From the
Receptors are internalized from the plasma membrane at
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
The
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
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.
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.
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).
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).
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
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
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
In addition to a tyrosine at the first
position of a
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.
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
The method of calculation was as
follows. Predicted potential for a
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
-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).
-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).
-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.
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`.
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
Fe
Tf or
Fe
Tf in the presence of 200-fold excess unlabeled Tf
to measure nonspecific binding. One six-well plate was used to measure
surface binding of
I
Tf 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.
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).
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.
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.
-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.
-turn, since the G31Y substitution was
chosen solely because it was predicted to be in position 1 of a
-turn.
-turn is sufficient to form an independent
internalization motif and are thus in agreement with those of Collawn
et al. (1993).
-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).
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.
-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
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.