Leucine-based Receptor Sorting Motifs Are Dependent on the Spacing Relative to the Plasma Membrane*

Carsten GeislerDagger §, Jes Dietrich, Bodil L. Nielsen, Jesper Kastrup, Jens Peter H. Lauritsenparallel , Niels Ødum§, and Mette D. Christensen**

From the Institute of Medical Microbiology and Immunology, University of Copenhagen, Panum Institute, DK-2200 Copenhagen, Denmark

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Many integral membrane proteins contain leucine-based motifs within their cytoplasmic domains that mediate internalization and intracellular sorting. Two types of leucine-based motifs have been identified. One type is dependent on phosphorylation, whereas the other type, which includes an acidic amino acid, is constitutively active. In this study, we have investigated how the spacing relative to the plasma membrane affects the function of both types of leucine-based motifs. For phosphorylation-dependent leucine-based motifs, a minimal spacing of 7 residues between the plasma membrane and the phospho-acceptor was required for phosphorylation and thereby activation of the motifs. For constitutively active leucine-based motifs, a minimal spacing of 6 residues between the plasma membrane and the acidic residue was required for optimal activity of the motifs. In addition, we found that the acidic residue of leucine-based motifs must be located amino-terminal to the dileucine sequence for proper function of the motifs and that residues surrounding the motifs affect the activity of the motifs. Thus, our observations suggest that the position, the exact sequence, and surrounding residues are major determinants of the function of leucine-based receptor sorting motifs.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

At least two types of leucine-based receptor sorting motifs can be described. One type is constitutively active, and receptors with this type of Leu-based motifs are sorted from the trans-Golgi network to late endosomes/lysosomes, e.g. the cation-dependent mannose 6-phosphate receptor, Limp-II, and the invariant chain of the major histocompatibility complex class II (Ii)1 (1-3). The other type of Leu-based motifs is activated following phosphorylation. Most receptors carrying this type of Leu-based motifs are expressed at the cell surface and become internalized after protein kinase activation, leading to phosphorylation and activation of the Leu-based motifs (4-6).

Among other receptors, the T cell receptor (TCR) is internalized following activation of protein kinase C (PKC). PKC-induced internalization of the TCR is mediated via the Leu-based motif S126DKQTLL132 in the cytoplasmic tail of the TCR subunit CD3gamma (5, 7) (amino acid numbering of human CD3gamma according to Ref. 8). The Leu-based motif in CD3gamma has been extensively characterized, and PKC-induced internalization of the TCR can be described as a two-step process. In the first step, recognition and phosphorylation of CD3gamma Ser126 by PKC, basic amino acids surrounding Ser126 are important (7). The phosphorylation of CD3gamma Ser126 most probably induces a conformational change of CD3gamma , leading to the second step, recognition and binding of clathrin-coated vesicle adaptor proteins to CD3gamma . In this step, CD3gamma Asp127, Leu131, and Leu132 constitute the binding motif for adaptor proteins (9).

Several studies have demonstrated that in addition to binding of Leu-based receptor sorting motifs, adaptor proteins have the capacity to bind tyrosine-based motifs (10-16). Furthermore, it has been shown that the position of Tyr-based sorting motifs influences the function of this type of receptor sorting motif. Thus, the YXRF motif of the transferrin receptor requires a spacing of at least 7 residues relative to the plasma membrane (PM) to function as an internalization motif (17), and the function of the YXXI sorting motif of lamp1 is also strictly dependent on the position within the cytoplasmic tail of lamp1 (18). Theoretically, the position of Leu-based motifs within the cytoplasmic tail of receptors may influence their activity at the trans-Golgi network and the cell surface (5). However, this possibility has not yet been experimentally addressed.

In this study, the role of the position of the CD3gamma Leu-based motif in receptor sorting was examined in the complete TCR and in chimeric CD4/CD3gamma molecules. We found that a minimal spacing of 7 residues between the PM and Ser126 was required for phosphorylation and activation of the CD3gamma Leu-based motif in the TCR. Furthermore, the phosphorylation-independent, constitutively active Leu-based motif in chimeric CD4/CD3gamma molecules required a minimal spacing of 6 residues between the PM and the acidic residue for optimal activity. Finally, we found that the acidic residue of Leu-based motifs must be located amino-terminal to the dileucine sequence for proper function of the motifs and that residues surrounding the motifs affect the activity of the motifs.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cells and Antibodies-- JGN cells, a TCR cell-surface negative variant of the human T cell line Jurkat that does not synthesize CD3gamma (19), were cultured in RPMI 1640 medium supplemented with 2 × 105 units/liter penicillin (Leo Pharmaceutical Products, Ballerup, Denmark), 50 mg/liter streptomycin (Merck, Darmstadt, Germany), and 10% (v/v) FCS (Life Technologies, Inc., Paisley, United Kingdom) at 37 °C in 5% CO2. The mouse mAb UCHT1 directed against human CD3epsilon was obtained purified and phycoerythrin (PE)-conjugated from Dakopatts A/S (Glostrup, Denmark). Anti-CD3epsilon mAb was used to determine TCR expression as anti-CD3epsilon staining directly correlates with expression of all other TCR peptides expressed at the cell surface, including mutated CD3gamma . The rat anti-mouse CD4 mAb L3T4 was obtained purified and PE-conjugated from Pharmingen (San Diego, CA). Rabbit anti-rat Ig was from Dakopatts A/S, and the phorbol ester phorbol 12,13-dibutyrate (PDB) was from Sigma. A 20 mM PDB stock solution was prepared in dimethyl sulfoxide (Sigma) and stored at -20 °C. Further dilutions of PDB were prepared in cell culture medium immediately before use.

Constructs, Transfection, and TCR Down-regulation-- All CD3gamma mutations and chimeric CD4/CD3gamma molecules were constructed as described previously (5, 9, 20) by polymerase chain reaction using Vent DNA polymerase containing 3' right-arrow 5' proofreading exonuclease activity (New England Biolabs Inc., Beverly, MA) and the plasmids pJ6T3gamma -2 (8) and pCD-L3T4.25 (21) as templates. The primers used for each mutant are listed in Table I. The CD3gamma polymerase chain reaction products were digested with XhoI and EcoRI and cloned into the 6.2-kilobase XhoI/EcoRI fragment of the expression vector pMH-Neo-CD3gamma WT. The CD4/CD3gamma polymerase chain reaction products were digested with XbaI and EcoRI and cloned into the expression vector pMH-Neo (22). Mutations were confirmed by DNA sequencing. Transfections were performed using the Bio-Rad Gene Pulser at a setting of 270 V and 960 microfarads with 40 µg of plasmid/2 × 107 cells. After 3-4 weeks of selection, G418-resistant clones were expanded and maintained in medium without G418. Approximately 30% of the clones expressed the transfected molecules. Clones expressing comparable levels of TCR were selected if possible. The expression level of the chimeric CD4/CD3gamma molecules varied considerably between the different constructs, and clones expressing the highest level of each construct were selected for further studies.

                              
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Table I
Primers used to make the CD3gamma mutations and the chimeric CD4/CD3gamma molecules

For TCR down-regulation, cells were adjusted to 2 × 105 cells/ml of medium (RPMI 1640 medium and 10% FCS) and incubated at 37 °C with various concentrations of the phorbol ester PDB. At the indicated times, cells were transferred to ice-cold phosphate-buffered saline containing 2% FCS and 0.1% NaN3 and washed twice. The cells were stained directly with PE-conjugated mAb UCHT1 and analyzed in a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA). Mean fluorescence intensity (MFI) was recorded and used in the calculation of percent anti-CD3 binding: ((MFI of phorbol ester-treated cells)/(MFI of untreated cells)) × 100%. For each construct, at least three different clones were analyzed.

CD3gamma Phosphorylation and Internalization of the TCR and Chimeric CD4/CD3gamma Molecules-- Phosphorylation assays were performed as described previously (5, 7). The phosphorylated CD3gamma chain with a molecular mass of 26-30 kDa was coprecipitated with CD3epsilon (20 kDa) using anti-CD3epsilon mAb UCHT1 and subsequently analyzed by SDS-polyacrylamide gel electrophoresis. For each construct, at least two different clones were analyzed.

To determine the internalization rates of TCR and chimeric CD4/CD3gamma molecules, cells were incubated in RPMI 1640 medium and 10% FCS at a cell density of 2 × 105 cells/ml at 37 or 4 °C with PE-conjugated anti-CD3epsilon or anti-CD4 mAb, respectively. At the times indicated, aliquots of the cell suspension were washed in ice-cold RPMI 1640 medium and 10% FCS, divided in two equal parts, and subsequently treated with 300 µl of 0.5 M NaCl and 0.5 M acetic acid (pH 2.2) for 10 s or left untreated. The fluorescence of the cells was measured in the FACSCalibur. The percentage of internalized mAb to cell-surface bound mAb was subsequently calculated using the following equation: ((HAR - CAR)/CT) × 100%, where HAR is the MFI of acid-treated cells incubated at 37 °C, CAR is the MFI of acid-treated cells incubated at 4 °C, and CT is the MFI of untreated cells incubated at 4 °C. For each construct, at least three different clones were analyzed.

Metabolic Labeling-- Metabolic pulse-chase labeling studies were performed as described previously (20) using [35S]methionine and [35S]cysteine (Promix, Amersham International, Buckinghamshire, United Kingdom). Labeled cells were lysed in 1% Nonidet P-40 lysis buffer (20 mM Tris-HCl (pH 8.0), 1 mM MgCl2, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 8 mM iodoacetamide, and 1% Nonidet P-40), precipitated with rat anti-mouse CD4 mAb and rabbit anti-rat Ig, and subsequently analyzed by SDS-polyacrylamide gel electrophoresis on 10% acrylamide gels under nonreducing conditions. Autoradiography of the dried gels was performed using Hyperfilm-MP (Amersham International). 14C-Labeled proteins from Amersham International were used as molecular mass markers.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Function of Leu-based Motifs Is Preserved When the Spacing Relative to the PM Is Increased-- To investigate whether an increase of the spacing between the PM and the Leu-based motif of CD3gamma influenced the function of the motif, the CD3gamma -QDGx2 and CD3gamma -QDGx3 constructs were made. In CD3gamma -QDGx2 and CD3gamma -QDGx3, the Leu-based motif was moved down from the PM by inserting 3 and 6 residues between the motif and the PM, respectively (Fig. 1A). These constructs and wild-type CD3gamma were separately transfected into the CD3gamma -negative Jurkat variant JGN (19), and TCR-positive transfectants were isolated. Both JGN-QDGx2 and JGN-QDGx3 cells down-regulated the TCR as efficiently as JGN-WT cells following PKC activation (Fig. 1, B and D). This demonstrated that the function of the CD3gamma Leu-based motif was not affected by increasing the spacing between the Leu-based motif and the PM with at least 6 residues.


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Fig. 1.   Function of Leu-based motifs is preserved when the spacing between the motifs and the PM is increased. A, schematic representation of the amino acid sequences in the cytoplasmic tails of the CD3gamma chains expressed in the indicated cell lines and a summation of the results from the TCR down-regulation analyses. B and C, TCR down-regulation of cells incubated with different concentrations of the PKC activator PDB for 1 h. TCR down-regulation was determined by staining the cells with PE-conjugated anti-CD3epsilon mAb followed by flow cytometry comparing MFI of PDB-treated cells with MFI of untreated cells. D, FACS histograms of untreated cells (white areas) and cells treated with PDB (110 nM) for 1 h (black areas). The cell line and the percent anti-CD3 binding following PDB treatment are given in the upper left corner of each histogram. The ordinate gives the relative cell number, and the abscissa gives the fluorescence intensity in a logarithmic scale in arbitrary units. MFI of the cell lines stained with irrelevant monoclonal antibodies varied between 2 and 6 arbitrary units (data not shown). E and F, internalization rates of the TCR in cells preincubated for 60 min at 37 °C in normal medium or in medium containing 12 nM PDB, respectively. The internalization rates were determined as described under "Experimental Procedures."

It has been suggested that a membrane-proximal versus a membrane-distal position of Leu-based motifs may influence their activity (5). To test the function of Leu-based motifs in a membrane-distal position, the CD3gamma -doWT, CD3gamma -doSA, and CD3gamma -doDA constructs were made using the previously described CD3gamma -LLAA as template (5). In CD3gamma -LLAA, Leu131 and Leu132 are mutated to alanines, and PKC-mediated TCR down-regulation is abolished in JGN-LLAA cells, although Ser126 phosphorylation is intact (Fig. 1, A, C, and D). The CD3gamma -doWT construct coded for the CD3gamma -LLAA chain with the wild-type Leu-based motif at the C-terminal end. The CD3gamma -doSA and CD3gamma -doDA constructs coded for the CD3gamma -LLAA chain with a mutated Leu-based motif corresponding to a Ser126 right-arrow Ala and an Asp127 right-arrow Ala mutation at the C-terminal end, respectively (Fig. 1A). These different types of Leu-based motifs were chosen as we have recently shown that they all function as sorting motifs in chimeric CD4/CD3gamma molecules (9). Thus, the SDKQTLL and ADKQTLL motifs are constitutively active, and the SAKQTLL motif is activated by phosphorylation in chimeric CD4/CD3gamma molecules. All of the transfectants expressed the TCR at comparable levels, suggesting that none of the motifs were constitutively active (Fig. 1D). Interestingly, PKC-mediated TCR down-regulation was reduced in JGN-doWT cells, whereas JGN-doDA cells down-regulated the TCR as efficiently as JGN-WT cells (Fig. 1, C and D). The CD3gamma -doSA construct did not express the serine corresponding to Ser126, and as expected, JGN-doSA cells did not down-regulate the TCR as a response to PKC activation (Fig. 1, C and D). To analyze whether the membrane-distal SDKQTLL and ADKQTLL motifs in the mutated TCR were constitutively active, the internalization rates of the TCR were measured. Both JGN-doWT and JGN-doSA cells had low spontaneous TCR internalization rates equal to those of JGN-WT and JGN-doDA cells, indicating that the SDKQTLL and ADKQTLL motifs were not constitutively active when positioned membrane-distal in CD3gamma (Fig. 1E). Furthermore, PDB treatment of the cells increased the TCR internalization rates equally in JGN-WT and JGN-doDA cells, but only slightly in JGN-doWT cells as compared with JGN-doSA cells (Fig. 1F). Thus, when positioned membrane-distal, the SAKQTLL version of Leu-based motifs functioned as efficiently in PKC-mediated TCR down-regulation as the membrane-proximal wild-type Leu-based motif.

Phosphorylation of the Leu-based Motif Is Critically Dependent on a Spacing of at Least 7 Residues between Ser126 and the PM-- To investigate whether a reduction of the spacing between the PM and the Leu-based motif of CD3gamma influenced the function of the motif, the CD3gamma -d1, CD3gamma -d2, CD3gamma -d3, and CD3gamma -d6 constructs were made. In these constructs, successive deletions of 1, 2, 3, and 6 residues stepwise reduced the spacing between the Leu-based motif and the PM (Fig. 2A). The constructs were separately transfected into JGN cells, and TCR-positive transfectants were isolated. The ability of the transfectants to down-regulate the TCR following PKC activation was tested. As shown in Fig. 2B, JGN-d1 and JGN-d2 cells down-regulated the TCR as efficiently as JGN-WT cells. However, PKC-mediated TCR down-regulation was abolished in JGN-d3 and JGN-d6 cells (Fig. 2, B and D). To determine whether phosphorylation of Ser126 was affected by reducing the spacing relative to the PM, phosphorylation assays were performed. CD3gamma was as efficiently phosphorylated in JGN-d1 and JGN-d2 cells as in JGN-WT cells following PKC activation. In contrast, phosphorylation of CD3gamma was abolished in JGN-d3 and JGN-d6 cells (Fig. 2C). These data indicate that the first step in PKC-mediated TCR internalization, namely recognition and phosphorylation of the Leu-based motif by PKC, is critically dependent on a spacing of at least 7 residues between Ser126 and the PM.


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Fig. 2.   Phosphorylation of the Leu-based motif is critically dependent on a spacing of at least 7 residues between Ser126 and the PM. A, schematic representation of the amino acid sequences in the cytoplasmic tails of the CD3gamma chains expressed in the indicated cell lines and a summation of the results from the CD3gamma phosphorylation and TCR down-regulation analyses. B, TCR down-regulation of cells incubated with different concentrations of PDB for 1 h. TCR down-regulation was determined by staining the cells with PE-conjugated anti-CD3epsilon mAb followed by flow cytometry comparing MFI of PDB-treated cells with MFI of untreated cells. C, phosphorylation analyses of CD3gamma from JGN-WT (lane 1), JGN-d1 (lane 2), JGN-d2 (lane 3), JGN-d3 (lane 4), and JGN-d6 (lane 5) cells treated with PDB (225 nM) for 10 min. D, FACS histograms of untreated cells (white areas) and cells treated with PDB (110 nM) for 1 h (black areas). The cell line and the percent anti-CD3 binding following PDB treatment are given in the upper left corner of each histogram. The ordinate gives the relative cell number, and the abscissa gives the fluorescence intensity in a logarithmic scale in arbitrary units. MFI of the cell lines stained with irrelevant monoclonal antibodies varied between 2 and 6 arbitrary units (data not shown).

Optimal Activity of Phosphorylation-independent Leu-based Motifs Is Dependent on a Spacing of at Least 6 Residues between the Acidic Amino Acid and the PM-- We have previously shown that the SDKQTLL motif functions as a constitutively active receptor sorting motif in chimeric CD4/CD3gamma molecules independently of phosphorylation and that the DXXXLL sequence constitutes a binding motif for adaptor proteins (9). Thus, chimeric CD4/CD3gamma molecules expressing the wild-type SDKQTLL motif or the ADKQTLL motif have high spontaneous internalization rates and are quickly degraded in the lysosomes. To determine the role of the spacing between the Leu-based motif and the PM for adaptor binding and receptor sorting, we took advantage of these observations. Constructs were produced in which the spacing between the DKQTLL motif and the PM was stepwise reduced by successive deletions of 1, 2, 4, 5, and 6 residues, respectively (Fig. 3A). These and the CD4/3-tS126 and CD4/3-SA constructs were separately transfected into JGN cells, and transfectants expressing the chimeric molecules were isolated. JGN-CD4/3-tS126 and JGN-CD4/3-d6 cells expressed the chimeras with high intensity at the PM; JGN-CD4/3-SA, JGN-CD4/3-d1, and JGN-CD4/3-d4 cells expressed the chimeras with low intensity; and JGN-CD4/3-d2 and JGN-CD4/3-d5 cells expressed the chimeras with intermediate intensity (Fig. 3B). To determine the activity of the Leu-based motif in the chimeras, the spontaneous internalization rates of the chimeras were measured. Three groups of the chimeras could be distinguished according to their internalization rates. Thus, the CD4/3-tS126 and CD4/3-d6 chimeras had low internalization rates; the CD4/3-SA, CD4/3-d1, and CD4/3-d4 chimeras had high internalization rates; and the CD4/3-d2 and CD4/3-d5 chimeras had intermediate internalization rates (Fig. 3C). Thus, the internalization rates of the chimeras inversely paralleled the cell-surface expression levels of the chimeras. Furthermore, pulse-chase metabolic labeling experiments demonstrated that highly expressed chimeras with low internalization rates were stable for at least 4 h, whereas weakly expressed chimeras with high internalization rates were degraded during the 4-h chase period (Fig. 3D). Taken together, these data demonstrated that a spacing of at least 6 residues relative to the PM is required for optimal activity of phosphorylation-independent Leu-based motifs. The results obtained with the CD4/3-d2 chimera indicated that parameters other than the spacing relative to the PM might also influence the activity of Leu-based motifs.


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Fig. 3.   Optimal activity of phosphorylation-independent Leu-based motifs is dependent on a spacing of at least 6 residues between the acidic amino acid and the PM. A, schematic representation of the amino acid sequences in the cytoplasmic tails of the chimeric CD4/CD3gamma molecules expressed in the indicated cell lines and a summation of the results from the internalization and pulse-chase metabolic labeling analyses. B, FACS histograms of the recipient JGN cells (white areas) and transfectants (black areas) stained with anti-CD4 mAb. The name of the transfectant analyzed is given in the upper left corner of each histogram. The ordinate gives the relative cell number, and the abscissa gives the fluorescence intensity in a logarithmic scale in arbitrary units. C, spontaneous internalization rates of the various chimeric CD4/CD3gamma molecules. The internalization rates were determined as described under "Experimental Procedures." D, pulse-chase metabolic labeling of JGN-CD4/3-tS126, JGN-CD4/3-SA, JGN-CD4/3-d4, and JGN-CD4/3-d6 cells pulsed for 30 min (lane 1) and chased for 1 (lane 2), 2 (lane 3), and 4 (lane 4) h. The chimeric CD4/CD3gamma molecules are seen as the broad dominating band surrounded by several fine unspecific bands.

A Basic Amino Acid at Position -1 Reduces the Activity of Phosphorylation-independent Leu-based Motifs-- We (9) and others (23, 24) have previously demonstrated that an acidic amino acid (Asp or Glu) positioned 4 or 5 residues N-terminal to the dileucine sequence is required for optimal activity of Leu-based motifs. In this study, the CD4/3-d2 chimera had a reduced internalization rate as compared with the CD4/3-SA, CD4/3-d1, and CD4/3-d4 chimeras (Fig. 3). In contrast to the CD4/3-SA, CD4/3-d1, and CD4/3-d4 chimeras, a basic amino acid (Arg) immediately preceded the DKQTLL motif of the CD4/3-d2 chimera. Thus, the possibility existed that the reduced internalization rate of the CD4/3-d2 chimera was not due to the altered spacing between the DKQTLL motif and the PM, but was caused by the Arg located immediately N-terminal to the DKQTLL motif. To test this hypothesis, the CD4/3-SR construct, in which Arg was substituted for Ser126, was made (Fig. 4A). FACS analyses demonstrated that the CD4/3-SR chimera was expressed at a higher level at the cell surface as compared with the CD4/3-SA chimera (Fig. 4B). In agreement with this, the CD4/3-SR chimera had a reduced internalization rate and was more stable as compared with the CD4/3-SA chimera in pulse-chase metabolic labeling experiments (Fig. 4, C and D). These data indicate that the presence of a basic amino acid immediately N-terminal to the acidic amino acid in Leu-based motifs reduces the activity of the motifs.


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Fig. 4.   A basic amino acid at position -1 reduces the activity of phosphorylation-independent Leu-based motifs. A, schematic representation of the amino acid sequences in the cytoplasmic tails of the chimeric CD4/CD3gamma molecules expressed in the indicated cell lines and a summation of the results from the internalization and pulse-chase metabolic labeling analyzes. B, FACS histograms of the recipient JGN cells (white areas) and transfectants (black areas) stained with anti-CD4 mAb. The name of the transfectant analyzed is given in the upper left corner of each histogram. The ordinate gives the relative cell number, and the abscissa gives the fluorescence intensity in a logarithmic scale in arbitrary units. C, spontaneous internalization rates of the various chimeric CD4/CD3gamma molecules. The internalization rates were determined as described under "Experimental Procedures." D, pulse-chase metabolic labeling of JGN-CD4/3-SA, JGN-CD4/3-SR, JGN-CD4/3-SDAA, and JGN-CD4/3-rev cells pulsed for 30 min (lane 1) and chased for 1 (lane 2), 2 (lane 3), and 4 (lane 4) h. The chimeric CD4/CD3gamma molecules are seen as the broad dominating band surrounded by several fine unspecific bands.

Functional Leu-based motifs are found both in type I (e.g. CD3gamma ) and type II (e.g. Ii) integral membrane proteins, indicating that adaptor proteins have the ability to bind Leu-based motifs regardless of their orientation relative to the PM. Thus, in type I integral membrane proteins, the acidic amino acid of the Leu-based motif is located membrane-proximal relative to the dileucine sequence, whereas in type II integral membrane proteins, the acidic amino acid of the Leu-based motif is located membrane-distal relative to the dileucine sequence. To test whether a laterally reversed Leu-based motif consisting of an acidic amino acid located membrane-distal relative to the dileucine sequence had the ability to function as a receptor sorting motif in type I integral membrane proteins, the CD4/3-rev construct was made (Fig. 4A). This construct was made using the previously described CD4/3-SDAA as template (9). In CD4/3-SDAA, Ser126 and Asp127 were mutated to alanines, which resulted in an inactive Leu-based motif (Fig. 4A). As for the CD4/3-SDAA chimera, the CD4/3-rev chimera was highly expressed at the cell surface, had a low internalization rate, and was stable for at least 4 h (Fig. 4, B-D). Thus, the laterally reversed Leu-based motif in CD4/3-rev did not function as an active receptor sorting motif. This indicated that although adaptor proteins have the ability to bind Leu-based motifs of both type I and II integral membrane proteins, the acidic amino acid of Leu-based motifs must be located N-terminal to the dileucine sequence for proper function of the motifs.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

This is the first study that defines the critical minimal spacing between Leu-based motifs and the PM required for proper function of Leu-based motifs. For phosphorylation-dependent Leu-based motifs, a minimal spacing of 7 residues between the PM and the phospho-acceptor was required for phosphorylation and thereby activation of the motifs. For constitutively active Leu-based motifs, a minimal spacing of 6 residues between the PM and the acidic amino acid was required for optimal activity of the motifs.

In contrast to defining a critical minimal spacing, an upper limit of the spacing between the Leu-based motifs and the PM was not found. Thus, increasing the spacing between the CD3gamma Leu-based motif and the PM by 6 residues did not affect PKC-mediated TCR down-regulation. Even when located at the C-terminal end of CD3gamma , the SAKQTLL version of the motif functioned as efficiently as the membrane-proximal wild-type motif. These results are supported by the presence of phosphorylation-dependent Leu-based motifs in gp130 (6) and the cation-independent mannose 6-phosphate receptor (25), which are located 139 and 155 residues from the PM, respectively (Table II). Taken together, these observations indicate that phosphorylation-dependent Leu-based motifs can be found several residues from the PM even in a membrane-distal position.

                              
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Table II
Spacing relative to the plasma membrane of phosphorylation-dependent and constitutively active Leu-based motifs

Reducing the spacing between the Leu-based motif and the PM to 6 residues completely abolished the first step in PKC-mediated TCR internalization, namely phosphorylation of the serine corresponding to Ser126. This observation is in good agreement with previous studies of the TCR demonstrating that CD3gamma Ser123, which is found 6 residues from the PM, is not phosphorylated by PKC, although it is placed in a PKC consensus phosphorylation site (5). As the PKC recognition site RXSXKQ (7) was conserved in all the analyzed constructs, our results indicate that a spacing of at least 7 residues between the PM and the phospho-acceptor is required for recognition/binding of PKC to its substrates. This is supported by the observation that the phospho-acceptor group of receptors phosphorylated by PKC is generally found more than 7 residues from the PM (26). One possible explanation for the requirement of a spacing of at least 7 residues for PKC-mediated phosphorylation might simply be that this minimal spacing is required to allow binding of the substrate in the binding cleft of the catalytic domain of PKC. This explanation is in agreement with results obtained from model building of the catalytic domain of PKC demonstrating a close contact between residues N-terminal of the phospho-acceptor position in the PKC pseudosubstrate and residues in the substrate-binding cleft of PKC (27).

The results obtained with the complete TCR indicated that the second step in PKC-mediated TCR internalization, namely adaptor binding and receptor internalization, was not affected by a reduction of the spacing between the Leu-based motif and the PM by 2 residues. How adaptor binding was affected in the complete TCR by a further reduction of the spacing could not be evaluated since this abolished phosphorylation and activation of the motif. To avoid the dependence of phosphorylation, we took advantage of the observation that the DKQTLL sequence functions as an active phosphorylation-independent Leu-based motif in chimeric receptors like Tac/CD3gamma and CD4/CD3gamma (9, 28). The data obtained with the chimeric CD4/CD3gamma molecules demonstrated that a spacing of at least 6 residues relative to the PM was required for optimal activity of the phosphorylation-independent Leu-based motif. A spacing of 5 residues allowed suboptimal activity of the motifs, whereas a spacing of 4 residues did not allow activity of the motif. These data concur with the observation that lysosomal sorting is abolished in mutated Limp-II molecules in which the Leu-based motif is spaced only 4 residues from the PM (2) and are furthermore supported by the identification of a Leu-based motif in the beta 2-adrenergic receptor located only 5 residues from the PM (Table II) (29). Constitutively active Leu-based motifs are also found in Limp-II, CD44, and the cation-dependent mannose 6-phosphate receptor with a spacing of 12, 36, and 59 residues relative to the PM, respectively (Table II) (1, 2, 30). Taken together, these observations indicate that constitutively active Leu-based motifs can be found with a spacing of 5 to several residues from the PM.

Previous studies have identified a critical minimal spacing of 6-7 residues between Tyr-based motifs and the PM for optimal function of Tyr-based motifs (17, 18). Whereas the µ-chains of the adaptor protein complexes are involved in binding to Tyr-based motifs, the adaptor chain(s) involved in binding Leu-based motifs has not been identified with certainty (16, 31-35). However, the similar requirements of a critical minimal spacing of Leu- and Tyr-based motifs relative to the PM indicate that binding of adaptor proteins to Leu- and Tyr-based motifs might share common mechanisms.

Like Tyr-based motifs, Leu-based motifs are found in both type I and type II integral membrane proteins (Table II). This indicates that adaptor proteins must be flexible in binding Tyr- and Leu-based motifs and that they have the ability to bind these motifs regardless of the orientation of the motifs relative to the PM. Despite this, our observations demonstrated that the acidic residue of Leu-based motifs must be located N-terminal of the dileucine sequence for proper function of Leu-based motifs. Furthermore, residues surrounding the Leu-based motif probably affect adaptor binding, as might be suggested from the observation that the presence of a basic amino acid immediately N-terminal to the Leu-based motifs reduces the activity of the motif. Thus, our observations suggest that the position, the exact sequence, and residues surrounding Leu-based receptor sorting motifs are major determinants of their function.

    ACKNOWLEDGEMENTS

We thank Dr. M. J. Crumpton for plasmid pJ6T3gamma -2 and Dr. D. R. Littman for plasmid pCD-L3T4.25.

    FOOTNOTES

* This work was supported in part by the Danish Cancer Society, the Novo Nordisk Foundation, the Danish Medical Research Council, the Danish Natural Science Research Council, Director Ib Henriksens Foundation, Gerda and Aage Haensch's Foundation, and Director Leo Nielsen and Wife Karen Magrethe Nielsen Foundation for Medical Basic Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Inst. of Medical Microbiology and Immunology, University of Copenhagen, Panum Inst., Bldg. 18.3, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark. Tel.: 45-3532-7880; Fax: 45-3532-7881; E-mail: cgtcr{at}biobase.dk.

§ Member of the Biotechnology Center for Cellular Communication.

Recipient of a Ph.D. scholarship from the University of Copenhagen.

parallel Recipient of a scholarship from the Danish Cancer Society.

** Recipient of a scholarship from the Danish Medical Research Council.

The abbreviations used are: Ii, invariant chain of the major histocompatibility complex class II; TCR, T cell receptor; PKC, protein kinase C; PM, plasma membrane; FCS, fetal calf serum; mAb, monoclonal antibody; PE, phycoerythrin; PDB, phorbol 12,13-dibutyrate; WT, wild-type; MFI, mean fluorescence intensity; FACS, fluorescence-activated cell sorting.
    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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