Role of the C terminus of the interleukin 8 receptor in signal transduction and internalization.

Interleukin 8 (IL-8) is a potent neutrophil chemoattractant and activator. Two IL-8 receptor subtypes, A and B, are expressed in neutrophils. In this work, we analyzed the role of the C terminus domain of the IL-8 receptor on the signal transduction and receptor internalization mechanisms. The IL-8 receptor A was tagged with an epitope corresponding to the monoclonal antibody 1D4 to monitor the localization of the IL-8 receptor. We demonstrated IL-8-dependent receptor internalization by monitoring the density of surface 125I-labeled IL-8 binding sites and by immunofluorescence microscopy. Truncation of the last 27 amino acids of the IL-8 receptor A severely impaired the IL-8-induced internalization of the receptor. Of importance was the observation that binding of IL-8 to receptors A and B triggered a dramatically faster rate of internalization of receptor B than receptor A, suggesting that the heterologous C termini among receptor subtypes modulate the rate of internalization of IL-8 receptors. However, substitution of the C terminus of the receptor subtype A for the C terminus of receptor B reduced the internalization rate of receptor A. Furthermore, we found that the rate of internalization of IL-8 receptor B triggered by IL-8 was faster than the one induced by the IL-8-related peptide, melanoma growth stimulatory activity. Studies with human neutrophils pretreated with 100 nM IL-8 for 5 min revealed a positive and a negative calcium response mediated by receptors A and B, respectively. In contrast, neutrophils pretreated with melanoma growth stimulatory activity showed positive calcium responses to both receptors A and B. These data suggest that the neutrophil responses mediated by IL-8 are modulated by the rate of internalization of receptors.

Interleukin 8 (IL-8) is a potent neutrophil chemoattractant and activator. Two IL-8 receptor subtypes, A and B, are expressed in neutrophils. In this work, we analyzed the role of the C terminus domain of the IL-8 receptor on the signal transduction and receptor internalization mechanisms. The IL-8 receptor A was tagged with an epitope corresponding to the monoclonal antibody 1D4 to monitor the localization of the IL-8 receptor. We demonstrated IL-8-dependent receptor internalization by monitoring the density of surface 125 I-labeled IL-8 binding sites and by immunofluorescence microscopy. Truncation of the last 27 amino acids of the IL-8 receptor A severely impaired the IL-8-induced internalization of the receptor. Of importance was the observation that binding of IL-8 to receptors A and B triggered a dramatically faster rate of internalization of receptor B than receptor A, suggesting that the heterologous C termini among receptor subtypes modulate the rate of internalization of IL-8 receptors. However, substitution of the C terminus of the receptor subtype A for the C terminus of receptor B reduced the internalization rate of receptor A. Furthermore, we found that the rate of internalization of IL-8 receptor B triggered by IL-8 was faster than the one induced by the IL-8-related peptide, melanoma growth stimulatory activity. Studies with human neutrophils pretreated with 100 nM IL-8 for 5 min revealed a positive and a negative calcium response mediated by receptors A and B, respectively. In contrast, neutrophils pretreated with melanoma growth stimulatory activity showed positive calcium responses to both receptors A and B. These data suggest that the neutrophil responses mediated by IL-8 are modulated by the rate of internalization of receptors.
Interleukin 8 (IL-8) 1 is a major mediator of transendothelial migration and activation of neutrophils at the site of inflammation (Oppenheim et al., 1991). Two IL-8 receptor subtypes, designated as type A and B, have been identified in human and rabbit neutrophils (Holmes et al., 1991;LaRosa et al., 1992;Thomas et al., 1991;Prado et al., 1994). These receptors belong to the superfamily of G-protein-coupled receptors. IL-8 receptor subtype A binds with high affinity to IL-8 and with low affinity to structurally related peptides MGSA (melanoma growth stimulating activity) and NAP-2 (neutrophil activating peptide-2) (Lee et al., 1992;LaRosa et al., 1992). In contrast, IL-8 receptor B binds with high affinity to IL-8 and MGSA and moderate affinity to NAP-2 (LaRosa et al., 1992;Lee et al., 1992). Termination of neutrophil responses at the site of injury is thought to be a key step in the regulation of the inflammatory process. The mechanisms of termination of receptor signals mediated by IL-8 are unknown. Studies with other Gprotein-coupled receptors have suggested that agonist binding to receptors triggers multiple structural and temporal changes of the receptor, leading to the generation of activation signals and termination signals for the phosphorylation, down-regulation, and internalization of receptors (Sibley et al., 1987). Several reports have also suggested that these signals are generated via independent pathways. For example, substitution of phosphorylated residues of ␣ 2a -adrenergic receptor by Ala abolished desensitization without affecting internalization of the receptor (Eason et al., 1995). Furthermore, it appears that different internalization pathways operate in various G-protein coupled receptors. Thus, C termini domains are involved in internalization of angiotensin II receptor (Thomas et al., 1995), ␤ 2 -adrenergic receptors (Liggett et al., 1993), parathormone receptor (Huang et al., 1995) and ␣ 1B -adrenergic receptors (Lattion et al., 1994), whereas the third intracellular loop regulates the internalization of muscarinic receptors (Pals-Rylaarsdam et al., 1995). The role of internalization in several G-protein-coupled receptors is unclear. Although internalization of the receptor into an intracellular compartment appears to play a role in the resensitization of the ␤ 2 -adrenergic receptors (Barak et al., 1994). The role of the C terminus in signaling and regulation of neutrophil chemoattractant receptors is unknown. Although recently Ben-Baruch et al. (1995) has shown that truncation of the distal C terminus of the IL-8 receptor B does not affect the IL-8-dependent chemotaxis in transfected CHO cells. In this study, we characterize the internalization profile of the IL-8 receptor subtypes, the role of the C-terminal tail in internalization, and the potential role of internalization of IL-8 receptors in regulating the IL-8-dependent neutrophil responses. Construction of Plasmids-The cDNAs encoding for the human and rabbit IL-8 receptors, types A and B, were subcloned into the pRc/CMV mammalian expression vector. F3RTag cDNA, encoding the rabbit IL-8 receptor A containing the 8-amino acid epitope for Mab 1D4 at the C terminus, was synthesized by PCR using the sense oligonucleotide, 5Ј-GAT ATC GAA TTC AAG CTT ACT GTG GCC GTA ATG GAA GTA AAC-3Ј (primer A) and the antisense oligonucleotide, 5Ј-GAT ATC GAA TTC TCT AGA TTA TGC AGG TGC CAC CTG AGA GGT TTC GAG ATT TGA AGG CAC GTT GGT-3Ј (primer B). The sense oligonucleotide contained a HindIII site, whereas the antisense oligonucleotide contained a XbaI site and a sequence encoding the epitope for the Mab 1D4 (ETSQVAPA) (Mackenzie, 1984). The PCR product (F3RTag) was digested with HindIII and XbaI and ligated to pRc/CMV. The tag sequence and the fidelity of the cloned PCR were confirmed by DNA sequencing.

Materials-Oligonucleotides
The F3RDel cDNA, encoding the rabbit IL-8 receptor A with 27 amino acids truncated at its C terminus, was synthesized by PCR using primer A and an antisense oligonucleotide, 5Ј-GAT ATC GAA TTC TCT AGA TTA TGC AGG TGC CAC CTG AGA GGT TTC GCG GGC CGC AAG CAT CTT-3Ј (primer C). Primer C was designed to contain a XbaI site, a nucleotide sequence encoding the Mab 1D4, and six amino acids from Arg-327 to Lys-322. The PCR product (F3RDel) was digested with HindIII/XbaI and ligated to pRc/CMV.
To construct the chimeric receptor, the 121-base pair PvuII/XbaI of 5B1a (rabbit IL-8 receptor B) encoding the last 39 amino acids was excised from pRC/CMV-5B1a and subcloned into the PvuII/XbaI-digested pRC/CMV-F3R (rabbit IL-8 receptor A) backbone. The fidelity of the chimeric receptor was confirmed by DNA sequencing.
Transfection and Ligand Binding of IL-8 Receptors-CHO cells were transfected with cDNA constructs using TransfectACE according to the manufacturer's instructions. Transfected CHO cells were selected in the presence of 500 g/ml G418 and by 125 I-labeled IL-8 binding. 125 I-Labeled IL-8 radioligand binding assays were performed as described .
Inositol Phosphate Determination-CHO cells were grown in 10-cm tissue culture dishes and incubated with 5 Ci/ml myo-[ 3 H]inositol for 16 to 24 h in inositol-free Dulbecco's modified Eagle's medium supplemented with 10% dialyzed fetal bovine serum. After labeling, cells were washed and detached with phosphate-buffered saline (PBS) containing 3 mM EDTA. Cells were pelleted and resuspended at a concentration of 4 ϫ 10 5 cells/ml in Dulbecco's PBS containing 0.9 mM CaCl 2 , 1 mg/ml glucose, 20 mM Hepes, pH 7.4, and 20 mM LiCl. Cells were stimulated with 100 nM IL-8 for 10 and 30 min. Incubations were terminated by removing medium and adding three volumes of chloroform/methanol. Extraction of total inositol phosphates on Dowex AG1-X8 formate form resin was performed as described (Blount and Krause, 1993).
Intracellular Calcium Measurements-Cells were detached, pelleted, and resuspended in a physiological buffer solution containing 140 mM NaCl, 5 mM KCl, 1 mM MgS0 4 , 1 mM CaCl 2 , 1 mM Na 2 HP0 4 , 5 mM glucose, 20 mM HEPES (pH 7.4), and 1 mg/ml bovine serum albumin. Cells were loaded with 5 M Indo-1AM for 1 h at room temperature. Cells were washed and resuspended at a density of 1 ϫ 10 7 cells/ml with buffer solution. Cells were stimulated with 100 nM IL-8, and fluores- cence intensity was measured at 37°C using an excitation wavelength of 330 nm and an emission wavelength of 405 nm (Grynkiewicz et al., 1985).
For studies of calcium responses in neutrophils, blood was drawn from healthy human donors and layered in a Mono-Poly Resolving Medium (ICN Biomedical, Inc., Aurora, Ohio). Neutrophils were isolated in accordance to the manufacturer's instructions, then suspended in physiological buffer solution at a concentration of 1 ϫ 10 7 cells/ml and loaded with 5 M Indo-1AM for 30 min at room temperature. Neutrophils were pretreated with 100 nM IL-8 or 100 nM MGSA at 37°C for 5 min, washed with ice-cold physiological buffer, and pelleted at 1000 ϫ g for 10 min at 4°C. Cells were incubated at 37°C at the indicated time points and then tested for agonist-dependent intracellular calcium rise. MGSA and IL-8 were added sequentially to the cuvette containing neutrophils to assess the responses mediated by IL-8 receptors B and A, respectively.
Immunofluorescence Microscopy-Immunofluorescent staining of IL-8 receptors in permeabilized CHO cells was carried out as described by Papkoff et al. (1983). In brief, CHO cells were grown on microscope slides for 24 h. Cells were then incubated at 37°C in the presence or absence of IL-8 for various times. Cells were fixed with 3% formaldehyde in the presence of 2% sucrose and permeabilized with an ice-cold solution containing 0.5% Triton X-100, 0.3 M sucrose, 0.003 M MgCl 2 , 0.05 M NaCl, and 0.01 M HEPES (pH 7.4) in PBS for 5 min. Permeabilized cells were incubated with 50 g/ml of monoclonal antibody (ID4) for 30 min. Antibody binding was detected by the addition of a 1:20 dilution of fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Boehringer Mannheim). Fluorescence was analyzed using a Zeiss fluorescence microscope with epi-illumination. Images were detected using a Canon camera and an ASA 400 Kodak film.
IL-8 Receptor Internalization-Stably transfected CHO cells were incubated for various times with 100 nM of IL-8 at 37°C. Cells were washed with ice-cold PBS containing 20 mM HEPES, pH 7.4, to remove unbound IL-8. To strip surface-bound IL-8, cells were treated with 0.2 M acetic acid (pH 3.0) containing 0.5 M NaCl for 5 min at 4°C. The number of binding sites was then determined by incubating the cells at 4°C for 2 h with 1 nM 125 I-labeled IL-8 either alone (total binding) or in the presence of 100 nM IL-8 (nonspecific binding). Cells were washed three times with ice-cold PBS and solubilized with 0.2% SDS. Cellassociated radioactivity was determined in a ␥ counter. Data were expressed as the percentage of specific binding in the absence of IL-8 pretreatment.

RESULTS AND DISCUSSION
The Epitope-tagged Receptor-An 8-amino acid epitope (tag) corresponding to the Mab 1D4 was fused to the C-terminal end of receptor A to monitor the localization of the IL-8 receptors. As shown in Fig. 1a, the tagged receptor exhibited a similar binding profile as the wild-type receptor. As expected for IL-8 receptor A, high affinity binding to IL-8 and very little binding to the structurally related peptide MGSA and NAP-2 were demonstrated. Most importantly, the activation of the tagged receptor was similar to the wild-type receptor, as shown by the IL-8-dependent inositol phosphate formation or IL-8-dependent rise in intracellular Ca 2ϩ assays (Fig. 1, b and c). These data indicate that the epitope tag fused at the C terminus of the receptor did not affect the binding site for IL-8 or the mechanism for receptor activation. These results are in agreement with previous studies indicating that fusion of epitope tags to the C-terminal end of the IL-8 receptor did not affect the binding to IL-8 (Gayle et al., 1993).
Internalization of the IL-8 Receptor A-Agonist binding to the IL-8 receptor A at 37°C reduced, in a time-dependent fashion, the number of surface 125 I-labeled IL-8 binding sites in CHO cells expressing the wild-type and the tagged receptor A (Fig. 2a). No major difference was observed in the time course of receptor sequestration among the wild-type and tagged receptors. These results have suggested that agonist binding triggers receptor internalization. By immunofluorescence microscopy, we determined the localization of the tagged IL-8 receptor A expressed in CHO cells pretreated with and without IL-8. As shown in Fig. 2b, untransfected CHO cells exhibited background fluorescence. CHO cells expressing the tagged IL-8 receptor showed fluorescence on the cell surface. On the other hand, cells expressing the tagged receptors pretreated with IL-8 exhibited fluorescence in the perinuclear region of the cell and low fluorescence in the cell surface. These findings indicate that the loss of surface 125 I-labeled IL-8 binding reflects receptor internalization.
Role of the C Terminus in Receptor Internalization-The C terminus domain of various G-protein-coupled receptors have been implicated in the mechanisms of G-protein activation, receptor desensitization, and internalization (Dohlman et al., 1991). To examine the role of the C terminus of the IL-8 receptor, CHO cells were transfected with a cDNA construct encoding the rabbit IL-8 receptor A deleted of its last 27 amino acids. All potential phosphorylation sites (Ser and Thr) at the C terminus were removed in the truncated receptor. CHO cells expressing the truncated receptor exhibited a binding profile similar to the wild-type receptor (Fig. 3a), showing that truncation of the last 27 amino acids did not affect the binding domain of the IL-8 receptor. The IL-8-dependent inositol phosphate production and the IL-8-dependent calcium rise were similar in both the wild-type and the mutant receptor (Fig. 3, b  and c), indicating that the mechanisms of receptor activation were unaffected by deletion of the last 27 amino acids of the IL-8 receptor A. These data are consistent with recent findings indicating that deletion of the distal region of the C terminus of FIG. 2. Internalization and localization of receptors in CHO cells. a, internalization of wild-type rabbit IL-8 receptor A and tagged receptors expressed in CHO cells. Cells expressing wild-type (q), tagged receptors (f) were incubated with 100 nM IL-8 at 37°C for the indicated times. Each point was determined in triplicate. b, localization of tagged receptors in CHO cells. Untransfected CHO cells (left panel) and transfected cells with epitope-tagged receptor cDNA (middle and right panels) are indicated by arrows. Cells transfected with tagged receptor cDNA were stimulated with or without 100 nM IL-8 for 60 min and were fixed, permeabilized, and stained to allow for receptor localization.
the IL-8 receptor did not affect the IL-8-dependent chemotaxis of transfected cells (Ben-Baruch et al., 1995). However, the IL-8-induced internalization of the truncated IL-8 receptor A was severely impaired (Fig. 4a). This result suggests that the distal C terminus domain is a major component of the internalization pathway of the IL-8 receptor. Since the homologous IL-8 receptor subtypes A and B exhibit high sequence diversity in their distal C termini, we examined whether IL-8 receptors shows different internalization profiles. Indeed, we found that the rate of internalization of receptor subtype B was dramatically faster than the receptor subtype A (Fig. 4c). After a 5-min exposure to 100 nM IL-8, more than 95% of IL-8 receptors B and less than 40% of receptors A were internalized. Of importance is that similar rates of IL-8-dependent internalization were observed among human and rabbit receptors of the same subtype. These results are in good agreement with a recent study in human neutrophils indicating that the IL-8-dependent internalization of receptor B is faster than receptor A . These results suggest that the C terminus domains of IL-8 receptors modulate the extent and rate of internalization of receptors. To examine this hypothesis, we created an IL-8 receptor chimera (A/B) in which the C terminus of IL-8 receptor A was substituted by the C terminus of IL-8 receptor B. As indicated in Fig. 4c, the chimeric receptor exhibited lower rates of internalization than the wild-type receptor A. This finding supports the idea that the C terminus tail of IL-8 receptor B cannot regulate the rate of internalization of IL-8 receptor A. It is possible that the C terminus of receptor B is not well coupled to the internalization pathway of the IL-8 receptor A.
To determine whether the type of ligand dictates the internalization profile of the receptors, we examined the MGSA versus IL-8-dependent internalization of the IL-8 receptor B. MGSA and IL-8 bind with similar high affinity to IL-8 receptor B, and both ligands are equally potent in inducing a Ca 2ϩ response in cells transfected with IL-8 receptor B (LaRosa et al., 1992;Lee et al., 1992). As shown in Fig. 5, MGSA triggered a slower rate of internalization of receptor B than IL-8. These data strongly support the idea that binding of IL-8 and MGSA to receptor B induces different structural changes of the receptor, leading to the generation of distinct internalization pathways. Previous studies with the ␤ 2 -adrenergic receptor system have suggested that internalization or sequestration is required for receptor resensitization (Barak et al., 1994). To examine the role of internalization of receptors in neutrophils, we monitored the agonist-dependent Ca 2ϩ responses in human neutrophils pretreated for 5 min with a saturating concentration of IL-8 (100 nM) or MGSA (100 nM). Under these conditions, IL-8-induced internalization of more than 95% of IL-8 receptor B but only less than 40% of IL-8 receptor A (Fig. 4b). On the other hand, MGSA induced internalization of less than 40% of IL-8 receptor B (Fig. 5). Following agonist treatment, neutrophils were incubated at 37°C and sequencially challenged with 100 nM MGSA and 100 nM IL-8 to induce calcium responses mediated by receptors B and A, respectively. As shown in Fig. 6a, MGSA and IL-8 induce a rise in intracellular Ca 2ϩ in untreated neutrophils. However, IL-8-pretreated neutrophils incubated for a short time (5 min) at 37°C do not respond to MGSA, but the neutrophils did respond to IL-8 (Fig. 6b). Longer incubation times (1 h) were needed to obtain a calcium response with MGSA (Fig. 6c). These results suggested that the negative response observed with MGSA is primarily due to the rapid IL-8-induced rate of internalization of all of the IL-8 receptors B (Fig. 4b). The response to IL-8 is probably mediated by IL-8 receptor A that did not get completely internalized by IL-8 (Fig.  4b). Consistent with this idea, MGSA-pretreated neutrophils, in which MGSA induces a slow rate of internalization of receptors B (Fig. 5), responded quickly to both MGSA and IL-8 (Fig.  6d). This study strongly supports the hypothesis that internalization of the IL-8 receptor is a major mechanism for modulating the neutrophil responses elicited by IL-8 and its functionally related peptides such as MGSA. FIG. 6. Ca 2؉ responses mediated by IL-8 receptors A and B in human neutrophils. Neutrophils were loaded with 5 M Indo-1AM as described under "Experimental Procedures." Neutrophils were pretreated with vehicle alone (a), with 100 nM IL-8 (b and c), or 100 nM MGSA (d) at 37°C for 5 min and then washed with ice-cold PBS. Pretreated neutrophils were incubated at 37°C for the indicated times and then challenged with 100 nM MGSA or 100 nM IL-8 to activate IL-8 receptors B and A, respectively.