©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Internalization Motif Is Created in the Cytoplasmic Domain of the Transferrin Receptor by Substitution of a Tyrosine at the First Position of a Predicted Tight Turn (*)

Bronislaw Pytowski (§) , Timothy W. Judge , Timothy E. McGraw (¶)

From the (1) Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Receptors are internalized from the plasma membrane at 10 times the rate of bulk membrane. The predominant model for the motif that promotes rapid internalization proposes a requirement for a tyrosine located in the first position of a tight turn. In this report we show that an internalization motif can be created de novo by substituting a tyrosine for the first or last residues of a tetrapeptide GDNS (residues 31-34) that is predicted to form a tight turn within the cytoplasmic domain of the human transferrin receptor. These substitutions restore wild-type levels of internalization to transferrin receptors that are poorly internalized due to missense mutations in the native internalization motif. The introduction of a tyrosine at the first or last position of the GDNS tetrapeptide in a transferrin receptor containing an unmodified wild-type internalization motif significantly increases the internalization rate above that of the wild-type receptor. Our results indicate that a functional novel internalization motif can be created by placing specific aromatic amino acids within the overall structure of an existing -turn in a cytoplasmic domain of a receptor.


INTRODUCTION

Identification of specific sequences in the cytoplasmic domains of membrane proteins that mediate rapid internalization through clathrin-coated pits has been an area of intense investigation (for reviews see Trowbridge et al. (1993) and Roth (1993)). Most attention has focused on tyrosine-based internalization motifs in which the minimum required sequence is a tyrosine at the first position of a -turn and a bulky hydrophobic amino acid at the fourth position (Collawn et al., 1990). Other features of the tyrosine-based internalization motifs are that their activity is independent of polypeptide chain polarity with respect to the plasma membrane and that there is a certain degree of flexibility in their distance from the plasma membrane (Jing et al., 1990; Collawn et al., 1990; Collawn et al., 1991; Jadot et al., 1992).

The -turn model in which internalization motifs share a common three-dimensional structure and chemistry is supported by direct evidence from two-dimensional NMR analysis (Bansal and Gierasch, 1991; Eberle et al., 1991) and by considerable mutagenesis data derived from studies of a number of internalized proteins (reviewed in Trowbridge et al. (1993)). Most commonly, the use of site-directed mutagenesis in structure-function studies of receptors involves the generation of loss-of-function mutant molecules through the removal of specific residues. In the human transferrin receptor (TR),() this experimental approach led to the delineation of the tetrapeptide YTRF, between amino acids 20 and 23 of the cytoplasmic domain, as the minimum sequence required for internalization ( i.e. the internalization motif) (Jing et al., 1990; Collawn et al., 1990; McGraw and Maxfield, 1990).

An alternative approach to defining the requirements for a functional internalization motif is to create, by mutagenesis, an internalization motif within a polypeptide stretch that does not normally promote internalization (Ktistakis et al., 1990; Lazarovits and Roth, 1988). In studies of this type, we have shown that the substitution of a tyrosine for serine 34 of the cytoplasmic domain of TR restores wild-type levels of internalization to a mutant TR in which tyrosine 20 of the native internalization motif has been replaced with a cysteine (McGraw et al., 1991). We originally chose position 34 because of the presence of neighboring charged residues, reasoning that this region of TR was likely to be on the surface of the molecule. Thus, it was difficult to interpret precisely why the tyrosine at position 34 creates an internalization motif.

In this report we present evidence that the introduction of a tyrosine at position 34 of TR creates a novel internalization signal, structurally independent from the native internalization motif. Analysis of the sequence of TR near position 34 by the method of Chou and Fasman (1978) predicts that residues 31-34 form a -turn. At position 34, the tyrosine occupies the ultimate position of the putative turn. Because the polarity of the -turn with respect to the membrane is not critical for its function, it is possible that substitution of a tyrosine at position 31 may also create an internalization motif. Consistent with this reasoning we have found that substitution of a tyrosine at position 31 (and, to a lesser extent, substitution of a phenylalanine) is able to promote rapid internalization of TR mutated in the native internalization motif. Finally, we demonstrate that the tyrosine and phenylalanine substitutions at position 31, when introduced in the context of a TR containing the native internalization motif, significantly increase the internalization rate above that of the wild-type TR. Thus, two independent internalization motifs on a single receptor molecule enhance the internalization rate.


MATERIALS AND METHODS

Ligands

Human apotransferrin (Sigma) was purified by Sephacryl S-300 chromatography. The preparation and iodination of diferric transferrin (Tf) as well as Fe and Fe loading of Tf have been described previously (McGraw et al., 1987).

In Vitro Mutagenesis

The mutations F13A,Y20C, Y20C,F23A, F13A,S34Y, F23A,S34Y, Y31, Y20C,G31Y, G31F, and Y20C,G31Y were prepared following published procedures for site-directed mutagenesis (Kunkel, 1985) using oligonucleotides purchased from Operon Technologies (San Pablo, CA). The oligonucleotides used were as follows: Y20C, 5`-GGTACATGACAATGG-3`; F13A, 5`-CTCCACCAGCCAAGTTAG-3`; A23 5`-GAGCCAGGCTGGCCCGGG-3`; S34Y, 5`-CCACATGTATGTTATCGC-3`; G31Y, 5`-GTAGATTACGATAACAGTCATG-3`; and G31F, 5`-GTAGATTTCGATAACAGTCATG-3`. All mutated codons are underlined. The deletions 3-59, 3-28, and 3-28 (S34Y) and 3-28 (G31Y) were prepared by polymerase chain reaction using oligonucleotides prepared at ImClone Systems, Inc. (New York). The oligonucleotides used in all deletion constructs were as follows: 1, 5`-TGACCATGATTACGAATT-3`; 2, 5`-CATCATTCTGAACTGCCA-3`; and 4, 5`-TCAACACACCAATTGCAT-3`. The variable oligonucleotides were as follows: 3-59, 5`-GTTCAGAATGATGAAAAGGTGTAGTGGA-3`; 3-28, 5`-GTTCAGAATGATGGTAGATGGCGATAAC-3`; and 3-28 G31Y, 5`-GTTCAGAATGATGGTAGATACGATAACA-3`.

Complete TR cDNA was reconstructed as described previously (McGraw et al., 1988). The presence of nucleotide substitutions was verified by sequencing the double-stranded DNA. The altered receptor cDNA was introduced into cells using Lipofectin (Life Technologies, Inc.). The plasmid pSV3-Neo was co-transfected into the cells as a selectable marker.

Cells

The TRVb (TR variant) cell line used in this study for transfection of cDNA is a derivative of Chinese hamster ovary cells that does not express functional hamster TR (McGraw et al., 1987). Following transfection, the cells were cultured in Ham's F12 medium supplemented with 5% fetal bovine serum, 14 m M NaHCO, 100 units/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml G418.

Internalization Rate

The rate of Tf internalization was determined using a modified In/Sur method (Wiley and Cunningham, 1982; McGraw and Maxfield, 1990). Cells grown in six-well clusters were washed and incubated with 3 µg/ml I-Tf in McCoy's 5A salts containing 20 m M Hepes, 26 m M sodium bicarbonate, pH 7.2 (medium 1). One six-well plate was used per time point. After incubation for 2, 4, 6, or 8 min, the incubation medium was removed, and the cells were rinsed once with medium 1, placed on ice, and flooded with 0.2 N acetic acid in 0.2 M NaCl prechilled to 4 °C. Following a 2-min incubation on ice the cells were washed 3 times with medium 2 (150 m M NaCl, 20 m M Hepes, pH 7.4, 1 m M CaCl, 5 m M KCl, 1 m M MgCl) prechilled to 4 °C, solubilized in 0.1% Triton X-100, 0.1 N NaOH, and counted in a counter. The acid wash removes surface-bound ligands; thus, cell-associated radioactivity following this wash has been internalized during the incubation at 37 °C. The amount of surface TR was determined by incubating a six-well plate of cells on ice with 3 µg/ml I-Tf in medium 2 for at least 2 h, followed by five washes with medium 2 prechilled to 4 °C. The internalization rate constant for Tf was determined as the slope of the ratio of acid-resistant Tf binding (internal) to steady-state surface binding versus time. A 200-fold excess of unlabeled Tf was added to two wells of each plate to determine nonspecific binding, which typically was less than 10% of the total counts/min. All data present have been corrected for nonspecific binding.

Iron Accumulation

The method for assaying the rate at which cells accumulate diferric Tf loaded with Fe or Fe has been described previously (McGraw et al., 1987). One six-well cluster of cells was used for each time point. Two wells from each plate were incubated with FeTf or FeTf in the presence of 200-fold excess unlabeled Tf to measure nonspecific binding. One six-well plate was used to measure surface binding of ITf as described above. Accumulated iron counts are corrected for the amount of steady-state surface TR to account for differences in iron accumulation attributable to different levels of TR expression. These data are then normalized to the number of TR expressed in cells expressing the wild-type human TR to allow for comparisons of the efficiency of iron accumulation among the different cell lines (McGraw and Maxfield, 1990).

Secondary Structure Predictions

Secondary structure prediction for peptide sequences was performed on a Microvax computer (Digital Equipment Corp., Maynard, MA) using the program Peptide Structure (Genetics Computer Group, Inc., Madison, WI). The specific algorithms used are referenced in the text.


RESULTS

A schematic of the cytoplasmic domain of the human TR, with the mutated residues discussed in this report noted, is shown in Fig. 1. cDNAs encoding the in vitro mutagenized TRs were transfected into TRVb cells, a variant of CHO cells that does not express functional endogenous TR (McGraw et al., 1987). This cell line allows for the characterization of the behavior of the mutated TR in a background free of endogenous hamster TR (McGraw et al., 1987). TRVb cells expressing the wild-type human TR, TRVb-1, were used as controls (McGraw et al., 1987). Where examined, the mutations studied in this report did not alter the rate at which internalized TR was returned to the cell surface (not shown). These findings are consistent with the proposal that receptors are returned to the cell surface by a default mechanism.

A Tyrosine at Position 34 Restores Rapid Internalization to Mutant TRs Containing Alanine Substituted for Phenylalanine at Position 13 or 23

We have previously shown that the internalization-defective phenotype of a TR in which the sole cytoplasmic tyrosine (Tyr-20) within the wild-type internalization motif YTRF (residues 20-23) has been mutated to a cysteine (Cys-20) can be restored by introducing a tyrosine at position 34 (McGraw et al., 1991). If the S34Y substitution functions by creating a novel, independent internalization motif, it would be expected to restore rapid internalization to other TR mutants that are slowly internalized due to mutations that affect the function of the native YTRF internalization motif. Two such mutations are alanine substitutions for the phenylalanines at positions 13 and 23. The substitution of alanine for phenylalanine 23 (F23A), which occupies the fourth position of the native internalization motif, reduces the TR internalization by 3-fold. This is to the same extent as mutation of tyrosine 20 (Collawn et al., 1990; McGraw et al., 1991). The substitution of alanine for phenylalanine 13 (F13A) reduces the internalization rate by 2-fold. However, since phenylalanine 13 is not part of the YTRF tetrapeptide, it is not understood why this mutation affects the activity of the native internalization motif (McGraw et al., 1991).

To address whether the internalization motif created by the S34Y substitution is functionally independent of the phenylalanyl residues at or near the wild-type motif, we created TR constructs containing the S34Y mutation together with either the F13A or the F23A mutation and determined the internalization rate constant for each mutant receptor (Wiley and Cunningham, 1982; McGraw and Maxfield, 1990). The double mutant F23A,S34Y is internalized twice as rapidly as the F23A mutant TR, indicating that the S34Y mutation is able to significantly (although not completely) restore rapid internalization to the F23A TR (Fig. 2, A and C). The F13A,S34Y mutant TR internalizes Tf at nearly twice the rate of the F13A mutant TR, indicating that the S34Y mutation almost completely restores a wild-type internalization to the F13A mutant TR (Fig. 2, B and C).


Figure 2: Comparison of Tf internalization rates of human TR mutants. The results presented in panels A and B are the means ± S.E. of at least eight independent experiments. These data are from studies of representative clonal lines expressing the various TR mutants. The internalization assay was performed as described under ``Materials and Methods.'' The y axis is the ratio of the Tf internalized to the amount of Tf bound to the surface of the cells at steady state. The slope of this plot is the internalization rate constant (Wiley and Cunningham, 1982). In panel C the mean internalization rate values ± S.E. for two independently isolated clonal lines expressing the F23A,S34Y ( A23Y34), F13A,Y20C ( A13Y34), F13A ( A13), F23A ( A23), or wild-type ( wt) TR are shown. In panel D the rates of Fe accumulation by cells expressing the F13A,F23A, F13A,Y20C, or F23A,S34Y TR are shown. The results are the mean values ± S.E. of at least three separate determinations. The data are presented as a percentage of the rate at which cells expressing the wild-type human TR accumulate Fe from diferric Tf. The rates were determined as described under ``Materials and Methods.''



Cells expressing the F13A,S34Y and F23A,S34Y double mutant TRs were further tested for the ability to accumulate iron from diferric Tf (Fig. 2 D). The iron accumulation rates are presented as a percentage of the rate of accumulation by wild-type TR expressed in TRVb CHO cells. Cells expressing the F23A,S34Y and F13A,S34Y double mutant TRs accumulate iron more rapidly than the corresponding F13A or F23A single mutations, in agreement with the measurements of the internalization rate. Together these findings demonstrate that the S34Y substitution creates an internalization motif that is able to restore rapid internalization to TRs with mutations within (F23A) or outside (F13A) the native internalization motif.

An Aromatic Amino Acid at Position 31 Restores Rapid Internalization to Y20C Mutant TR

We next sought to determine whether the S34Y internalization motif conforms to the canonical aromatic amino acid-based internalization motif in which an aromatic amino acid occupies position 1 of a -turn (Collawn et al., 1990). Since the structure of the TR cytoplasmic domain has not been determined we employed the Chou-Fasman (Chou and Fasman, 1978, 1979) and Garnier-Osguthorpe-Robson (Garnier et al., 1978) methods of secondary structure prediction to predict the probable location of -turns. These two methods agree in predicting a -turn near position 34 (not shown). Since it is known that the peptide polarity of tyrosine-based internalization motifs with respect to the membrane does not affect activity of the internalization motif (Collawn et al., 1991; Jadot et al., 1992), either the tetrapeptide from 34 to 37 or the tetrapeptide from 31 to 34 of the TR should be a -turn. The tetrapeptide from 31 to 34, with a tyrosine at 34, is predicted by Chou-Fasman analysis to be a -turn, whereas the tetrapeptide from 34 to 37, with a tyrosine at 34, is not (). Furthermore, the native TR sequence from 31 to 34 is also predicted to adopt a -turn structure. Thus, if the S34Y substitution functions as an internalization motif because S34Y occupies position 4 of a -turn located at positions 31-34, then substitution of an aromatic amino acid for the glycine at position 31 (residue 1 of the predicted -turn) should also create a functional internalization motif.

To test this prediction, we constructed double mutants that place a tyrosine or a phenylalanine at position 31 in the internalization-defective Y20C mutant TR. The results from a representative experiment measuring the internalization rate constants for the wild-type, Y20C,G31Y, Y20C,S34Y, and Y20C TRs are shown in Fig. 3 A, and a summary of the internalization rate constant measurements for independently isolated clonal lines is shown in Fig. 3 B. These results demonstrate that substitution of a tyrosine for the glycine at position 31 is able to restore rapid internalization to the internalization-defective Y20C mutant TR. The Y20C,G31Y TR internalizes Tf as rapidly as the wild-type TR and the Y20C,S34Y mutant TR. Thus, G31Y substitution creates a functional internalization motif within the cytoplasmic domain of the TR. A double mutant TR containing a phenylalanine at position 31 and a cysteine at position 20 is also more rapidly internalized than the Y20C mutant TR, albeit slower than the wild-type or Y20C,G31Y TR (Fig. 3, B and C).


Figure 3: Comparison of Tf internalization rates of wild-type TR and TR mutants Y20C ( C20), Y20C,G31Y ( C20Y31), Y20C,S34Y ( C20Y34), and Y20C,G31F ( C20F31). In panel A the results of a representative experiment measuring the internalization rate of clonal lines expressing the wild-type, Y20C, Y20C,G31Y, or Y20C,S34Y TR are shown. The results are means ± S.D. of four measurements. The values have been corrected for nonspecific Tf binding. In panel B the mean internalization rate values ± S.E. for two independently isolated clonal lines expressing either the Y20C,G31Y or Y20C,G31F are compared with the values measured for clonal lines expressing the wild-type ( wt), Y20C,S34Y, or Y20C mutant TR. The internalization rate constants were derived from rate experiments illustrated in panel A. In panel C a representative experiment measuring the iron accumulation is shown. The values are the mean ± S.D. of four measurements. The data are corrected for nonspecific Tf binding. The Fe values have been normalized to 2 ng of Tf binding per well of cells.



Examination of iron accumulation rates of cells expressing the Y20C,G31Y or Y20C,G31F mutant TR revealed that these cells accumulate iron from diferric Tf more rapidly than cells expressing the Y20C mutant (Fig. 3 C). The rates for iron uptake for cells expressing the Y20C,G31Y and Y20C,S34Y TR are greater than the rate characteristic of the wild-type receptor, while cells expressing the Y20C,G31F TR accumulate iron more slowly than cells expressing the wild-type TR but faster than cells expressing the Y20C TR. These results are in agreement with the Tf internalization rate measurements and support the conclusion that the G31Y substitution and to a lesser degree the G31F substitution create a functional internalization motif.

A TR Containing an Aromatic Amino Acid at Position 31 and a Tyrosine at Position 20 Is More Rapidly Internalized than the Wild-type TR

If the internalization motif created by substitution of an aromatic amino acid at position 31 is functional and independent of the native internalization motif then the G31Y substitution in the context of the wild-type TR may result in a receptor that is more rapidly internalized than the wild-type TR because of the presence of two internalization motifs ( e.g. Collawn et al. (1993)). To test this hypothesis, we produced constructs encoding the G31Y and G31F mutant TRs and examined the effect of these mutations on the internalization rate constant and the rate of iron accumulation from diferric Tf. Direct measurement of Tf internalization demonstrates that the G31Y and G31F TRs are internalized more rapidly than the wild-type TR (Fig. 4, A and B), and cells expressing these TRs accumulate iron more rapidly than cells expressing the wild-type TR (Fig. 4 C). Therefore, the G31Y and G31F substitutions create an internalization motif that, when placed in the context of the native TR cytoplasmic domain, acts additively with the native internalization motif to accelerate the rate of Tf endocytosis. Internalization Motif Created at Positions 31-34 Is Not Functional When Amino-proximal Sequences of the TR Are Deleted-The regions to the amino and carboxyl sides of the native internalization motif can be deleted without altering TR internalization and can be moved to different locations within the cytoplasmic domain of the TR and still function (Collawn et al., 1990; Jing et al., 1990). To determine whether the internalization motif created by either the G31Y or S34Y substitutions requires sequences located on its amino-terminal side, residues between positions 2 and 29 were deleted from the wild-type TR (3-28 TR), the G31Y TR (3-28 G31Y TR), and the S34Y TR (3-28 S34Y TR). As a control, the behavior of a TR in which the sequences between positions 2 and 60 were deleted (3-59 TR) was also examined (Collawn et al., 1993; Johnson et al., 1993). Both the 3-28 TR and the 3-59 TR were internalized at 10-20% of the rate of the wild-type TR (Fig. 5) (Johnson et al., 1993). The substitution S34Y or G31Y in the context of the 3-28 TR was unable to restore wild-type levels of internalization to the 3-28 TR (Fig. 5). This finding demonstrates that the internalization motif created by substitution of aromatic residues at position 31 or 34 does not function when the amino-proximal sequences are deleted.


Figure 4: Comparison of Tf internalization rates of wild-type TR and TR mutants G31Y ( Y31) and G31F ( F31). In panel A the results of a representative experiment measuring the internalization rate of clonal lines expressing the wild-type, G31Y, or G31F TR are shown. The results are means ± S.D. of four measurements. The values have been corrected for nonspecific Tf. In panel B the mean internalization rate values ± S.E. for two independently isolated clonal lines expressing either the G31Y or G31F TR are compared with the values measured for the wild-type ( wt) TR. In panel C a representative experiment measuring the iron accumulation is shown. The values are the mean ± S.D. of four measurements. The data are corrected for nonspecific Tf binding. The Fe values have been normalized to 2 ng of Tf binding per well of cells.



Endocytic Phenotype of TR Expressing the F13A,Y20C and Y20C,F23A Mutations

The 3-59 TR is internalized more slowly than TRs mutated at residue Tyr-20, Phe-23, or Phe-13 (Fig. 5). One interpretation of this finding would be that these mutant TRs contain residual structural features at the native internalization motif that may allow them to interact with clathrin-coated pits more efficiently than the TR in which the entire cytoplasmic domain has been deleted. To address this question, we examined the relative contributions of the Tyr-20, Phe-23, and Phe-13 residues to the function of the native internalization motif of TR by constructing pairwise site-specific substitutions. This approach to structure-function studies can be used to investigate the functional interrelationship of residues in a given protein. In general, if two residues contribute discrete components to the free energy of interaction between two proteins the effect of their simultaneous removal will be additive. If, on the other hand, the effect of the double mutant is equal to that of one of the individual mutations, the mutated residues are likely to comprise a single recognition motif that requires both residues to function (Wells, 1990). The effect of the F23A,Y20C and F13A,Y20C substitutions on the internalization of the TR is demonstrated in Fig. 6 A. The internalization rates of cells expressing the Y20C,F23A TR are not significantly different from that of the F23A TR. Our inability to detect further reduction in the internalization rate constant in the double mutant is not a reflection of the sensitivity of the assay since reductions in internalization rate below that measured for the Y20C and F23A mutant TRs can be reliably measured (Fig. 5). Cells expressing the F13A,Y20C mutant TR internalize Tf at a rate significantly slower than the rates of cells expressing the F13A mutant TR but identical to that obtained with the Y20C mutation alone (Fig. 6 B). Thus, the effect of the combinatorial loss of phenylalanine 23 and tyrosine 20 is nonadditive and equal, indicating that the relative contributions of Phe-23 and Tyr-20 to the function of TR are similar. While the combinatorial loss of phenylalanine 13 and tyrosine 20 is also nonadditive, the effect of the individual substitutions is not equal, with the absence of tyrosine 20 defining the internalization rate of the double mutant.


Figure 5: Comparison of the internalization rates, relative to wild type, of TR deletion mutants and deletion mutants containing the G31Y ( Y31) or S34Y ( Y34) substitution. The internalization rates of cells expressing the 3-28 and 3-59 TR or the 3-28 TR containing either the S34Y or G31Y mutations are shown. The internalization rate of the F23A ( A23) mutant TR is shown for comparison. The data are the means of at least three measurements ± S.E. The wild-type TR internalization rate measured in this set of experiments was 0.13 ± 0.01 min(± S.E.). The rate constants were measured as described under ``Materials and Methods.''




Figure 6: Comparison of Tf internalization rates of wild-type TR and TR mutants F23A ( A23), Y20C,F23A ( C20A23), F13A ( A13), and F13A,Y20C ( A13C20). In panel A the results of a representative experiment measuring the internalization rate of clonal lines expressing either the wild-type, F23A, or Y20C,F23A TR are shown. The results are means ± S.D. of four measurements. The values have been corrected for nonspecific Tf. In panel B the results of a representative experiment measuring the internalization rate of clonal lines expressing the wild-type, F13A, or F13A,Y20C TR are shown. The results are means ± S.D. of four measurements. The values have been corrected for nonspecific Tf. The data for the wild-type TR internalization in panel B are the same as in panel A and are presented in both panels to illustrate the degree of slowed internalization induced by the various mutations. In panel C the mean internalization rate values ± S.E. for two independently isolated clonal lines expressing either the F13A,Y20C or Y20C,F23A TR or control lines expressing the wild-type ( wt), F13A, or F23A TR are shown.




DISCUSSION

In this report we sought to further understand how the substitution of a tyrosine for the native serine at position 34 of the human TR is able to restore the internalization-defective phenotype of a TR mutant in which tyrosine 20 has been changed to cysteine. We have shown that the S34Y mutation is able to restore rapid internalization to slowly internalized F23A and F13A TR. These findings indicate that the S34Y substitution creates an independent internalization motif within the cytoplasmic domain of the TR.

If the S34Y substitution creates an internalization motif that conforms to the requirements for tyrosine-based internalization motifs, then S34Y should be in the first position of a -turn. The conditions for a predicted -turn are met by the tetrapeptide GDNS (amino acids 31-34) of the wild-type receptor sequence, and the introduction of a tyrosine at either position 31 or 34 has little effect on the predicted conformation of this tetrapeptide. Thus, in the S34Y substitution we placed an aromatic residue at position 4 of a likely -turn. Since the internalization motifs function without regard to the polarity with the membrane, substitution of a tyrosine in position 1 of the turn, that is for glycine 31, should also create an internalization motif. We find that the G31Y substitution restores the internalization-defective phenotype of the Y20C mutant TR. Therefore, the G31Y substitution, similar to the S34Y mutation, creates a novel internalization motif. The G31F substitution is also able to increase internalization of the Y20C mutant TR, albeit to a lesser degree than the G31Y or S34Y mutations.

The conclusion that the G31Y and G31F mutations create an independent internalization motif is strengthened by the observation that placing these substitutions in the context of the wild-type TR augments the rate of internalization by approximately 2-fold. The increased rate of internalization of the G31Y and G31F TRs suggests that the native and novel internalization motifs can function additively in promoting internalization. Thus, these results provide a conceptual basis for understanding how the substitution of a tyrosine at position 34 creates a novel TR internalization motif. Furthermore, they provide new experimental evidence for the proposal that the minimum required sequence for efficient internalization is a tyrosine at the first position of a -turn, since the G31Y substitution was chosen solely because it was predicted to be in position 1 of a -turn.

In a recent study the complete YTRF internalization motif of the native TR was substituted for the amino acids at positions 31-34 of the TR (Collawn et al., 1993). In agreement with our findings, it was found that the TR with two YTRF internalization motifs (at native position and at positions 31-34) is more rapidly internalized than the wild-type TR and that substitution of YTRF at positions 31-34 restores wild-type rates to internalization-defective receptors in which the native YTRF has been replaced with PPGY (Collawn et al., 1993). Our results indicate that placement of a tyrosine at either the first or fourth position of a -turn is sufficient to form an independent internalization motif and are thus in agreement with those of Collawn et al. (1993).

In addition to a tyrosine at the first position of a -turn other amino acids of the -turn can significantly influence the internalization rate (for reviews see Trowbridge et al. (1993) and Roth (1993)). These additional requirements may explain why, in a previous study, we found that a phenylalanine at position 34 cannot create an internalization motif (McGraw et al., 1991) whereas, in the present study, a phenylalanine at position 31 does function. Different requirements for tyrosine or phenylalanine in rapid internalization have been previously noted. In a study of the influenza virus hemagglutinin, an internalization signal created by mutagenesis had a strict requirement for tyrosine (Lazarovits and Roth, 1988). By contrast, in the internalization motifs of the low density lipoprotein receptor (NPVY) and the TR (YTRF) the tyrosine can be replaced with phenylalanine or tryptophan without loss of efficiency (Davis et al., 1987; McGraw and Maxfield, 1990).

Our results showing that the measured internalization rate of the Y20C,F23A TR is the same as that resulting from either the Y20C or the F23A mutations alone are consistent with these residues both being equally important in the formation of the internalization motif (Collawn et al., 1990). The combinatorial loss of both Tyr-20 and Phe-23 does not reduce the internalization rate to the level observed when large portions of the cytoplasmic domain are deleted, i.e. 3-59 or 3-28 TRs. In both of these constructs the native internalization motif is deleted. These results demonstrate that simultaneous mutation of the tyrosine and phenylalanine of the YTRF motif does not completely remove all the information that promotes rapid internalization from the plasma membrane. These findings do not agree with a previous study reporting that the Y20A,F23A TR is internalized with the same efficiency as the 3-59 TR (Collawn et al., 1990). This difference could be due to different experimental techniques employed to measure TR internalization (steady-state TR distribution and rate of iron accumulation (Collawn et al. 1990) versus measurement of rate of Tf uptake) (this report; Johnson et al., 1993), the different mutations used, or differences in cell types studied. However, both sets of data agree that the majority of information required for rapid internalization is removed by mutating Tyr-20, Phe-23, or both, simultaneously.

Regarding different methods of characterizing the internalization behavior of various TR mutants, it is interesting to note that the magnitude of the defect in internalization of all the mutant cell lines examined in this study was consistently smaller when assayed by iron accumulation than by the measurement of rate of internalization of Tf. These observations confirm our previous findings (McGraw and Maxfield, 1990). The explanation for this phenomenon is not clear.

In conclusion, a functional internalization motif can be created de novo by substituting a tyrosine at the first position of a tetrapeptide sequence strongly predicted to form a -turn. This finding provides additional support for the proposal that one class of internalization motifs is an aromatic amino acid (most often tyrosine) in the first position of a -turn.

  
Table: Predicted potential for -turn formation at position 34

The method of calculation was as follows. Predicted potential for a -turn comprising four residues starting at position i is calculated from the values of turn frequencies ( f) and is given by the equation, p= f f f f. Conformational parameters |N7 P|N8, |N7 P|N8, and |N7 P|N8 are the averages of the frequencies of the four residues in the -turns, -helix, and -sheet, respectively. The values for f, |N7 P|N8, |N7 P|N8, and |N7 P|N8 are derived from analysis of 29 solved protein structures (Chou and Fasman, 1979). A -turn is predicted if P> 0.75 10, |N7 P|N8 > 1.0, and |N7 P|N8 < |N7 P|N8 > |N7 P|N8. The native GDNS tetrapeptide (positions 31-34 of the TR) has a high predicted potential for formation of a -turn, as do the tetrapeptides in which tyrosine has been substituted for glycine 31 or for serine 34.



FOOTNOTES

*
This work was supported by research grants from the Council for Tobacco Research and the American Heart Association, New York City affiliate. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: ImClone Systems, Inc., 180 Varick St., New York, NY 10014.

Junior Investigator of the American Heart Association, New York affiliate. To whom correspondence should be addressed: Dept. of Pathology, College of Physicians and Surgeons of Columbia University, 630 West 168th St., New York, NY 10032. Tel.: 212-305-8545; Fax: 212-305-5498.

The abbreviations used are: TR, transferrin receptor; Tf, diferric transferrin.


ACKNOWLEDGEMENTS

We acknowledge the expert technical assistance of Cathy Ferrone and Tracy Shevell. We are grateful to Drs. Kenneth Dunn, Amy Johnson, and Lester Johnson for critical reading of the manuscript and to Dr. James Farmar (ImClone Systems, Inc.) for the kind preparation of the oligonucleotides.


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