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Characterization of a Dipeptide Motif Regulating IFN- Receptor 2 Plasma Membrane Accumulation and IFN- Responsiveness¹

Sergio D. Rosenzweig^2,*, Owen M. Schwartz, Margaret R. Brown, Thomas L. Leto^* and Steven M. Holland^3,*,

^* Laboratory of Host Defenses, Laboratory of Clinical Infectious Diseases, and Biological Imaging Facility, National Institutes of Allergy and Infectious Diseases; and Clinical Immunology Laboratory, Warren Grant Magnuson Clinical Center, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The IFN-R complex is composed of two IFN-R1 and two IFN-R2 polypeptide chains. Although IFN-R1 is constitutively expressed on all nucleated cells, IFN-R2 membrane display is selective and tightly regulated. We created a series of fluorescent-tagged IFN-R2 expression constructs to follow the molecule’s cell surface expression and intracellular distribution. Truncation of the receptor immediately upstream of Leu-Ile 255–256 (254X) created a receptor devoid of signaling that overaccumulated on the cell surface. In addition, this truncated receptor inhibited wild-type IFN-R2 activity and therefore exerted a dominant negative effect. In-frame deletion (2552) or alanine substitution (LI255–256AA) of these amino acids created mutants that overaccumulated on the plasma membrane, but had enhanced function. Single amino acid substitutions (L255A or I256A) had a more modest effect. In-frame deletions upstream (2532), but not downstream (2572), of Leu-Ile 255–256 also led to overaccumulation. A truncation within the IFN-R2 Jak2 binding site (270X) led to a mutant devoid of function that did not overaccumulate and did not affect wild-type IFN-R2 signaling. We have created a series of novel mutants of IFN-R2 that have facilitated the identification of intracellular domains that control IFN-R2 accumulation and IFN- responsiveness. In contrast to IFN-R1, not only dominant negative, but also dominant gain-of-function, mutations were created through manipulation of IFN-R2 Leu-Ile 255–256. These IFN-R2 mutants will allow fine dissection of the role of IFN- signaling in immunity.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Some receptors are internalized continuously (ligand-independent endocytosis), whereas others remain exposed on the cell surface until ligand is bound, after which they become susceptible to endocytosis (ligand-dependent endocytosis). Regions regulating receptor fate comprise a short part of the cytoplasmic tail and are usually located close to the plasma membrane (1, 2). IFN-R1 and IFN-R2 belong to the class 2 cytokine receptor family, molecules devoid of intrinsic phosphatase or kinase activities; both chains share similar genomic and protein structures, and overall homology is close to 50% (3). However, the IFN-R1 intracellular domain contains 221 amino acids, whereas that of IFN-R2 contains only 66 amino acids.

The IFN-R1 intracellular domain has a leucine-isoleucine (LI)⁴ dipeptide at positions 270–271 that is thought to be involved in receptor endocytosis (4). Truncations in the IFN-R1 intracellular domain upstream of this dipeptide lead to plasma membrane overaccumulation of the mutated receptor (4, 5). In humans, heterozygous mutations in the proximal intracellular domain that truncate the molecule and delete the LI dipeptide lead to accumulation of the mutant receptor and exert a dominant negative effect on IFN- responsiveness (5). Plasma membrane overaccumulation, preserved IFN- binding capacity, and disruption of the IFN-R complex stoichiometry in the absence of a functional IFN-R1 intracellular domain are thought to underlie the dominant negative effect (4, 5).

Although IFN-R1 is constitutively expressed on all nucleated cells and recycles after IFN- stimulation, IFN-R2 membrane display is more difficult to detect and appears to be tightly regulated. An endocytosis or recycling domain in IFN-R2 has been hypothesized. Intracellular stores of IFN-R2 are usually significantly higher than those present on the cell surface, but the factors regulating receptor display and fate are not completely understood (3, 6, 7, 8, 9, 10, 11, 12). Based on the structural homologies between IFN-R1 and IFN-R2, we designed different IFN-R2 expression constructs to explore the only LI determinant (255–256) in the intracellular domain of IFN-R2.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Constructs

The cDNA sequences for the IFN-R2 mature protein (MP) and signal peptide (SP) were separately cloned into the plasmid-enhanced GFP-C1 vector (BD Clontech, Palo Alto, CA), resulting in the construct 5'... SP/GFP/MP ... 3', expressing a GFP-IFN-R2 fusion protein.

American Type Culture Collection (Manassas, VA) IFNGR2 was used as template (ATCC 79923; EcoRI-EcoRI cloned in pBluescript vector). We engineered an EcoRI site 3 bp upstream of the first codon of the IFNGR2 MP sequence (forward primer, 5'-CGCCAGACCCGAATTCCCAGCTGCTGCCCGCTCCTCAGCAC-3' (the first codon of the IFNGR2 MP sequence is underlined); reverse primer, downstream of the ATCC IFNGR2 distal EcoRI cloning site, 5'-GAGCGCGCGTAATACGACTCACTATAGGG-3'). The resulting PCR product and the pEGFP-C1 vector were EcoRI digested (Invitrogen Life Technologies, Carlsbad, CA), purified (Geneclean II; Bio 101, Carlsbad, CA) and ligated with the TaKaRa ligation kit following the producer’s recommendations (TaKaRa, Shuzo, Japan). DH5--competent bacteria were transformed and grown on Luria Bertoni broth/kanamycin plates. Colonies were selected and checked for insertion and orientation by PCR. Using the same IFNGR2 cDNA American Type Culture Collection clone as template, Eco47III sites were introduced by PCR on either side of the IFNGR2 SP cDNA sequence (forward primer, 5'-CGCCGCCGAGCGCTCCGGGGCCATG-3' (the first codon of the IFNGR2 SP cDNA sequence is underlined); reverse primer, 5'-GTACAGGCGTAGCGCTGGCTGCTGAGG-3'; Eco47III site created nine amino acids into the IFNGR2 MP cDNA sequence). The resulting PCR product and the GFP/IFNGR2 MP construct were Eco47III digested (Invitrogen Life Technologies), and the products were ligated to create the construct SP/GFP/IFNGR2. The inserts and boundaries were sequenced and confirmed to be appropriate. Expression of the GFP-IFN-R2 fusion protein could be detected by immunoblot using anti-GFP or anti-IFN-R2 Ab (Fig. 1).

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Detection of recombinant WT GFP-IFN-R2 fusion proteins in HEK 293 transfected cells. HEK 293 cells were transiently transfected with pEGFP-C1 empty vector (EV) and four different SP/GFP/IFNGR2 construct clones (1–4). Immunoblots were run in parallel. The pEGFP-C1 EV and the four SP/GFP/IFNGR2 clones expressed GFP (left). Only the SP/GFP/IFNGR2 clones express IFN-R2 protein (right). The same m.w. fusion proteins were detected with either anti-GFP mAb or anti-IFNR2 Ab.

Mutations 254X, 270X, LI255–256AA, L255A, I256A, 2532, 2552, and 2572 were separately introduced in the SP/GFP/IFNGR2 construct by site-directed mutagenesis following the manufacturer’s protocol (QuikChange Site Directed Mutagenesis Kit; Stratagene, La Jolla, CA). These mutations lead to the following mutant constructs: SP/GFP/254X, a stop codon was introduced after the fourth amino acid of the IFN-R2 intracellular domain, immediately upstream of the LI dipeptide (fifth and sixth intracellular amino acids); SP/GFP/270X, a stop codon was introduced after the 20th amino acid of the IFN-R2 intracellular domain (downstream of the LI motif, within the Jak2 binding site); SP/GFP/2532, the sequence for amino acids arginine-glycine 253–254 (third and fourth intracellular amino acids) was deleted in-frame from the IFN-R2 intracellular domain; SP/GFP/2552, the sequence for amino acids leucine-isoleucine 255–256 (fifth and sixth intracellular amino acids) was deleted in-frame from the IFN-R2 intracellular domain; SP/GFP/2572, the sequence for amino acids lysine-tyrosine 257–258 (seventh and eighth intracellular amino acids) was deleted in-frame from the IFN-R2 intracellular domain; SP/GFP/LI255–256AA, the sequence for amino acids leucine 255 and isoleucine 256 was substituted with the sequence for alanine (fifth and sixth intracellular amino acids); SP/GFP/L255A, the sequence for amino acid leucine 255 was substituted with an alanine sequence (fifth intracellular amino acid); and SP/GFP/I256A, the sequence for amino acid isoleucine 256 was substituted with an alanine sequence (sixth intracellular amino acid).

All constructs were sequenced in their entirety to confirm the integrity of the relevant inserts and mutations. No mutations other than those specifically introduced were detected. Mutations individually introduced by site-directed mutagenesis into the wild-type (WT) construct resulted in the mutant constructs shown in Fig. 2.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Design of WT and mutant IFN-R2 expression constructs. , signal peptide; with EGFP inside, EGFP cassette; thick vertical lines, IFNGR2 mature protein coding sequence; , transmembrane domain; , Jak2 binding site.

HEK 293 (American Type Culture Collection, CRL-1573) cells were grown in DMEM (Invitrogen Life Technologies) with 10% FCS (Gemini Bio-Products, Woodland, CA), 2 mM L-glutamine (HyClone, Logan, UT), and antibiotics (Pen/Strep; Invitrogen Life Technologies). Cells were split every 3–4 days. SV40-transformed fibroblasts from an IFN-R2-deficient patient (13) were grown in Eagle’s MEM with 10% FCS (Gemini Bio-Products, Woodland, CA), 2 mM L-glutamine (HyClone), 1 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO), and antibiotics (Pen/Strep; Invitrogen Life Technologies).

Cells were seeded in six-well plates 24 h before transfections (1–2 x 10⁵ cells/well) and transfected over 8 h in OptiMEM (Invitrogen Life Technologies) with 5 µl of Lipofectin (Invitrogen Life Technologies) and 1 µg of DNA (HEK 293) or 2 µg of DNA (IFN-R2-deficient fibroblasts), following the lipofectin adherent-cell transient-transfection protocol. In WT-only or mutant-only experiments, 1 µg of the relevant vector was cotransfected with 1 µg of an irrelevant vector (pEGFP-C1). In WT-mutant combination experiments, 1 µg of WT vector was cotransfected with 1, 0.1, or 0.01 µg of the mutant vector, and pEGFP-C1 was added to bring the total amount of DNA to 2 µg. After transfection, cells were grown for 48 or 72 h before evaluation. Where indicated, cells were stimulated with IFN- (Actimmune; Intermune, Brisbane, CA; 1000 U/ml) for 15 min before harvest.

Based on microscopic evaluation of GFP expression, single-construct transfection efficiency varied between 15 and 25% for all constructs tested. -Galactosidase and pRL-Renilla were also used to normalize transfection efficiency, and no significant differences were found between the IFNGR2 expression vectors (not shown).

Human embryo kidney 293 cells (HEK 293) and SV40-transformed IFN-R2-deficient fibroblasts were transiently transfected with WT, mutant, or combinations of WT and mutant constructs. GFP-IFN-R2 fusion protein accumulation and distribution patterns were evaluated in HEK 293 cells, whereas functional studies were conducted in IFN-R2-deficient fibroblasts. IFN- responsiveness was evaluated by phospho-STAT1 (P-STAT1) accumulation (14).

Immunoblotting

Total cell lysates from transfected cells were prepared 48 or 72 h after transfection. Samples were resolved on 10% bis-Tris precast gels (Novex; Invitrogen Life Technologies) and transferred to 0.2-µm pore size polyvinylidene difluoride membranes (Invitrogen Life Technologies). Membranes were preblocked overnight at 4°C in a 5% blocking grade nonfat dry milk solution (Bio-Rad, Hercules, CA), probed for 1 h with the primary Ab, washed three times, and then reprobed for 30 min with HRP-conjugated secondary Ab. Blots were developed with the ECL Plus kit (Amersham Biosciences, Little Chalfont, U.K.) according to the manufacturer’s instructions. Where indicated, blots were stripped for 30 min at 50°C in 50 ml of a stripping solution (10 mM 2-ME, 2% SDS, and 62.5 mM Tris), reblocked, and reprobed.

Total cell lysates from WT and mutant transiently transfected HEK 293 cells were digested with EndoH (New England Biolabs, Beverly, MA) following the producer’s protocol. Samples were resolved in 10% bis-Tris precast gels (Novex; Invitrogen Life Technologies).

Primary Abs. Polyclonal rabbit-anti-human P-STAT1 (Tyr701) Ab (Cell Signaling, Beverly, MA) was used at a 1/1000 dilution. Mouse anti-human STAT1 mAb (Transduction Laboratories, Lexington, KY) was used at a 1/5000 dilution. Mouse anti-GFP mAb (Living Colors; BD Biosciences, Palo Alto, CA) was used at a 1/1000 dilution. Polyclonal rabbit-anti-human IFN-R2 Ab (Preclinical Biopharmaceutics Laboratory, Piscataway, NJ) was used at a 1/1000 dilution. Mouse anti-human -actin mAb (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a 1/5000 dilution.

Secondary Abs. Anti-mouse HRP-conjugated Ab (Amersham Biosciences) was used at a 1/10,000 dilution. Anti-rabbit HRP-conjugated Ab (Amersham Biosciences) was used at a 1/5,000 dilution.

Densitometry

Immunoblot films were digitized (Astra 1220U scanner, Photoshop 6.0 software, Adobe Systems, San Jose, CA) and analyzed with National Institutes of Health Image 1.63 software.

Confocal microscopy

Images were collected on a TCS-SP2 AOBS confocal microscope (Leica Microsystems, Mannheim, Germany) using a x63 oil immersion objective NA 1.32 at different zoom factors. GFP was excited using an argon laser at 488 nm, and Alexa Fluor was excited using a krypton laser at 568 mm. Differential interference contrast images were collected simultaneously with the fluorescence images using the transmitted light detector. Z stacks of images were collected using a step increment of 0.203 µm between planes. All pictures were taken at identical settings.

Transiently transfected HEK 293 cells were incubated with the anti-GFP Alexa Fluor-conjugated Ab for 1 h at 37°C (Molecular Probes, Eugene, OR), after which the Ab-containing medium was replaced with fresh medium. Confocal microscopy was performed without cell permeabilization.

IFN- binding assays

Recombinant human IFN- (PeproTech, Rocky Hill, NJ) was labeled with Cy5 (Amersham Biosciences, Piscataway, NJ) following the manufacturer’s instructions with some alterations. Briefly, IFN- was diluted in PBS, pH 7.4, the pH was adjusted to 9.3 with 1 M sodium carbonate, and IFN- was aliquoted into three equal fractions. Lyophilized Cy5 dye was reconstituted in PBS, pH 7.4, and the appropriate amounts were added to separate fractions of the IFN- to deliver 0.5, 1, and 2x normal dye/protein labeling ratios. The reaction was incubated at room temperature for 30 min, and the unconjugated dye was separated from the Cy5-IFN- conjugates by gel filtration. The resulting product, 0.5x Cy5-IFN-, was used to asses the ligand binding capacity of the WT and mutant IFN-R2 constructs.

HEK 293 cells were transiently transfected with WT and mutant IFNGR2 vectors. Cells were harvested 48 h after transfections, washed, and exposed to 0.5x Cy5-IFN- (0.5 µg/10⁶ cells) for 15 min at 4°C. The Cy5 geometric mean channel was analyzed by flow cytometry in GFP-positive (successfully transfected) cells. To verify that this labeled IFN- was able to bind the authentic receptor, we performed flow cytometry-based competition experiments with unlabeled IFN- mainly as previously described (15). Briefly, untransfected HEK 293 cells were incubated with 0.5x Cy5-IFN- (0.02 µg/10⁶ cells) for 15 min at 4°C. The 0.5x Cy5-IFN- was competed off with increasing amounts of unlabeled IFN- (0.002, 0.02, 0.5, 2.5, and 10 µg/10⁶ cells), and the bound Cy5 geometric mean channel was determined for each concentration.

Statistical analysis

Densitometry results from the different transfection conditions were analyzed by t test (two-sample, assuming equal variances; Excel statistical software; Microsoft, Redmond, WA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cellular accumulation

Variable levels of GFP-IFNGR2 fusion protein accumulation were detected with the different constructs tested (Fig. 3). Comparable levels of accumulation were seen in cells transfected with the WT vector or with mutants SP/GFP/2572, SP/GFP/L255I, SP/GFP/I256A, and SP/GFP/270X. Constructs devoid of the LI255–256 dipeptide, SP/GFP/254X, SP/GFP/2552, and SP/GFP/LI255–256AA, showed significantly higher accumulation levels. Mutant SP/GFP/2532 also showed increased levels of accumulation (Fig. 3).

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Detection of WT and mutant IFN-R2 fusion proteins in HEK 293 transfected cells. HEK 293 cells were transiently transfected with WT and mutant constructs, and GFP-IFN-R2 fusion protein accumulation was evaluated with anti-GFP mAb. Representative GFP-fusion protein accumulation patterns are shown. Densitometry was determined based on the GFP/-actin ratio. *, p < 0.05 compared with WT SP/GFP/IFNGR2.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. a, Accumulation and localization of GFP-IFN-R2 fusion proteins detected by confocal microscopy. HEK 293 cells were transiently transfected with pEGFP-C1 empty vector, WT SP/GFP/IFNGR2, or IFN-R2 mutant vectors. Untransfected cells are also shown with both phase and fluorescent microscopy. b, Flow cytometry-based binding analysis. One million HEK 293 cells (untransfected or transfected with different constructs) were stimulated with 0.5 µg of Cy5-IFN- during 15 min at 4°C. After gating in the GFP-positive cells (cells efficiently transfected with the GFP-tagged vectors), Cy5 geometric means were evaluated. Ten thousands events were acquired in a FACScan flow cytometer and analyzed using CellQuest (BD Biosciences, Mountain View, CA). A representative experiment is shown. c, Competition assay. One million HEK293 cells were simultaneously stimulated with Cy5-IFN- (0.02 µg) and increasing amounts of unconjugated IFN- (0.002, 0.02, 0.5, 2.5, and 10 µg) during 15 min at 4°C. Twenty-five thousand ungated events were acquired in a FACScan flow cytometer. Cy5 geometric mean channels were analyzed using CellQuest (BD Biosciences).

View larger version (44K): [in this window] [in a new window]   FIGURE 5. Confocal microscopy: internalization detection of cell-surface-expressed GFP fusion proteins. HEK 293 cells were transiently transfected with different vectors. After 48 h, cells were exposed during 1 h at 37°C to an Alexa Fluor-labeled anti-GFP Ab (1 µg/ml concentration). Cells were washed before confocal visualization. Alexa Fluor should be detected intracellularly in cells undergoing internalization of the cell surface-expressed GFP fusion protein. a, WT SP/GFP/IFNGR2-transfected cells show cell surface and intracellular detection of GFP (green; left panel). Alexa Fluor was also detected in the cell surface and intracellularly (red; right panel). b, SP/GFP/254X-transfected cells show cell surface and intracellular detection of GFP (green; left panel). Alexa Fluor is almost exclusively detected in the cell surface (red; right panel). c, SP/GFP/LI255–256AA-transfected cells show cell surface and intracellular detection of GFP (green; left panel). Alexa Fluor is almost exclusively detected in the cell surface (red; right panel).

Cell surface-expressed N-glycosylated proteins, such as IFN-R2, become EndoH resistant in the Golgi. Resistance to EndoH, a high mannose cleaving enzyme, helps to identify proteins properly processed in the Golgi. When digested with EndoH, the WT GFP-IFN-R2 fusion protein was predominantly EndoH-sensitive (Fig. 6), indicating that most of it had not left the Golgi, as shown in Fig. 4a. In contrast, the plasma membrane-overaccumulated fusion proteins produced by the mutants SP/GFP/254X and SP/GFP/LI255–256AA yielded more EndoH-resistant than EndoH-sensitive proteins (Fig. 6), suggesting that the majority of the fusion proteins produced by these vectors had made their way through the Golgi to the cell surface. As shown in Figs. 4a and 6, EndoH-resistant bands roughly correlated with the levels of cell surface-accumulated fusion proteins, whereas the EndoH-sensitive bands corresponded to the intracellular fusion proteins.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. EndoH digestion susceptibility of WT and mutant IFN-R2 fusion proteins. Ten micrograms of total cell lysates from transfected HEK 293 cells were digested with EndoH. EndoH (–), undigested samples; EndoH (+), digested samples. Densitometry was used to determine the EndoH resistant/EndoH sensitive ratio.

After determining that the WT construct SP/GFP/IFNGR2 restored IFN- responsiveness to transiently transfected IFN-R2-deficient fibroblasts, we examined the ability of our mutant constructs to do so. At the same time, we performed cotransfections of WT and mutant constructs to look for recessive or dominant effects (Fig. 7). As expected, constructs SP/GFP/254X (devoid of all but four amino acids of the intracellular domain) and SP/GFP/270X (devoid of the terminal portion of the intracellular domain, including part of the Jak2 binding site) could not alone support STAT1 phosphorylation after IFN- stimulation. In contrast, constructs SP/GFP/I256A and SP/GFP/2572 (not shown), supported normal phospho-STAT1 accumulation. More interestingly, the overaccumulating constructs SP/GFP/LI255–256AA and SP/GFP/2552 (not shown) showed significantly increased levels of phospho-STAT1 accumulation. Mutants SP/GFP/L255A and SP/GFP/2532 also supported increased levels of phospho-STAT1 accumulation, but to a lesser extent than SP/GFP/2552 and SP/GFP/LI255–256A (not shown).

View larger version (43K): [in this window] [in a new window]   FIGURE 7. IFN- responsiveness of IFN-R2-deficient fibroblasts transiently transfected with WT and mutant constructs. Cells were stimulated with IFN- (1000 U/ml) for 15 min before harvesting. Representative phospho-STAT1 (P-STAT1) accumulation patterns are shown. Densitometry was used to determine the phospho-STAT1/STAT1 ratio. *, p < 0.005 compared with SP/GFP/IFNGR2.

Although both chains of the IFN-R complex are needed for signaling, the extracellular domain of IFN-R1 is the only site of ligand binding, although the association of IFN-R2 may stabilize it (17). Because our mutants altered surface accumulation of IFN-R2, we examined whether there were any effects on IFN- binding conferred by these using a flow cytometry-based approach that allowed us to determine binding of IFN- specifically to transfected cells (15). The transiently transfected WT (SP/GFP/IFNGR2), dominant negative (SP/GFP/254X) and gain-of-function (SP/GFP/LI255–256AA) IFN-R2 mutants did not differ in terms of IFN- binding (Fig. 4b). The specificity of the assay was confirmed by the successful competition of Cy5-IFN- binding by unlabeled IFN- (Fig. 4c).

Dominant negative effects

SP/GFP/270X was incapable of supporting IFN- signaling when transfected alone. However, in equal cotransfections of WT SP/GFP/IFNGR2 to SP/GFP/270X, IFN- stimulation yielded WT levels of phospho-STAT1 accumulation (Fig. 7), indicating recessive function for SP/GFP/270X. In contrast, equal cotransfections of WT SP/GFP/IFNGR2 and SP/GFP/254X showed potent inhibition of IFN- signaling, indicating a dominant negative effect for the truncated receptor SP/GFP/254X (Fig. 7). Similar inhibitory effects were seen at low (1/10) and high (5:1, 10:1, and 100:1) WT:mutant transfection ratios (Fig. 8a). Interestingly, when the transfected cell incubation time was shortened from 72 to 48 h, which allowed for demonstrably less fusion protein accumulation, the inhibitory effect was not seen at 10:1 or 100:1 WT:mutant transfection ratios (Fig. 8b). These results suggest that the SP/GFP/254X dominant negative effect is accumulation dependent.

View larger version (21K): [in this window] [in a new window]   FIGURE 8. IFN- responsiveness of IFN-R2-deficient fibroblasts transiently transfected with different ratios of SP/GFP/254X. IFN-R2-deficient fibroblasts were cotransfected with different ratios of WT:SP/GFP/254X vectors (1:10, 1:1, 5:1, 10:1, and 100:1), with pEGFP-C1 added to maintain DNA at 2 µg for all transfections. Cells were harvested 72 h (a) or 48 h (b) after transfection. Cells were stimulated with IFN- (1000 U/ml) for 15 min immediately before harvest. Densitometry was used to determine the phospho-STAT1 (P-STAT1)/STAT1 ratio.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
IFN-R2 is thought to be involved in the control of many aspects of IFN- responsiveness, which, in turn, is involved in immunity, autoimmunity (18, 19, 20, 21, 22, 23), and tumor control (24, 25, 26, 27, 28). Manipulation of the intracellular domain of IFN-R2, most importantly the region around LI255–256, significantly modified receptor accumulation and IFN- responsiveness, enabling us to create both dominant negative and dominant gain-of-function receptors. In this manuscript we show that the effects of these mutants on receptor accumulation and, in turn, function are associated with impaired receptor internalization due to disruption of the critical intracellular LI motif. In WT-vector-transfected cells, the Alexa Fluor-labeled anti-GFP Ab bound to cell surface-expressed WT fusion protein was detected intracellularly after 1-h incubation. The fact that this happened without IFN- stimulation emphasizes that IFN-R2 recycling is independent of ligand binding (9). In contrast, virtually no intracellular anti-GFP Ab was detected in cells transfected with vectors where the LI255–256 dipeptide structure was not preserved. These results suggest that constructs with the dipeptide LI255–256 are capable of undergoing receptor internalization, whereas those without it are not.

By simultaneously binding clathrin and specific amino acid sequences on the cytosolic portion of transmembrane proteins, the adapter proteins 1–4 determine which proteins will be specifically included or excluded from budding transport vesicles (reviewed in Ref. 2). The adapter protein complex recognizes signals for sorting of transmembrane proteins to endosomes and lysosomes in the intracellular domains. The adapter protein complexes recognize different types of sorting signals, such as tyrosine-based (e.g., YXXØ, where Y is a tyrosine, and Ø a bulky hydrophobic amino acid) and dileucine-based motifs (e.g., leucine-leucine or LI) (2, 29). A segment at the proximal region of intracellular IFN-R2 contains features of both these motifs: 251-KYRGLIK-257 (tyrosine-based sorting signal in bold; dileucine-based sorting signal underlined). The first Leu of a dileucine-based sorting signal is generally invariant, and its substitution usually greatly decreases the potency of the signal; the second Leu can be replaced by an Ile without loss of activity (2, 29). This is consistent with our findings: whereas Ala substitution of Leu 255 (SP/GFP/L255A) caused increased receptor accumulation (Figs. 3 and 4a), Ala substitution of Ile 256 (SP/GFP/I256A) did not.

Interestingly, deleting the two amino acids upstream of L255 I256 (R253 and G254, 2532) also caused overaccumulation of the receptor. However, the presence of R253 and G254 in the mutant truncated at 254 (254X) was not sufficient to maintain receptor at normal levels. The mutant construct SP/GFP/254X, with only four intracellular amino acids, overaccumulated in the plasma membrane and exerted a dominant negative effect on IFN- response. Bach et al. (30) created an IFNGR2 mutant construct devoid of all but three intracellular domain amino acids (IC) and showed plasma membrane overaccumulation and inability to support IFN- signaling. They did not explore possible dominant negative effects.

Intracellular, but more significantly cell surface, overaccumulation of GFP-IFN-R2 fusion proteins occurred with the truncated SP/GFP/254X and the LI-substituted SP/GFP/LI255–256AA. The fact that the Alexa Fluor anti-GFP Ab remained surface-localized in these constructs, in contrast with the WT vector, suggests that the affected pathway primarily involves receptor internalization (Fig. 5, b and c). As seen in the regulation of selected membrane proteins, such as the growth hormone receptor (GHR), ubiquitination helps regulate protein internalization and protein degradation (31). The GHR is a class 2 cytokine receptor constitutively bound to Jak2, as is IFN-R2. Interestingly, mutations in the GHR ubiquitin-dependant endocytosis motif result in altered receptor endocytosis, plasma membrane overaccumulation, and altered receptor degradation, somewhat resembling the effects seen with our IFN-R2-overaccumulating mutants (31, 32). Ubiquitination may facilitate the internalization process as an adaptor between lysine residues in the membrane protein and the adapter protein molecules, or to clathrin itself (32). Moreover, degradation through the proteasome requires ubiquitination.

The mechanisms involved in the dominant negative and gain-of-function effects exerted by our constructs were analyzed. Using a flow cytometry-based binding assay (15), we ruled out that the dominant negative effect of SP/GFP/254X was associated with impaired IFN- binding to the IFN-R complex and that the dominant gain-of-function effect was associated with increased IFN- binding to its receptor complex.

The mechanism proposed for IFN-R1 dominant negative mutations has been overaccumulation of the mutant receptor, leading to competition with WT IFN-R1 for binding of ligand and participation in the authentic receptor complex. This mechanism could also be hypothesized for the early intracellular truncation mutants of IFN-R2, such as SP/GFP/254X (4, 5, 30). In contrast to SP/GFP/254X, mutant constructs that overaccumulated on the plasma membrane, but retained functional intracellular domains, such as SP/GFP/2552 and SP/GFP/LI255–256AA, showed increased IFN- responsiveness both alone and in cotransfection with the WT IFN-R2. In retrospect, a gain-of-function effect was demonstrated, but not remarked, by Bach et al. (30) in a chimeric receptor created by fusion of their IC mutant and murine Jak2 (-JAK2). This vector was devoid of the L255-I256 motif, but had preserved function by virtue of the Jak2 moiety. Interestingly, this construct led to higher MHC class I expression and nitrate production than WT constructs in response to human IFN-. Therefore, it appears that mutants without signaling capacity that overaccumulate on the cell surface exert dominant negative effects, whereas those that overaccumulate with a preserved functional intracellular domain exert an enhancing or gain-of-function effect.

Our construct with an intact LI255–256 motif, but a truncated Jak2 binding site (SP/GFP/270X), did not overaccumulate, signal, or exert a dominant effect over the WT construct. Hence, mutants incapable of supporting signaling that do not overaccumulate appear to have no effect on IFN- signaling by WT receptors, thereby exerting a recessive effect.

Under normal conditions, IFN-R1 is in excess of IFN-R2 on the plasma membrane. Mutations leading to cell surface overaccumulation of functional IFN-R2 molecules may simply provide more of the limiting factor for IFN-R complex assembly, IFN-R2. This would allow the formation of an increased number of IFN-R complexes and thereby increase signal transduction potential.

In summary, the IFN-R2 LI255–256 motif plays a critical role in the receptor’s fate, by participation in the internalization process. We have developed overaccumulating dominant negative (SP/GFP/254X) and dominant gain-of-function (SP/GFP/2552 and SP/GFP/LI255–256AA) mutants through manipulation of the IFN-R2 intracellular domain, particularly the dipeptide LI255–256. Although overaccumulation and cell surface expression correlate with the activities of these constructs, they may not be sufficient or even necessary for eliciting the demonstrated dominant effects. Along these lines, we have recently reported a mutation in the IFN-R2 transmembrane domain (791delG) in which the mutant receptor was not expressed on the cell surface, did not overaccumulate, and yet still exerted a dominant negative effect (33). Taken together, these data suggest that there are physiologically distinct and clinically significant, but unrecognized, aspects of IFN- signaling awaiting elucidation. These IFN-R2 mutants will allow fine dissection of IFN- signaling.

	Acknowledgments

 
We are grateful to Dr. Juan S. Bonifacino for his constructive comments, and to Larry Lantz for preparation of the Cy5-conjugated IFN-.

	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 the National Institutes of Health Fogarty International Center and the Fogarty International Research Collaboration Award, Grant R01TW006644 (to S.D.R.).

² Current address: Servicio de Inmunologia, Hospital Nacional de Pediatria J. P. Garrahan, Combate de los Pozos 1881, Ciudad de Buenos Aires 124), Argentina.

³ Address correspondence and reprint requests to Dr. Steven M. Holland, Building 10, Room 11N103, 10 Center Drive, MSC 1886, Bethesda, MD 20892. E-mail address: smh{at}nih.gov

⁴ Abbreviations used in this paper: LI, leucine-isoleucine; EGFP, enhanced GFP; GHR, growth hormone receptor; MP, mature protein; SP, signal peptide; WT, wild type; pGFP, plasmid GFP.

Received for publication July 30, 2003. Accepted for publication June 30, 2004.

	References

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