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The Journal of Immunology, 2006, 176: 5418-5425.
Copyright © 2006 by The American Association of Immunologists

Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Individual Carboxyl-Terminal Serine and Threonine Residues1

Ross M. Potter, Diane C. Maestas, Daniel F. Cimino and Eric R. Prossnitz2

Department of Cell Biology and Physiology and University of New Mexico Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adaptation, defined as the diminution of receptor signaling in the presence of continued or repeated stimulation, is critical to cellular function. G protein-coupled receptors (GPCRs) undergo multiple adaptive processes, including desensitization and internalization, through phosphorylation of cytoplasmic serine and threonine residues. However, the relative importance of individual and combined serine and threonine residues to these processes is not well understood. We examined this mechanism in the context of the N-formyl peptide receptor (FPR), a well-characterized member of the chemoattractant/chemokine family of GPCRs critical to neutrophil function. To evaluate the contributions of individual and combinatorial serine and threonine residues to internalization, desensitization, and arrestin2 binding, 30 mutant forms of the FPR, expressed in the human promyelocytic U937 cell line, were characterized. We found that residues Ser328, Ser332, and Ser338 are individually critical, and indeed sufficient, for internalization, desensitization, and arrestin2 binding, but that the presence of neighboring threonine residues can inhibit these processes. Additionally, we observed no absolute correlation between arrestin binding and either internalization or desensitization, suggesting the existence of arrestin-independent mechanisms for these processes. Our results suggest C-terminal serine and threonine residues of the FPR represent a combinatorial code, capable of both positively and negatively regulating signaling and trafficking. This study is among the first detailed analyses of a complex regulatory site in a GPCR, and provides insight into GPCR regulatory mechanisms.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adaptation processes regulating G protein-coupled receptors (GPCRs)3 ensure an efficient response to ligands while preventing damage to the host. Adaptation usually consists of two processes: desensitization, which attenuates activity of cellular signaling machinery; and internalization, which removes receptors from the cell surface ( 1, 2, 3, 4, 5). GPCR adaptation is initiated by phosphorylation of cytoplasmic Ser and Thr residues by GPCR kinases (GRKs), protein kinase A, or protein kinase C ( 6, 7, 8, 9). The primary role of serine/threonine phosphorylation is thought to be the binding of the cytoplasmic protein arrestin, which blocks further interactions with G proteins and in many systems initiates receptor internalization as well as additional signaling pathways ( 2).

One of the most studied chemoattractant/chemokine GPCRs, the human N-formyl peptide receptor (FPR) is expressed on leukocytes, predominantly neutrophils and monocytes. FPR ligands include short, mostly hydrophobic peptides characteristic of those secreted or shed by bacteria, most notably formyl-Met-Leu-Phe (fMLF). Ligand binding to the FPR induces coupling to and activation of inhibitory guanine nucleotide binding (Gi) proteins, which in turn activate multiple effectors, including phospholipase C, resulting in calcium mobilization. Unlike other extensively investigated GPCRs such as the beta2 adrenergic and angiotensin 1A receptors, FPR internalization does not require arrestin, despite the fact that the FPR forms stable endosomal complexes with arrestin ( 10). FPR internalization is, however, dependent upon receptor phosphorylation ( 11). The FPR is the first GPCR for which it has been shown that arrestin binding is required for receptor recycling to the cell surface ( 12). Because of its differences from other model GPCR systems, investigation of the FPR offers unique insights into GPCR function and regulation.

The C terminus of the FPR contains 11 serine and threonine residues located at Ser319, Thr325, Ser328, Thr329, Thr331, Ser332, Thr334, Thr336, Ser338, Thr339, and Ser342 (Table I). Previous studies by our laboratory have shown that of these, eight residues located between and including Ser328 and Thr339 are critical for internalization and desensitization of the FPR ( 11). These eight residues are arranged as two clusters of four serines or threonines ( 6). Acidic residues precede each cluster, which is characteristic of GRK-mediated phosphorylation sites ( 13).


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  		Table I. Wild-type and mutant FPR C-terminal serine and threonine residues

 
In a prior study, five mutant forms of the FPR were characterized in an effort to address the role of groups of receptor phosphorylation sites in receptor internalization and desensitization (see Table I) ( 11). The {Delta}ST mutant replaced all serine and threonine residues between Ser319 and Ser342 with alanine or glycine residues. Similarly, the {Delta}A mutant replaced the four residues of the first serine/threonine cluster (located between Ser328 and Ser332 and designated the A site), and {Delta}B replaced the four residues of the second serine/threonine cluster (located between Thr334 and Thr339 and designated the B site). {Delta}C and {Delta}D essentially divided the A site by replacing only the first two ({Delta}C) or second two ({Delta}D) residues. The {Delta}ST mutant fails to become phosphorylated in response to ligand binding and neither internalizes nor desensitizes. {Delta}A, {Delta}B, {Delta}C, and {Delta}D all internalize similarly to the wild-type FPR; however, {Delta}B desensitizes to a reduced extent, whereas {Delta}A desensitizes to an extent comparable to {Delta}ST. {Delta}C and {Delta}D desensitize similarly to the wild-type receptor. Because certain serine or threonine residues are crucial for desensitization but not internalization, these processes likely proceed by different mechanisms. These studies also suggested that phosphorylation of residues within the A site appear to be specifically crucial to the process of desensitization ( 11). Subsequent studies further revealed that {Delta}A is notably deficient in its ability to bind arrestin2, providing the first evidence that arrestin binding was not required for FPR internalization ( 14, 15). Because of the number of potential phosphorylation sites and the complex nature of the resulting patterns of phosphorylation, a more detailed analysis of the functional roles of individual FPR phosphorylation sites was undertaken.

In this study, we tested the function of 30 individual and combined C-terminal serine and threonine site mutants of the FPR in desensitization, internalization, and arrestin binding. In constructing these mutant receptors, we began with a {Delta}ST template and selectively restored combinations of Ser and/or Thr residues. Our results indicate that residues Ser328, Ser332, and Ser338 are critical to desensitization, internalization, and arrestin colocalization. Other residues appear to play weaker roles in these processes and in some cases may be inhibitory. These results suggest that Ser/Thr residues within the C terminus of GPCRs are the basis of a combinatorial code, in that the presence of specific combinations of residues, leading to differential phosphorylation patterns by GRKs, both positively and negatively regulate the signaling and trafficking of the receptor. With the crystal structure of this receptor, as well as the specific peptide sequences favored by members of the GRK family as phosphorylation targets, as yet undetermined, these findings provide insight into the differential roles of serine and threonine residues in the FPR C terminus and their importance in receptor regulation. This study represents the first detailed analysis of a complex GRK-regulated site in a GPCR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

fMLF, RPMI 1640, FBS, and N-formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys (6pep) were purchased from Sigma-Aldrich. 6pep-fluorescein, Alexa546, and indo-1/AM were obtained from Molecular Probes. 6pep-Alexa546 was prepared as described previously ( 15, 16, 17). PBS and HBSS were obtained from Invitrogen Life Technologies.

Construction and expression of site-directed mutants in U937 cells

The cDNA encoding the FPR was obtained from a human HL-60 granulocyte library ( 18). The {Delta}ST mutant in which all 11 of the serine and threonine residues have been replaced (S319A, T325G, S328A, T329A, T331A, S332G, T334G, T336G, S338G, T339A, S342G), as well as the {Delta}A mutant (S328A, T329A, T331A, S332G), the {Delta}B mutant (T334G, T336G, S338G, T339A), the {Delta}C mutant (S328A, T329A), and the {Delta}D mutant (T331A, S332G), have been described earlier ( 19). The new mutants generated in this study were created via site-directed mutagenesis of the {Delta}ST plasmid. Plasmid DNA was transfected into U937 cells using a lipid-based method (Effectene; Qiagen) according to the manufacturer’s instructions. Briefly, cells were plated overnight, transfected with 1 µg of plasmid DNA, and then selected for 3–4 wk in G418 at a final concentration of 1 mg/ml. Surviving cells were pooled, and the expression levels were analyzed by flow cytometry following incubation with 10 nM N-fNleLFNleYK-FITC on ice. Where necessary, transfected U937 cells were sorted using a MoFlo Cytometer (DakoCytomation). Cells were grown in RPMI 1640 (supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES (pH 7.4), and 10% FBS) at 37°C in a humidified atmosphere of 5% CO2.

Flow cytometry

Cells (5 x 105) were harvested by centrifugation, washed once with PBS, and resuspended to 106 cells/ml in ice-cold PBS. Binding was conducted in 0.5 ml with 6pep-fluorescein at 10 nM. Cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences) for fluorescent intensity and gated on forward and side scatter to exclude debris and dead cells. Nonspecific binding was determined in the presence of 1 µM N-fMLF and was almost identical with the level of fluorescence observed with vector-transfected cells.

Receptor internalization

Receptor internalization was determined as the agonist-dependent loss of FPR from the cell surface ( 11). Wild-type and mutant FPR-transfected U937 cells were harvested, washed, and resuspended in PBS as described above. Cells were then stimulated with 1 µM fMLF for the indicated time at 37°C and washed three times with PBS. Remaining cell surface receptors were determined with 10 nM 6pep-fluorescein. Ligand-stained cells were then analyzed for fluorescent intensity on a FACSCalibur flow cytometer as described above. Receptor internalization is expressed as the percentage decrease of the cell surface receptors of the treated cells relative to the untreated cells.

Desensitization

Desensitization of calcium mobilization was determined as follows. Cells (1 x 107) were collected by centrifugation and resuspended at 5 x 106 cells/ml in HBSS. The cells were incubated with 5 µM indo-1/AM for 30 min at 37°C with gentle rocking, washed once with HBSS, and resuspended to a concentration of ~106 cells/ml in HBSS. The cells were then divided into two samples: one was stimulated with 1 µM fMLF for 8 min, whereas the other was treated with buffer only. The cells were then washed three times with HBSS at room temperature to remove surface-bound fMLF and resuspended in 1.5 ml of HBSS. The mobilization of intracellular Ca2+ in treated and untreated cells in response to a 40 nM fMLF stimulation was determined using a Quantimaster QM 2000-6 spectrofluorometer (Photon Technologies International) detecting the ratio of fluorescence at 400 and 490 nm, as described previously ( 19). Desensitization is expressed as the percentage decrease of the response of the treated cells relative to the untreated cells.

Confocal microscopy and arrestin colocalization

U937 cells expressing FPR or mutant receptor were transiently transfected as follows. Cells were harvested by centrifugation and resuspended to a concentration of 2 x 107 cells/ml in RPMI 1640. Arrestin2 plasmid DNA (pEGFP-N1, 25 µg) was added to 400 µl of cells, and the cells were transferred to a 4-mm gap electroporation cuvette and pulsed (200 V/2000 µF for an exponential decay t1/2 of 50–55 msec) using a BTX 600 Electro Cell Manipulator (Genetronics). Cells were allowed to rest 10 min before being transferred to 10 ml of complete RPMI 1640 medium. Following transfection, cells were incubated for 24–48 h at 37°C/5% CO2.

Approximately 5 x 106 transiently transfected cells were harvested by centrifugation, washed once with PBS, resuspended in 1 ml of PBS, and divided into two samples. One sample was warmed to 37°C while the other was maintained on ice. Both samples were then treated with 10 nM (final) 6pep-Alexa546 for 10 min, at which time 0.5 ml of ice-cold 2% paraformaldehyde was added. The cells were then pelleted by gentle centrifugation and resuspended in 0.5 ml of ice-cold 2% paraformaldehyde. The cells were incubated at room temperature for 30 min and washed three times in PBS. After the third wash, the cells were resuspended in 12 µl of Vectashield (Vector Laboratories) before being mounted on glass slides with coverslips. Confocal fluorescent images were acquired at room temperature using a Zeiss LSM510 system equipped with argon and HeNe lasers for excitation at 488 and 543 nm. Samples were viewed using the 60 x 1.3 oil immersion lens.

The Profile function of the Zeiss LSM 510 software package was used to quantitate the ratio of arrestin2-enhanced GFP (EGFP) per 6pep-Alexa546-labeled receptor cluster. Because wild-type and mutant receptors organize into clusters following ligand stimulation ( 17), it is possible to position an analysis line through a receptor cluster and determine the average levels of peak fluorescent intensity for both 6pep-Alexa546 (representing FPR) and arrestin2-EGFP. A background value, obtained from the average intensities from a whole-cell line drawn through areas lacking receptor clusters, was subtracted from these values. The resulting corrected values were then normalized to values obtained from cells expressing wild-type receptor prepared and examined on the same day as the mutant receptor-expressing cells. Approximately 30 total receptor clusters (from 6 to 10 cells prepared on two different days) were analyzed per mutant.

Statistical analysis

Graphs were prepared using Prism software (GraphPad). Statistical analysis was performed on JMP-In Software (SAS Institute). Comparison of means was done using the Tukey-Kramer Honest Significant Difference test with an {alpha} value of 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
To study the functional significance of phosphorylatable residues in the processes of FPR internalization, desensitization, and arrestin binding, we constructed FPR mutants that would allow the characterization of individual C-terminal serine and threonine residues in specific combinations. Beginning with a {Delta}ST background, which lacks all serines and threonines in the FPR C terminus, we selectively restored specific serine or threonine residues. For purposes of clarity, numbers 1 through 8 were assigned to the following residues within the central C-terminal region of the FPR: Ser328, Thr329, Thr331, Ser332, Thr334, Thr336, Ser338, and Thr339. In this study, we designate a mutant by the Ser or Thr residues that are restored to the {Delta}ST template. Following this convention, the A site consists of residues 1 through 4, or Ser328, Thr329, Thr331, and Ser332; whereas the B site consists of residues 5 through 8, or Thr334, Thr336, Ser338, and Thr339. Similarly, mutant {Delta}A can be referred to as mutant 5678, whereas mutant {Delta}B can be referred to as mutant 1234.

Based on our earlier results with the {Delta}A, {Delta}C and {Delta}D mutants, which suggested that residues within the A site (Ser328, Thr329, Thr331, and Ser332) play a vital role in receptor desensitization ( 11), our first set of experiments was designed to examine whether particular serine or threonine residues in this region play an important role in desensitization. To this end, we subdivided the {Delta}C and {Delta}D mutants into their components by designing four mutants in which one residue from the A site was combined with all residues within the B site. These mutants were designated 15678 (Ser328 plus Thr334–Thr339), 25678 (Thr329 plus Thr334–Thr339), 35678 (Thr331 plus Thr334–Thr339), and 45678 (Ser332 plus Thr334–Thr339; Table II).


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  		Table II. C termini of A site mutant receptors

 
The mutant receptor constructs were stably expressed in U937 cells, a promyelocytic cell line that does not normally express the FPR but which previous studies have shown can process the transfected wild-type FPR in terms of phosphorylation, desensitization, and internalization with a similar proficiency to the endogenous FPR expressed in neutrophils ( 11). These same studies have shown that modifications to the cytoplasmic C terminus of the FPR do not affect the receptor’s affinity for FPR ligands, nor do they detrimentally affect receptor-mediated calcium mobilization. In addition, because our prior studies demonstrated that C-terminal phosphorylation is required for internalization and desensitization, we assume that mutant FPRs capable of such processes are at least partially phosphorylated following ligand binding ( 11). Flow cytometric analysis of transfected cells indicated that, for most mutants, >90% of all cells expressed receptor (data not shown). Where necessary, transfected U937 cells were sorted by flow cytometry to insure that a uniform population of cells expressing mutant receptor would be used in subsequent experiments.

To compare the rate of receptor internalization, U937 cells expressing {Delta}ST, {Delta}A, {Delta}C, {Delta}D, 15678, 25678, 35678, and 45678 as well as wild-type FPR were treated with saturating concentrations of 1 µM fMLF and incubated at 37°C for varying times, after which the cells were analyzed by flow cytometry for remaining surface receptors. As shown in Fig. 1A, mutants 15678, 25678, 35678, and 45678 internalized at rates comparable to the wild-type receptor. After 60 min of fMLF treatment, the surface receptors of all four mutants as well as wild-type FPR were ~15–25% of the untreated level. Surface receptors of the {Delta}ST-expressing cells were reduced slightly to ~85% of the untreated level after 60 min. A comparison of surface receptor levels 10 min after ligand treatment reveals that the reductions in surface expression of 15678, 25678, 35678, and 45678 were not significantly different from that observed with wild-type receptor or the original mutants {Delta}A, {Delta}C, and {Delta}D (p < 0.05) (Fig. 1B). These data agree with earlier results indicating that even partial phosphorylation within the FPR C terminus, as defined by {Delta}A, is sufficient for wild-type levels of receptor internalization ( 11).


Figure 1
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  		FIGURE 1. Mutants 15678, 25678, 35678, and 45678 internalize comparably but differentially desensitize and bind arrestin. A, To examine the effect of individual A site residues expressed in conjunction with all four B site residues, wild-type FPR and mutant receptors {Delta}ST, 15678, 25678, 35678, and 45678 were expressed in U937 cells. To measure internalization rates, cells were treated with 1 µM fMLF for the indicated times, washed, treated with 10 nM 6pep-FITC, then analyzed by flow cytometry for remaining surface FPR. Data represent the mean ± SE for three independent experiments. B, Receptor desensitization and arrestin2 binding were measured as described in Materials and Methods. Values represent the mean ± SE for a minimum of two independent experiments done in duplicate (desensitization) or at least 30 receptor clusters from two independent experiments (arrestin2 binding), and are compared with the values for internalization after 10 min.

 
We next assessed the ability of mutant and wild-type receptors to desensitize following treatment with fMLF. U937 cells stably expressing mutant or wild-type receptors were loaded with 5 µM indo-1/AM and stimulated either with 1 µM fMLF or buffer. Calcium mobilization in response to a subsequent treatment of 40 nM fMLF (a concentration slightly greater than the EC50 in untreated cells) was then monitored. As shown in Fig. 1B, mutants {Delta}A, {Delta}C, and {Delta}D displayed desensitization similar to our earlier study, with the exception that {Delta}C desensitized to a slightly lower extent in our current experiments. Receptor 15678 desensitized to an extent similar to the wild-type receptor, whereas receptors 35678 and 45678 desensitized at intermediate levels similar to {Delta}C, and receptor 25678 desensitized at a significantly reduced level (<50% of the wild-type FPR). These data suggest that the presence of residue 1 plays a supportive role in the desensitization of the FPR, whereas the presence of residues 3 and 4 may have a smaller effect on desensitization. The fact that mutant receptor 25678 did not desensitize significantly greater than {Delta}A suggests that addition of residue 2 alone does not enhance desensitization.

Recently published data have suggested that phosphorylation alone may be sufficient to decrease affinity of G protein for the FPR ( 14). Therefore, we chose to examine arrestin binding as an independent factor in receptor processing. This was accomplished by using confocal fluorescence microscopy to directly measure the ability of arrestin to colocalize with mutant receptors following ligand stimulation. U937 cells stably expressing wild-type or mutant receptors were transiently transfected with arrestin2-EGFP. After 24–48 h, the cells were treated with fluorescent ligand and visualized by confocal fluorescence microscopy. The Profile function of the Zeiss LSM 510 software package was used to quantitate the level of arrestin2-EGFP colocalization with 6pep-Alexa546-labeled receptor clusters. Because wild-type and all mutant receptors (including {Delta}ST) organize into clusters following ligand stimulation, one can analyze receptor clusters and determine the average levels of peak fluorescent intensity for both 6pep-Alexa546 (representing FPR) and arrestin2-EGFP. To eliminate day-to-day variations, all mutants were compared with wild-type receptors prepared on the same day and analyzed in parallel. Of the original mutant receptors, {Delta}C and {Delta}D bound arrestin2 to an extent comparable to wild-type receptor. Binding of arrestin by {Delta}A was not significantly different from {Delta}ST. The 35678 receptor also bound arrestin2 comparably to wild-type, whereas mutants 15678, 25678, and 45678 exhibited slightly reduced arrestin2 binding (Fig. 1B).

Having established the individual roles of residues within the A site, we next sought to examine the importance of serine/threonine residues within the B site, based upon mutants derived from the FPR mutant {Delta}C. This mutant has available phosphorylation sites at residues 3 (Thr331) and 4 (Ser332) from the A site as well as all four serine and threonine residues ( 5, 6, 7 and 8) from the B site. {Delta}C internalizes and desensitizes comparably to the wild-type receptor ( 11). Restoring Thr331 and Ser332 as well as one or more residues from the B site to the {Delta}ST template essentially deconstructed the {Delta}C mutant. Following our naming convention, the new mutants were designated 345, 347, 348, 3456, 3478, 3467, 3458, 34567, and 23567 (this latter mutant represents a hybrid to compare the effects of sites 34 vs 23; Table III).


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  		Table III. C termini of {Delta}C-based mutant receptors

 
The resulting {Delta}C-based mutant receptors varied widely in terms of internalization rate and extent (Fig. 2A). Receptors 345, 3458, 3467, 3478, and 34567 all internalized to an extent not significantly different from the wild-type receptor (>75% of wild type; p < 0.05). Receptors 347, 348, and 3456 internalized much more slowly than wild type, having internalization half-times of 5.2 min, 12.3 min, and 4.9 min, respectively, compared with the ~2 min observed for the wild-type receptor. Despite their lower rates of internalization, after 60 min 348 and 3456 internalized ~70% of their receptors, whereas 347 internalized only 30% of its surface receptors after 60 min. Thus, there appears to be no correlation between the rate and extent of receptor internalization for the FPR. This lack of correlation raises the possibility that rapid recycling of 347 to the surface results in higher equilibrium levels of cell surface receptors than 348 and 3456.


Figure 2
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  		FIGURE 2. {Delta}C-based mutants differ in rates of internalization and arrestin binding, but not desensitization. A, The regulatory contributions of residues within the {Delta}C mutation were examined by designing nine mutant receptors, each having three, four, or five of the {Delta}C mutations in various combinations. Internalization rates for {Delta}C-based mutant receptors were determined as described above. B, Desensitization and arrestin2-binding levels were determined for the {Delta}C-based mutant receptors as described above, and are compared with internalization rates after 10 min.

 
The internalization data for the {Delta}C-based mutants suggest that the location of phosphorylation sites may be as important to receptor internalization as the number of sites. Receptors 3456, 3478, 3467, and 3458 all have four available phosphorylation sites. However, 3456 and 3478, which have the first and second pair of residues of the B site respectively, do not internalize as rapidly or to the same extent as receptors 3467 and 3458, which restore residues at the center or extremities of the B site. Also, a comparison of the receptors 3456, 3467, and 34567 reveals that addition of a single residue can result in a marked change in internalization. As seen in Fig. 2B, the addition of the site 5 (Thr334) to mutant 3467 resulted in a ~15% relative decrease in mean internalization after 10 min; however, addition of the residue 7 residue (Ser338) to 3456 results in a ~40% relative increase in internalization.

Desensitization studies of the {Delta}C-based mutant receptors showed that receptors 3467 and 3458 had the highest levels of desensitization, although no receptor was significantly different from another (p < 0.05; Fig. 2B). Despite containing one additional potential phosphorylation site, 34567 desensitized 24% less than receptor 3467, reinforcing the idea that the location of available serine or threonine residues may be more crucial than their absolute number. Despite displaying similar overall levels of desensitization, the {Delta}C-based mutant receptors differed substantially in their colocalization with arrestin2 (Fig. 2B). Receptors 3467 and 3458 bound arrestin2 comparably to the wild-type receptor. Similar to the internalization and desensitization data, 34567 bound arrestin2 significantly <3467 (p < 0.05). All other mutant receptors bound arrestin to a lesser extent than wild type, mutants 347, 348, 3478, and 23567 significantly so (p < 0.05). It is noteworthy that 3478 and 3467 desensitize comparably, yet 3467 binds arrestin2 2-fold >3478. This suggests that arrestin binding is not the sole mechanism responsible for FPR desensitization. In addition, the discordance between receptor internalization and desensitization in mutants 347 and 348 confirm earlier conclusions drawn from the {Delta}A mutant that internalization and desensitization can occur through independent mechanisms. Finally, direct comparison of the 23567 and 34567 mutants also suggests the residue 4 may be important for arrestin binding.

Experimental results from the {Delta}C-based mutant receptors suggest that addition of a single threonine residue, namely residue 5 (Thr334) to receptor 3467, can result in a significant decrease in arrestin2 colocalization and smaller decreases in internalization and desensitization. Given this suggestion that the presence of a Ser or Thr residue at a certain site may suppress internalization, desensitization, and arrestin2 binding, we hypothesized that removal of certain residues from the {Delta}A mutant (which possesses serine and threonine residues at sites 5, 6, 7, and 8) could rescue its ability to desensitize. Consequently, receptor mutants 56, 78, 67, 58, and 567 were created and stably transfected into U937 cells (Table IV). Internalization rates for 56, 78, 67, 58, and 567 were as varied as the {Delta}C-based mutant receptors (Fig. 3A). Receptors 56 and 58 displayed decreased internalization rates and lower extents of internalization compared with wild-type receptor, with half-times of 8.1 and 3.2 min, respectively (Fig. 3A). Receptors 56 and 58 also internalized significantly less than the {Delta}C-based receptor mutants 3456 and 3458 (p < 0.05). Receptors 78, 67, and 567 internalized comparably to wild-type receptor. This result is similar to that obtained with the mutant receptors 3478, 3467, and 34567, which they resemble. As with the internalization results, receptors 56 and 58 desensitize and bound arrestin2 poorly (Fig. 3B). However, the mutations carried by receptors 78, 67, and 567 successfully "rescued" the {Delta}A mutant, because these mutants demonstrated desensitization and arrestin binding significantly greater than {Delta}A.


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  		Table IV. C termini of B site mutant receptors

 

Figure 3
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  		FIGURE 3. Mutants 56 and 58 fail to internalize, desensitize, or bind arrestin. A, To assess the regulatory function of B site residues, we designed a series of five receptors having two or three residues from the B site in different orders. Internalization rates were determined as described above. B, Desensitization and arrestin2-binding levels were determined for the B site mutant receptors as described above, and are compared with internalization rates after 10 min. 

 
All three of the mutant receptors that rescued the {Delta}A mutation contained residue 7 (Ser338), suggesting that isolating residue 7 from the other residues associated with the {Delta}A mutation increased desensitization and arrestin binding. Based on this observation, a series of single serine/threonine residue mutant receptors were created to evaluate the individual contribution of each site to internalization, desensitization, and arrestin2 colocalization. These receptors possess only one serine or threonine residue restored to the {Delta}ST template and are designated 1–8 (Table V). Based on our previous results demonstrating that {Delta}A (5678) does not desensitize or bind arrestin, we hypothesized that no single serine or threonine residue would likely be sufficient for desensitization or arrestin binding. Internalization assays showed that receptors 2, 3, 5, 6, and 8 behaved comparably to {Delta}ST with <20–30% internalization after 10 min (Fig. 4A). Of these, receptor 6 had the highest level of internalization, with ~35% of receptors internalizing 60 min. Unexpectedly, receptors 1, 4, and 7 demonstrated wild-type levels of internalization.


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  		Table V. Single-site mutant FPR receptors

 

Figure 4
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  		FIGURE 4. Single-site mutants 1, 4, and 7 function comparably to wild-type FPR. A, Based on findings that suggested individual serine or threonine residues could have inhibitory effects on other residues, mutant receptors having a single serine or threonine residue from either the A or B site were designed and expressed in U937. Internalization rates were determined as described above. B, Desensitization and arrestin2-binding levels were determined for the single-site mutant receptors as described above, and are compared with internalization rates after 10 min. 

 
The results from desensitization and arrestin2 colocalization experiments mirrored those of the internalization experiments (Fig. 4B). Again, receptors 1, 4, and 7 behaved similarly to wild-type receptor. As in the internalization assays, receptor 6 bound arrestin2 significantly better than {Delta}ST, however its desensitization rate was not significantly different. These results indicate that residues 1, 4, and 7 can individually support internalization, desensitization, and arrestin binding, with residue 6 possibly playing a minor role in these functions.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Previous work by our laboratory has shown that FPR internalization and desensitization are dependent upon the phosphorylation of different domains of the C terminus. Specifically, internalization of the FPR requires available serine/threonine residues in either the A site (Ser328 to Thr334) or the B site (Thr336 to Thr339), whereas desensitization requires available serine/threonine residues at either Ser328-Thr329 or Thr331-Ser332 within the A site in addition to the B site (Thr334–Thr339) residues. Not only did this finding suggest that distinct mechanisms may be responsible for desensitization and internalization of the FPR, it also raised the question of which serine or threonine residues within these regions regulate the processes of desensitization and internalization. A more detailed analysis of individual and combinatorial serine and threonine residues within the FPR C terminus was undertaken to answer these questions.

The results of the present study indicate that, of the serine and threonine residues defined by {Delta}C and {Delta}D, sites 1, 3, and 4 play dominant roles in the processes of desensitization and arrestin2 binding, whereas site 2 plays a minor contributory role. Deconstruction of the {Delta}C mutant provided the first clue that the location of serine/threonine residues, and not just the number of available residues, is important in internalization, desensitization, and arrestin2 binding. The different levels of internalization and arrestin2 binding observed in three- and four-site mutants support this conclusion. With the {Delta}C mutants we also observed that a particular site could have an inhibitory effect on regulatory processes. This was evidenced by the addition of site 5 (Thr334) to mutant 3467, which resulted in decreases in internalization, desensitization, and arrestin2 binding. Furthermore, the increased level of arrestin2 binding of mutant 3467 as compared with mutant 3478, despite similar levels of desensitization, indicates that arrestin2 binding alone may not be completely responsible for receptor desensitization. Overall, a number of mutants demonstrate a lack of absolute correlation between the extent of arrestin binding and the extent of either receptor internalization or desensitization, suggesting the existence of additional and distinct mechanisms.

Deconstruction of the {Delta}A mutant gave additional evidence that individual serine/threonine residues may have significantly positive or negative effects on internalization, desensitization, and arrestin2 binding. Mutants 56 and 58, both of which contain residue 5 (Thr334), displayed decreased levels of internalization, desensitization, and arrestin2 binding compared with mutants 78, 67, and 567. Similar to what was observed in mutants 3467 and 34567, the addition of site 5 (Thr334) to mutant 67 resulted in a decrease in arrestin2 binding. The observation that isolating site 7 from the other serine or threonine residues of the {Delta}A mutant-rescued desensitization led to the design of single-site receptor mutants. Of the eight single-site mutants, three (mutants 1, 4, and 7) demonstrated wild-type levels of internalization, desensitization, and arrestin2 binding. Our results indicate that a serine residue at position 1 (Ser328), 4 (Ser332), or 7 (Ser338) is sufficient for efficient receptor desensitization as well as arrestin2 binding. Conversely, the presence of residues 5 (Thr334) and 8 (Thr339) seems to have an inhibitory effect on these processes in the absence of residue 7 (Ser338).

The positive roles of sites 1, 4, and 7 were apparent, but not as absolute, in many of the more complex mutants. An example of the complex interactions between serine and threonine residues in the FPR C terminus can be seen when the data obtained for mutants having an intact residue 7 (Ser338) is examined. When residue 7 is present alone in the C terminus, the mutant receptor internalizes, desensitizes, and binds arrestin2 comparably to wild type. The addition of residue 8 does not significantly affect this functionality; however, the further addition of both residues 5 and 6 inhibits desensitization and arrestin binding of the receptor. Similarly, the {Delta}A mutant receptor, which possesses residues 5, 6, 7, and 8, does not desensitize or bind arrestin, but this functionality is fully restored upon addition of residues 1 or 3. Clearly, the interplay between C-terminal serine and threonine residues of the FPR represents a complex regulatory system governing FPR function.

Recent work by our laboratory has suggested that the pattern of phosphorylation sites within the FPR C terminus regulates arrestin affinity for receptor, its ability to induce changes in agonist affinity, as well as the intracellular trafficking pattern of internalized receptor ( 15, 16, 17). Oakley et al. (20, 21) have shown that receptor/arrestin interactions regulate the trafficking of the beta2-adrenergic and vasopressin V2 receptors. These interactions are likely based on C terminus phosphorylation patterns, because exchanging the C terminus of the beta2-adrenergic receptor for that of the vasopressin V2 receptor results in a trafficking pattern typical of the vasopressin V2 receptor ( 20, 21). These observations on the regulation of receptor function by receptor/arrestin interactions are consistent with our current results demonstrating both positive and negative effects of individual Ser/Thr residues in the FPR C terminus.

In addition to the FPR, a complex regulatory role for serine/threonine phosphorylation has also been reported for the M2 muscarinic receptor. Like the FPR, the M2 muscarinic receptor is phosphorylated at two sites of serine and threonine residues. In contrast to the FPR, these sites are located in the third cytoplasmic loop of the receptor and not the carboxyl tail ( 22, 23). In a study using phosphorylation-deficient mutants of the M2 muscarinic receptor, Lee et al. ( 24) observed a complex relationship between receptor phosphorylation and internalization/arrestin interaction. Clearly, the adaptive processes of internalization, desensitization, and arrestin2 binding are dependent upon the pattern of receptor phosphorylation and not merely the number of phosphorylation sites.

It is noteworthy that residues 1, 4, and 7, which, when present in isolation are able to promote internalization, desensitization, and arrestin binding, represent the only three serines present in the central region of the FPR C terminus. Previous work has shown that threonine residues of the FPR C terminus are indeed phosphorylated upon ligand binding ( 19). The fact that threonine residues are phosphorylated in the wild-type receptor suggests that these residues may be involved in either negative regulatory functions or other aspects of receptor function/regulation. Recent evidence consistent with the hypothesis that differences in phosphorylation patterns result in specific receptor functions comes from publications by Kim et al. ( 25) and Ren et al. (26). Their results, based on manipulations of GRK and arrestin expression, suggest that altering GPCR phosphorylation patterns results in distinct arrestin-mediated signaling functions, specifically that GRK 4/5 phosphorylation of the angiotensin II and the V2 vasopressin receptor stimulates arrestin-mediated ERK1/2 activation, whereas GRK 2/3 phosphorylation does not ( 25, 26). Although the phosphorylation sites favored by different GRKs are not well understood, the authors of both studies speculate that GRKs may have member-specific patterns of phosphorylation that in turn produce specific conformational changes in arrestin, consequently affecting its function. In effect, the phosphorylation pattern could instruct arrestin as to what function to perform. These conclusions support our finding that internalization, desensitization, and arrestin binding are regulated by differential phosphorylation patterns in the FPR C terminus. Furthermore, these results combined with our own suggest that agonist-induced phosphorylation of GPCR cytoplasmic domains constitute a combinatorial code, in that specific patterns of phosphorylation produce differing regulatory effects on receptor signaling and trafficking. Expanding upon the results of Ren et al. ( 26) and Kim et al. ( 25), it is likely that the mutant FPR receptors examined in our study could represent phosphorylation patterns of different members of the GRK family, and this in turn could provide the basis for the differing levels of internalization, desensitization, and arrestin2 binding.

As one of the first extensive studies of individual Ser/Thr residues in a GPCR, this study provides insight into the structural requirements necessary for receptor internalization, desensitization, and arrestin interaction. Our results indicate that the pattern of serine and threonine residues, and not simply their number, has a significant effect on receptor regulation. Until a high-resolution crystal structure of the FPR C terminus is available (particularly in a complex with arrestin), improvements to the current understanding of GPCR-arrestin interactions will rely upon similar mutant studies of the FPR and other GPCRs.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by National Institutes of Health Grant AI36357 (to E.R.P.) and a Geis Foundation Fellowship from the University of New Mexico Cancer Research and Treatment Center/Spatiotemporal Modeling Center (to R.M.P.). Flow cytometry data and confocal images in this study were generated in the Flow Cytometry and Fluorescence Microscopy Facilities, respectively, at the University of New Mexico Health Sciences Center, which received support from National Center for Research Resources (NCRR) 1 S10 RR14668, National Science Foundation MCB9982161, NCRR P20 RR11830, National Cancer Institute R24 CA88339, the University of New Mexico Health Sciences Center, and the University of New Mexico Cancer Center. Back

2 Address correspondence and reprint requests to Dr. Eric R. Prossnitz, Department of Cell Biology and Physiology, University of New Mexico, Albuquerque, NM 87131. E-mail address: eprossnitz{at}salud.unm.edu Back

3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; GRK, GPCR kinase; FPR, N-formyl peptide receptor; fMLF, N-formyl-met-leu-phe; 6pep, N-formyl-Nleu-Leu-Phe-Nleu-Tyr-Lys; EGFP, enhanced GFP. Back

Received for publication November 9, 2005. Accepted for publication February 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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