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

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.

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 cationdependent mannose 6-phosphate receptor, Limp-II, and the invariant chain of the major histocompatibility complex class II (Ii) 1 (1)(2)(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 S 126 DKQTLL 132 in the cytoplasmic tail of the TCR subunit CD3␥ (5, 7) (amino acid numbering of human CD3␥ according to Ref. 8). The Leu-based motif in CD3␥ 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 CD3␥ Ser 126 by PKC, basic amino acids surrounding Ser 126 are important (7). The phosphorylation of CD3␥ Ser 126 most probably induces a conformational change of CD3␥, leading to the second step, recognition and binding of clathrin-coated vesicle adaptor proteins to CD3␥. In this step, CD3␥ Asp 127 , Leu 131 , and Leu 132 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 CD3␥ Leu-based motif in receptor sorting was examined in the complete TCR and in chimeric CD4/CD3␥ molecules. We found that a minimal spacing of 7 residues between the PM and Ser 126 was required for phosphorylation and activation of the CD3␥ Leu-based motif in the TCR. Furthermore, the phosphorylation-independent, constitutively active Leu-based motif in chimeric CD4/CD3␥ 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
Cells and Antibodies-JGN cells, a TCR cell-surface negative variant of the human T cell line Jurkat that does not synthesize CD3␥ (19), were cultured in RPMI 1640 medium supplemented with 2 ϫ 10 5 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% CO 2 . The mouse mAb UCHT1 directed against human CD3⑀ was obtained purified and phycoerythrin (PE)-conjugated from Dakopatts A/S (Glostrup, Denmark). Anti-CD3⑀ mAb was used to determine TCR expression as anti-CD3⑀ staining directly correlates with expression of all other TCR peptides expressed at the cell surface, including mutated CD3␥. 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 Ϫ20°C. Further dilutions of PDB were prepared in cell culture medium immediately before use.
Constructs, Transfection, and TCR Down-regulation-All CD3␥ mutations and chimeric CD4/CD3␥ molecules were constructed as described previously (5,9,20) by polymerase chain reaction using Vent DNA polymerase containing 3Ј 3 5Ј proofreading exonuclease activity (New England Biolabs Inc., Beverly, MA) and the plasmids pJ6T3␥-2 (8) and pCD-L3T4.25 (21) as templates. The primers used for each mutant are listed in Table I. The CD3␥ 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-CD3␥WT. The CD4/CD3␥ 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 g of plasmid/2 ϫ 10 7 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/ CD3␥ molecules varied considerably between the different constructs, and clones expressing the highest level of each construct were selected for further studies.
For TCR down-regulation, cells were adjusted to 2 ϫ 10 5 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% NaN 3 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)) ϫ 100%. For each construct, at least three different clones were analyzed.
CD3␥ Phosphorylation and Internalization of the TCR and Chimeric CD4/CD3␥ Molecules-Phosphorylation assays were performed as described previously (5,7). The phosphorylated CD3␥ chain with a molecular mass of 26 -30 kDa was coprecipitated with CD3⑀ (20 kDa) using anti-CD3⑀ 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/ CD3␥ molecules, cells were incubated in RPMI 1640 medium and 10% FCS at a cell density of 2 ϫ 10 5 cells/ml at 37 or 4°C with PEconjugated anti-CD3⑀ 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 l 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:   . Labeled cells were lysed in 1% Nonidet P-40 lysis buffer (20 mM Tris-HCl (pH 8.0), 1 mM MgCl 2 , 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). 14 C-Labeled proteins from Amersham International were used as molecular mass markers.

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 CD3␥ influenced the function of the motif, the CD3␥-QDGx2 and CD3␥-QDGx3 constructs were made. In CD3␥-QDGx2 and CD3␥-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 CD3␥ were separately transfected into the CD3␥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 CD3␥ Leu-based motif was not affected by increasing the spacing between the Leu-based motif and the PM with at least 6 residues.
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 CD3␥-doWT, CD3␥-doSA, and CD3␥-doDA constructs were made using the previously described CD3␥-LLAA as template (5). In CD3␥-LLAA, Leu 131 and Leu 132 are mutated to alanines, and PKC-mediated TCR down-regulation is abolished in JGN-LLAA cells, although Ser 126 phosphorylation is intact (Fig. 1, A, C, and D). The CD3␥-doWT construct coded for the CD3␥-LLAA chain with the wild-type Leu-based motif at the C-terminal end. The CD3␥-doSA and CD3␥-doDA constructs coded for the CD3␥-LLAA chain with a mutated Leubased motif corresponding to a Ser 126 3 Ala and an Asp 127 3 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/CD3␥ molecules (9). Thus, the SDKQTLL and AD-KQTLL motifs are constitutively active, and the SAKQTLL motif is activated by phosphorylation in chimeric CD4/CD3␥ 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 CD3␥-doSA construct did not express the serine corresponding to Ser 126 , and as expected, JGN-doSA cells did not downregulate 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 CD3␥ (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 membranedistal, 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 Ser 126 and the PM-To investigate whether a reduction of the spacing between the PM and the Leu-based motif of CD3␥ influenced the function of the motif, the CD3␥-d1, CD3␥-d2, CD3␥-d3, and CD3␥-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 TCRpositive 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 Ser 126 was affected by reducing the spacing relative to the PM, phosphorylation assays were performed. CD3␥ was as efficiently phosphorylated in JGN-d1 and JGN-d2 cells as in JGN-WT cells following PKC activation. In contrast, phosphorylation of CD3␥ 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 Ser 126 and the PM.
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/CD3␥ molecules independently of phosphorylation and that the DXXXLL sequence constitutes a binding motif for adaptor proteins (9). Thus, chimeric CD4/CD3␥ 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 Leubased 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.
A Basic Amino Acid at Position Ϫ1 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 Ser 126 , 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.
Functional Leu-based motifs are found both in type I (e.g. CD3␥) 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, Ser 126 and Asp 127 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 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 CD3␥ Leu-based motif and the PM by 6 residues did not affect PKCmediated TCR down-regulation. Even when located at the Cterminal end of CD3␥, 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.
Reducing the spacing between the Leu-based motif and the PM to 6 residues completely abolished the first step in PKCmediated TCR internalization, namely phosphorylation of the serine corresponding to Ser 126 . This observation is in good agreement with previous studies of the TCR demonstrating that CD3␥ Ser 123 , 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 RX-SXKQ (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-termi-nal 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/CD3␥ and CD4/CD3␥ (9,28). The data obtained with the chimeric CD4/CD3␥ 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 ␤ 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 -chains of the adaptor protein complexes are involved in binding to Tyrbased motifs, the adaptor chain(s) involved in binding Leubased motifs has not been identified with certainty (16,(31)(32)(33)(34)(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 Tyrand 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.