger intracellular domains. Related chemoattractant receptors share higher homology in their intracellular domains as compared to their extracellular domains, suggesting that they may share common mechanisms of signal transduction and receptor regulation(4) . A comparison of the carboxyl-terminal tail of three chemoattractant receptors is presented in Fig. 1. The overall homology between any pair of these receptors is approximately 50% with regions as high as 75% over stretches of 17-19 amino acids. The highest homology is in the central third of this domain, whereas the lowest is in the last third of the carboxyl-terminal tail. Of note is the almost complete lack of serine and threonine residues in the first half of the tail sequences and preponderance of these residues in the second half of the tail. Preceding and within the Ser/Thr-rich region are numerous conserved acidic residues, less common in the other portions of the tail.

Given the recent observations of ligand-dependent phosphorylation of the FPR (23, 24) and the determination of high expression levels of human GRK2 in peripheral blood leukocytes and HL60 cells(20) , we asked whether the Ser/Thr-rich carboxyl terminus of the FPR could serve as an effective substrate for an endogenous neutrophil kinase. For this purpose we utilized a fusion protein containing the carboxyl-terminal 47 amino acids of the FPR expressed at the carboxyl-terminal end of glutathione S-transferase (GST-FPR). We have previously shown that such a fusion protein is capable of interacting with G(i) proteins and that the amino-terminal two-thirds of this region of the FPR is of critical importance(25) .

GST-FPR bound to glutathione-agarose beads was incubated with a neutrophil cytosol extract followed by extensive washing and subsequent incubation in the presence of [-P]ATP (Fig. 2, panel B). Analysis by SDS-polyacrylamide gel electrophoresis demonstrated the presence of a phosphorylated protein (lane 1) corresponding to the position of the Coomassie Blue-stained fusion protein (Fig. 2, panel A, lane 1). The specificity of the phosphorylation (Fig. 2, panels B and C) was demonstrated by the negligible phosphorylation of the glutathione S-transferase protein lacking the 47-amino acid FPR tail (but containing the sequence IVTD in place of the FPR tail, lane 2). We next attempted to determine the class of kinase responsible for the observed phosphorylation through the use of specific kinase inhibitors. Phosphorylation of GST-FPR by the neutrophil extract kinase activity (Fig. 2, panel B) was carried out in the presence of staurosporine to inhibit protein kinase C (lane 3), H9 to inhibit protein kinase A (lane 4), heparin to inhibit G protein-coupled receptor kinases (lane 5), and herbimycin A to inhibit tyrosine kinases (lane 6). Of these, the only inhibitor to have an effect was heparin, suggesting the involvement of a G protein-coupled receptor kinase. To test this possibility directly, purified GRK2 was substituted for the crude neutrophil extract in the phosphorylation assay (Fig. 2, panel C). The results demonstrate an identical pattern of phosphorylation as observed with the crude extract, namely specific phosphorylation of the GST-FPR fusion protein as compared with the GST-only construct and inhibition by heparin but not the other kinase inhibitors.


Figure 2: Phosphorylation of the immobilized FPR carboxyl terminus. Fusion protein, either containing or lacking the carboxyl-terminal 47 amino acids of the FPR (lanes 1, 3, 4, 5, and 6 or lane 2, respectively), was bound to glutathione-agarose beads and purified by extensive washing. Bound fusion protein was then incubated with either a crude neutrophil extract (panel B) or GRK2 (panel C), in a mixture containing 20 mM Tris-Cl, pH 7.2, 2 mM EDTA, 7 mM MgCl(2) and washed extensively. The beads were then incubated with 1-2 µCi of [-P]ATP in a final volume of 50 µl of the same buffer at 30 °C for 60 min. Prior to the addition of [-P]ATP, the following additions were made: staurosporine (300 nM final, lane 3); H9 (20 µM final, lane 4); heparin (1 µM final, lane 5), and herbimycin A (2 µM final, lane 6). Reactions were quenched with SDS sample buffer followed by electrophoresis on a 12.5% polyacrylamide gel. The gel was stained with Coomassie Blue (panel A) and subsequently exposed to x-ray film for visualization of the P-labeled proteins (panels B and C). The two arrows to the right of each panel indicate the positions of the GST-FPR fusion protein (upper arrow) and the GST fusion protein lacking the FPR carboxyl terminus (lower arrow). The data are representative of three experiments.



To compare the activity found in the neutrophil extract with that of a purified G protein-coupled receptor kinase, we determined the sensitivity of these two activities to the polyanionic inhibitor heparin. GST-FPR bound to agarose beads was assayed for phosphorylation in the presence of increasing concentrations of heparin (Fig. 3). Both activities were inhibited by heparin with IC values of 0.1-1 µM. These values are very similar to the IC for heparin previously reported using light-activated rhodopsin as the substrate (35) and suggest that the activity observed in the neutrophil extract may be due to a G protein-coupled receptor kinase. We next determined if the characteristics of the glutathione-agarose-bound fusion proteins extended to the eluted, soluble fusion proteins. Following elution with reduced glutathione, the GST-FPR and GST proteins were dialyzed extensively to remove free glutathione and exchange the buffer. Fig. 4shows that the GST-FPR protein is also a substrate for GRK2 in an entirely soluble system (lane 1). As with the glutathione-agarose-bound fusion protein, the protein lacking the carboxyl terminus of the FPR was not significantly phosphorylated (Fig. 4, lane 3). Phosphorylation carried out under these conditions was also sensitive to heparin (lane 2), but not the other protein kinase inhibitors characterized in Fig. 2(data not shown). Prolonged phosphorylation with increased amounts of GRK2 yielded GST-FPR fusion protein which was phosphorylated to an extent of 2-3 mol of phosphate/mol of fusion protein. This compares to the maximal levels of phosphorylation observed with the beta-adrenergic receptor using similarly isolated GRK2, assayed in the absence of additional stimulatory factors, such as G protein beta subunits(22) .


Figure 3: Sensitivity of phosphorylation of the FPR carboxyl terminus to heparin. Fusion protein bound to glutathione-agarose beads was assayed for phosphorylation by either the crude neutrophil extract (bullet) or GRK2 () as described in the legend to Fig. 2. Heparin was added at the indicated concentration prior to the addition of [-P]ATP. Quantitation of the P-labeled proteins was carried out with a Molecular Devices PhosphorImager. The data represent the mean and standard error from three experiments.




Figure 4: Phosphorylation of the soluble GST-FPR by GRK2. Fusion protein (0.5 µM), either containing or lacking the carboxyl-terminal 47 amino acids of the FPR (GST-FPR or GST, respectively) was incubated with GRK2 (62.5 nM) and heparin (1 µM) as indicated in a mixture containing 20 mM Tris-Cl, pH 7.2, 2 mM EDTA, 7 mM MgCl(2), and 0.1 mM [-P]ATP (1.5 fmol/cpm) in a final volume of 30 µl at 30 °C for 60 min. Reactions were quenched with SDS sample buffer followed by electrophoresis on a 12.5% polyacrylamide gel. The gel was stained with Coomassie Blue (upper panel) and subsequently exposed to x-ray film for visualization of the P-labeled proteins (lower panel). The positions of molecular mass standards (in kilodaltons) is indicated on the left.



The expressed portion of GST itself contains a total of 14 serine and threonine residues, 4 of which exist in an environment resembling the preferred site for phosphorylation of peptides by GRK2, being located 1-3 amino acids carboxyl-terminal to an acidic residue(36) . Despite this abundance of potential GRK2 phosphorylation sites in the primary sequence of GST, none of these sites serves as a substrate for the kinase (Fig. 4, lane 3). This suggests that the serine and threonine residues in the carboxyl terminus of the FPR are presented in a unique fashion, with phosphorylation being dependent on the three-dimensional structure of the protein. We tested this hypothesis by assaying phosphorylation of GST-FPR fusion protein which had been denatured by boiling and rapid cooling. Denaturation resulted in a reduction in the level of phosphorylation by about 90% (Fig. 5A). This confirms that more than simply the primary amino acid sequence of the FPR is involved in the recognition by GRK2. In fact, this result indicates that the three-dimensional structure is absolutely required and that the FPR carboxyl terminus in the incorrect conformation cannot be phosphorylated by GRK2. Results similar to those with GST were obtained when comparing maltose-binding protein to a fusion protein of the FPR carboxyl terminus to maltose-binding protein (not shown), further attesting to the specificity of the reaction. Examination of the sequence of the carboxyl terminus of the FPR reveals that both serine and threonine residues are in a position to be potential sites of phosphorylation. Analysis of the phosphoamino acid content of the phosphorylated GST-FPR protein by acid hydrolysis followed by thin layer chromatography revealed that 65% of the phosphoamino acids consisted of phosphothreonine, whereas 35% consisted of phosphoserine (Fig. 5B). As expected, no phosphotyrosine was detected. These results indicate that phosphorylation occurs in proportion to the content of serine and threonine in the carboxyl terminus of the FPR (see Fig. 1).


Figure 5: Characteristics of phosphorylation of the soluble GST-FPR by GRK2. A, effect of denaturation on GST-FPR phosphorylation. GST-FPR was either incubated at room temperature (lane 1) or heated to 100 °C for 5 min followed by rapid cooling on ice (lane 2) and analyzed for phosphorylation by GRK2 as described under ``Experimental Procedures.'' Reactions were terminated with SDS sample buffer followed by electrophoresis on a 12.5% polyacrylamide gel. The gel was stained with Coomassie Blue (upper panel) and subsequently exposed to x-ray film for visualization of the P-labeled fusion proteins (lower panel). B, analysis of phosphoamino acid content of phosphorylated GST-FPR. GST-FPR, phosphorylated by GRK2, was transferred from an SDS gel to Immobilon P membrane, and the fusion protein band was excised and acid-hydrolyzed. The sample was resuspended with unlabeled phosphoserine, phosphothreonine, and phosphotyrosine, spotted onto a cellulose thin layer plate, and separated by chromatography. Unlabeled phosphoamino acid standards were visualized with ninhydrin, whereas P-labeled phosphoamino acids were visualized by exposure to x-ray film. Phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) migrated 4.2 cm, 5.1 cm, and 5.8 cm, respectively, from the origin. Relative quantitation of the P-labeled phosphoamino acids was carried out with a Molecular Devices PhosphorImager.



We next examined the kinetic parameters of the phosphorylation of the GST-FPR fusion protein. Phosphorylation proceeded linearly for 3-4 h after which no further phosphorylation was observed (data not shown). Incubation of the fusion protein with increased amounts of GRK2 resulted in proportional increases in the level of phosphorylation (as described above), suggesting that under our assay conditions phosphorylation ceased due to exhaustion of the kinase after about 4 h. We also determined the effect of concentration of the fusion protein on the rate of phosphorylation (Fig. 6A). Phosphorylation by GRK2 reached a plateau with about 5 µM GST-FPR. Detailed analysis of the kinetic parameters revealed that the carboxyl terminus of the FPR is phosphorylated with a K(m) of 1.5 ± 0.5 µM and a V(max) of 3.0 ± 0.7 nmol/min/mg of GRK2 (Fig. 6B). This K(m) value is approximately 6-fold higher than that obtained for the agonist-bound state of the beta-adrenergic receptor and approximately 4-fold lower than that determined for the phosphorylation of activated rhodopsin by GRK2(15) . In addition to GRK2, we also tested the ability of three other purified G protein-coupled receptor kinases to phosphorylate the FPR carboxyl terminus. GRK3, which bears strong homology to GRK2, was active to approximately 50% the level (similar K(m), reduced V(max)) of GRK2, whereas GRK5 (37) and GRK6 (32) displayed no significant activity. The fact that two related G protein-coupled receptor kinases were incapable of phosphorylating the FPR carboxyl terminus further attests to the specificity of the reaction between the GST-FPR and GRK2.



Figure 6: Kinetics of phosphorylation of the FPR fusion protein by G protein-coupled receptor kinases. A, phosphorylation of GST-FPR by related G protein-coupled receptor kinases. Fusion protein containing the carboxyl terminus of the FPR (at the indicated concentration) was incubated with one of the following GRKs (at a final concentration of 10 nM): circle, GRK2; bullet, GRK3; , GRK5; or down triangle, GRK6 in a mixture containing 20 mM Tris-Cl, pH 7.2, 2 mM EDTA, 7 mM MgCl(2), and 0.1 mM [-P]ATP (1.5 fmol/cpm) at 30 °C for 30 min. Following electrophoresis on a 12.5% polyacrylamide gel and exposure to x-ray film for visualization, the P-labeled proteins were cut out of the dried gel and quantitated by liquid scintillation counting. Data are representative of two experiments. B, Scatchard analysis of the kinetic parameters of phosphorylation by GRK2. Fusion protein containing the carboxyl-terminal 47 amino acids of the FPR was incubated with GRK2 (62.5 nM) in the above buffer with 0.1 mM [-P]ATP (1.5 fmol/cpm) in a final volume of 30 µl at 30 °C for 30 min. Reactions were quenched with SDS sample buffer and analyzed as above. Data are plotted as v/F versus V, where v is the velocity of the reaction (nmol/min/mg of GRK2) and F is the free concentration of GST-FPR (µM). Data are representative of six experiments with similar results.



GRK2 has been shown to exhibit preferential phosphorylation of serine and threonine residues in an environment containing acidic residues (36) . Examination of the sequence of the carboxyl terminus of the FPR (Fig. 1) suggested that the serine and threonine residues at positions 328, 329, 331, 332, 334, 336, 338, and 339 were candidates for phosphorylation and that the 2 acidic residues at positions 326 and 327 may play a role in the interaction with GRK2. We therefore created mutations at these and the other sites shown in Fig. 7. The mutant GST-FPR fusion proteins were purified and analyzed for their ability to be phosphorylated by GRK2. The results are summarized in Table 1.


Figure 7: Sequences of mutant GST-FPR fusion proteins. The amino acid sequences of the wild type carboxyl terminus and mutant carboxyl termini are shown using the one-letter codes. Only the changed amino acids are indicated for the mutant proteins. The asterisk (*) represents the introduction of a stop codon to generate a truncated form of the protein.





Mutants 4, 5, 6, 8, and 9 were designed to determine which serine and threonine residues were involved in the phosphorylation by GRK2. The K(m) values for these mutants are not significantly different from that of the wild type, suggesting that they do not contribute significantly to the binding interaction between the carboxyl terminus of the FPR and GRK2. The effects of these mutations are evident in the V(max) values. Mutant 9, which replaces Thr, Thr, Ser, and Thr with alanine residues, undergoes approximately 50% of the phosphorylation compared to the wild type fusion protein. Mutant 8, which arose spontaneously during the mutagenesis to produce mutant 9, retains the threonine residue at position 336 and displays an increase in phosphorylation (15%) compared to mutant 9 suggesting that in total these four potential sites represent approximately 50% of the phosphorylation sites within the carboxyl terminus of the FPR sequence. The remaining likely sites of phosphorylation, Ser, Thr, Thr, and Ser, were replaced in mutant 4. Although we expected to see only at most a 45-50% reduction in the extent of phosphorylation (i.e. the complement of that observed with mutant 9), mutant 4 showed almost no phosphorylation (an 83% reduction in the V(max) as compared to the wild type). We further analyzed this region by subdividing mutant 4 into mutants 5 and 6, in which either only the first two mutations or second two mutations were made. Mutant 5, in which only Ser and Thr were replaced, exhibited phosphorylation almost equal to that of mutant 4, in which 4 residues were replaced. We would then expect that phosphorylation might not occur at residues Thr and Ser. However, when only these 2 residues were replaced (mutant 6), a level of phosphorylation (37%) intermediate between mutant 5 (23%) and mutant 9 (53%) was observed. We believe these results are most consistent with a mechanism in which phosphorylation occurs in a sequential manner beginning with residues Ser and Thr, proceeding through residues Thr and Ser, before reaching residues Thr, Thr, Ser, and Thr.

To investigate this possibility further, we attempted to mimic the phosphorylation of certain serine and threonine residues by mutating them to negatively charged glutamate residues, which spatially resemble phosphoserine and phosphothreonine. Mutants 11, 12, and 13 correspond to mutants 5, 6, and 4, respectively, with glutamine replacing the original serine or threonine. Again, there is no significant effect on the K(m) of the phosphorylation reaction. There is, however, in all three cases, an increase in the V(max) of the reaction, indicating a stimulation of phosphorylation. These values are not corrected for the fact that the number of potential phosphorylation sites is reduced by 2 in the case of mutant 11 and 12 and by 4 in the case of mutant 13. If the level of phosphorylation of mutant 13 is compared to that of the corresponding alanine- and glycine-substituted mutant (mutant 4), then the increase in phosphorylation due to the presence of the negatively charged residues is 6-fold. If a similar comparison is made between mutants 11 and 5, the increase in phosphorylation due to the presence of the negatively charged residues is 8-fold. Thus, we can speculate that when either or both Ser and Thr become phosphorylated by GRK2, the resulting intermediate is a superior substrate for further phosphorylation events. This is consistent with the sequential phosphorylation suggested by the results described above.

Given the apparent importance of negatively charged residues in the activation of GRK2, we also examined endogenous charged residues within the carboxyl terminus of the FPR. Mutants 1, 2, 3, and 7 replace charged residues with alanine and glycine residues, whereas mutant 10 removes the terminal 10 amino acids from the fusion protein. Whereas mutant 1 displayed K(m) and V(max) values almost identical with that of the wild type fusion protein, mutant 2 possessed an increased K(m) and somewhat decreased V(max). At low substrate concentrations (K(m)), the combined effect would result in significantly lower rates of phosphorylation as compared to the wild type protein. Mutants 3 and 7, which replace acidic residues within the phosphorylated region, were severely impaired in their ability to be phosphorylated. Both mutants exhibited approximately 5-fold decreases in their V(max) values. The K(m) of mutant 3 was similar to that of the wild type fusion protein, whereas the K(m) of mutant 7 was lower than that of the wild type fusion protein. These results confirm that acidic residues in the vicinity of the phosphorylation sites play a critical role in determining the activity of the kinase. The deletion construct, mutant 10, displayed an increased K(m), similar to mutant 2, but, in contrast to mutant 2, displayed a V(max) greater than that of the wild type protein. This suggests that residues within the terminal 10 residues contribute to the recognition of the substrate but may also sterically interfere with catalysis, resulting in a higher V(max) upon their removal.

Based on results described above which suggest that the activity in the neutrophil extract may be due to a G protein-coupled receptor kinase, such as GRK2, we compared the profile of activity of the neutrophil kinase to that of purified GRK2 using the collection of mutants described in Fig. 7and Table 1. In addition to the 13 mutants, the wild type GST-FPR and control GST fusion proteins were bound to glutathione-agarose beads and purified. Phosphorylation was assayed on the fusion proteins bound to the beads as described in Fig. 2. The results indicate a high degree of correlation between the relative amounts of phosphorylation of the fusion proteins by the two sources of kinase (Fig. 8). Since protein amounts could only be estimated by densitometry of the Coomassie Blue-stained gels and only a single concentration of fusion protein was used, the results do not take into account the effects of K(m) on the level of phosphorylation. This will affect comparing the levels of phosphorylation between mutants to some extent, but not comparing phosphorylation of a given mutant by the neutrophil extract versus purified GRK2. In each case, very similar levels of phosphorylation were observed for the two kinase sources. The exceptions were mutants 11, 12, and 13 which showed greater phosphorylation by the neutrophil extract as compared to the purified GRK2. However, phosphorylation of these mutants by the GRK2 under these conditions did produce considerably greater phosphorylation than seen with the wild type fusion protein, as observed with kinetic analyses of the soluble fusion protein mutants 11, 12, and 13. Together, these results provide still greater evidence that GRK2 or a very closely related kinase may be responsible for the phosphorylating activity observed in the neutrophil extract.


Figure 8: Comparison of the phosphorylation by neutrophil extract kinase and GRK2 of wild type and mutant FPR carboxyl termini. Fusion proteins representing the wild type or mutant fusion proteins described in Fig. 7were bound to glutathione-agarose beads and assayed for phosphorylation as described in the legend to Fig. 2using either a crude neutrophil extract (cross-hatched bars) or purified GRK2 (open bars). Reactions were quenched with SDS sample buffer followed by electrophoresis on a 12.5% polyacrylamide gel and subsequently exposed to x-ray film for visualization. Quantitation of the P-labeled proteins was carried out with a Molecular Devices PhosphorImager. Data are from three preparations of fusion protein with experiments performed in duplicate.




DISCUSSION

The results presented in this paper demonstrate for the first time that the carboxyl terminus of the FPR serves as an effective substrate for a neutrophil kinase, possibly the G protein-coupled receptor kinase, GRK2. Six results address the specificity of this phosphorylation.

First, the phosphorylation of the fusion protein lacking the carboxyl terminus of the FPR was negligible compared to that of the protein containing the FPR tail, despite the existence of numerous potential phosphorylation sites for GRK2 within the primary sequence of the GST. This indicates that the recognition of the phosphorylation site(s) within the FPR tail by the kinase is unique under our assay conditions.

Second, of the protein kinase inhibitors tested, only the G protein-coupled receptor kinase specific inhibitor, heparin, was able to prevent phosphorylation. Furthermore, the kinase activity of the neutrophil extract and purified GRK2 exhibited similar sensitivity to heparin (IC 100-300 nM), suggesting the former activity may be due to GRK2. Casein kinase II, GRK5, and GRK6, which are also sensitive to heparin, exhibit dissimilar IC values of approximately 10, 1, and 15 nM, respectively.

Third, the tertiary structure of the FPR carboxyl terminus is critical for its recognition by GRK2. This is evidenced by the large reduction in activity of the GRK2 toward denatured GST-FPR as compared to the native protein. The residual phosphorylation observed could either be a result of a low affinity interaction with the denatured GST-FPR or a result of incomplete denaturation of the GST-FPR.

Fourth, phosphorylation of GST-FPR is specific to GRK2 and the closely related GRK3, but does not occur with the more distant G protein-coupled receptor kinases, GRK5 and GRK6.

Fifth, the K(m) of the phosphorylation reaction (1.5 µM) approached the affinity of GRK2 for the beta(2)-adrenergic receptor itself, further indicating that the interaction of GRK2 with the carboxyl terminus of the FPR is highly specific.

Sixth, mutagenesis revealed that even the alteration of a single amino acid (e.g. D333A) can result in almost complete loss of phosphorylation.

Studies with synthetic peptides of lengths between 20 and 30 amino acids (approximately one-half of the size of the carboxyl-terminal tail of the FPR) corresponding to regions of the betaadrenergic receptor that serve as substrates for GRK2 demonstrate K(m) values of 1-5 mM(38) . This difference in K(m) between beta-adrenergic receptor and receptor-derived peptides may reflect the requirement for multiple interactions with numerous domains of the beta-adrenergic receptor which occurs only with the liganded receptor. Our results indicate that the carboxyl terminus of the FPR contains sufficient structural information to allow a high affinity interaction with GRK2, although additional intracellular domains may further increase the affinity of GRK2 for the FPR. The low observed V(max) for this reaction, as compared to that of the intact beta-adrenergic receptor (V(max) = 78 nmol/min/mg(15) ), may suggest that additional interactions are required to stimulate maximal activity of the kinase. Such a conclusion is supported by recent experiments demonstrating that agonist-activated receptors can increase the V(max) of phosphorylation of synthetic peptides by GRK2, suggesting that the overall topology of the activated receptor is more important than the substrate's specific primary amino acid sequence in the stimulation of GRK2 activity(39) . One possible explanation of our results is that, whereas the beta-adrenergic receptor upon binding agonist ``assembles'' a recognition site for GRK2 out of multiple low affinity sites, the FPR contains a pre-existing high affinity site that is presented upon the binding of ligand to the receptor. The presence of a pre-existing high affinity site for phosphorylation could also explain the unique form of class desensitization displayed by chemoattractant receptors where, for example, exposure of neutrophils to C results in the desensitization of the cell to fMLP and other chemoattractants but not to unrelated agonists(10, 40) .

Our results indicate that the serine and threonine residues within both regions encompassed by mutant 4 and mutant 9 are phosphorylated by GRK2. However, if the phosphorylation of these serine and threonine residues were random and independent, it would be expected that mutants 4 and 9 would both show approximately 50% of the level of phosphorylation compared to the wild type sequence. The fact that replacement of the upstream serine and threonine residues resulted in a greater reduction in phosphorylation of the remaining serine and threonine residues suggests that a hierarchy of phosphorylation exists, in which phosphorylation of carboxyl-terminal serine and threonine residue(s) is dependent on the prior phosphorylation of amino-terminal residue(s). Further evidence in favor of this mechanism is provided by mutants 5 and 6, which subdivide the serine and threonine residues of mutant 4. Here, replacement of the 2 amino-terminal-most serine and threonine residues results in a similar reduction in phosphorylation as mutant 4, in which 4 serine and threonine residues are replaced (80% reduction). Replacement of the next 2 serine and threonine residues results in a level of phosphorylation (63% reduction) between those of mutants 4 and 9 (47% reduction). The simplest explanation of these results invokes a sequential mechanism of phosphorylation beginning with residues 328 and/or 329, followed by residues 331 and/or 332, and finally residues 334 through 339.

Our results also indicate that the acidic residues at positions 326, 327, and 333 play a critical role in the stimulation of GRK2 activity by the receptor. Since acidic residues appear important in the interaction between GRK2 and synthetic peptides(36) , it is possible that phosphorylation of serine and threonine residues juxtaposed to asparagine and glutamine residues creates a new acidic phosphoserine or phosphothreonine recognition sequence for the phosphorylation of a subsequent carboxyl-terminal serine or threonine. Such a mechanism has been proposed for glycogen synthase kinase-3, which appears to use newly generated phosphoserine residues as recognition sites to phosphorylate subsequent serine residues in a hierarchical manner(41) . We attempted to test whether the introduction of a negative charge at potential sites of phosphorylation would stimulate the activity of the GRK2. For this purpose, mutants equivalent to mutants 4, 5, and 6 were created substituting glutamate residues for the original serine and threonine residues. The greatest effect was seen with the replacement of the first 2 serine and threonine residues with glutamine. This resulted in a 2-fold increase relative to the wild type fusion protein and an 8-fold increase relative to the alanine-substituted mutant. These results provide very strong supporting evidence that the first phosphorylation step greatly enhances the subsequent phosphorylation events. Sequential phosphorylation has also recently been shown to occur in the phosphorylation of rhodopsin by rhodopsin kinase, but it was not clear whether other GRKs possess a similar catalytic mechanism (42, 43, 44) . Our results suggesting a conserved mechanism of phosphorylation between multiple members of the class of GRKs are of particular significance in light of the amino acid sequence homology between members of the class of G protein-coupled receptor kinases (31) .

We and others have previously shown that the carboxyl terminus of the FPR interacts with G proteins(25, 45) . We utilized both a fusion protein, similar to that used here, and synthetic peptides to map regions of the FPR involved in the interaction with G proteins. Our results showed that the second intracellular loop and portions of the carboxyl terminus could interrupt binding of the FPR to G proteins. Within the carboxyl terminus of the FPR, the most effective region comprised the first third of the carboxyl terminus (Phe-Arg), the region juxtaposed to the membrane as predicted from receptor modelling. Also effective was a region representing the central portion of the carboxyl terminus (Ser-Leu), corresponding to the region demonstrated here to be phosphorylated. A peptide representing the terminal 12 amino acids of the FPR was ineffective in disrupting the FPR-G protein interaction. Our results presented here provide for a simple mechanism whereby phosphorylation of a region of the FPR involved in binding to G proteins could prevent this interaction and result in an uncoupling of the activated FPR from G protein. As with rhodopsin and the beta-adrenergic receptor systems, the subsequent binding of an arrestin-like protein could further potentiate the uncoupling.

In conclusion, the novel findings presented in this paper expand the family of known substrates for G protein-coupled receptor kinases to include a member of the chemoattractant class of hematopoietic cell receptors. We also obtained evidence that phosphorylation of the FPR carboxyl terminus by GRK2 is the result of a high affinity interaction and proceeds in a hierarchical manner. Together, these results provide a basis for further experiments to examine the mechanism of action of G protein-coupled receptor kinases using intact chemoattractant receptors and to determine the exact involvement of G protein-coupled receptor kinases in the desensitization and regulation of the entire class of chemoattractant receptors.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant AI36357 (to E. R. P.), Grants GM46572 and AI33503 (to R. D. Y.), and Grant GM44944 (to J. L. B.). This is Publication No. 8207-IMM from the Department of Immunology, the Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Immunology, IMM12, The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-4549; Fax: 619-554-6123.

(^1)
The abbreviations used are: G protein, guanine nucleotide-binding regulatory protein; GRK, G protein-coupled receptor kinase; FPR, N-formyl peptide receptor; fMLP, N-formyl-methionyl-leucyl-phenylalanine; GST, glutathione S-transferase.


ACKNOWLEDGEMENTS

We acknowledge Jorge Gomez for helpful discussions and Priya Kunapuli and Robert Loudon for preparations of GRK5 and GRK6, respectively. We would also like to acknowledge the contribution of the General Clinical Research Center of the Scripps Research Institute (NIH M01RR00833).

Addendum-At the time of submission of this manuscript, Takano et al.(46) published a report describing a mutant of the platelet-activating factor receptor in which Ser/Thr residues in the carboxyl-terminal tail were replaced with Ala. This mutant more potently activated multiple signal transduction pathways, leading to the conclusion that desensitization had been affected. Furthermore, a synthetic peptide corresponding to this region was phosphorylated by beta-adrenergic receptor kinase 1 (GRK2). These results provide corroborating evidence for our contention that GRKs may be involved in the desensitization of chemoattractant receptors.


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