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(Received for publication, October 17, 1994) From the
We have characterized the internalization and down-regulation of
the insulin receptor and nine receptors with mutations in the
transmembrane (TM) domain and/or flanking charged amino acids to define
the role of this domain in receptor cycling. When expressed in Chinese
hamster ovary cells, all had normal tetrameric structure and normal
insulin-stimulated autophosphorylation/kinase activity. Replacement of
the TM domain with that of the platelet-derived growth factor receptor,
insertion of 3 amino acids, and substitution of Asp for Val The insulin receptor is a member of the family of receptor
tyrosine kinases and consists of two In this study, we have examined the role of the transmembrane domain
and flanking charged amino acids of the insulin receptor in
ligand-dependent receptor internalization utilizing a series of nine
mutant insulin receptors in which these domains were modified by in
vitro mutagenesis. Although these mutants have normal
insulin-stimulated receptor kinase activity and an intact juxtamembrane
region, using a combination of biochemical and morphological
techniques, we find that several of these mutants exhibit decreased
insulin-stimulated internalization and one exhibits accelerated
internalization. Furthermore, these mutations in the transmembrane
domain affect receptor down-regulation and receptor-mediated insulin
degradation. Thus, the TM domain and the flanking charged amino acids
provide an important determinant of ligand-dependent internalization of
the insulin receptor and subsequent receptor down-regulation and ligand
degradation.
The internalization rate constant (K
Figure 1:
Amino acids sequences of transmembrane
domains in normal and mutant insulin receptors. Mutated sequences are underlined.
CHO cells stably transfected with cDNAs for normal and mutant
insulin receptors expressed between 2.5
Figure 2:
Autophosphorylation of TM domain mutant
insulin receptors in intact cells. Confluent CHO cells expressing
mutant and wild-type insulin receptors were incubated with
10
Figure 3:
Internalization of
The K
Figure 4:
Rate constant for insulin
internalization. The K
Figure 5:
Surface redistribution of
With respect to the second step of internalization, the anchoring in
clathrin-coated pits, the TM/c-neu and ++++exo/cyt
mutants exhibited a reduced association with these surface areas when
all surface-associated autoradiographic grains localizing
Figure 6:
Association of
Figure 7:
Insulin degradation in CHO cells. CHO
cells expressing mutant and wild-type (WT) receptors were
incubated with
Figure 8:
Down-regulation of mutant receptors
(preincubation time course). CHO cells were preincubated with or
without 10
Loss of cell-surface receptor was also estimated by changes in
binding of labeled anti-receptor antibody (Fig. 9). After
preincubation with 10
Figure 9:
Down-regulation as measured by
anti-receptor antibody binding. CHO cells were preincubated with or
without 10
Internalization of membrane receptors may play a critical
role in their cellular function, as for example with the receptor for
many nutrient-related components such as low density lipoprotein and
transferrin, or may play a more regulatory role, as in the case of most
hormone receptors. Evidence that the insulin receptor is involved in
ligand-stimulated internalization has been present for over 15 years,
but only recently have the structural determinants of this process
begun to be elucidated. The present results clearly show the importance
of the transmembrane domain of the insulin receptor in ligand-dependent
receptor internalization as demonstrated by a decreased receptor
internalization in the TM/c-neu mutant and an increased internalization
in the TM-AA/GP mutant. Our data also indicate that the 3 positively
charged amino acids on the cytoplasmic flank of the transmembrane
domain are another determinant of receptor internalization since
replacement of this sequence with uncharged amino acids (+exo/cyt
and ++++exo/cyt mutations) results in impaired
internalization. These findings could be confirmed measuring These determinants of
insulin-dependent receptor internalization are independent of receptor
kinase activation or structure of the juxtamembrane region, both of
which have been reported to be critical components in receptor
internalization(10, 11, 12, 13, 14, 15, 16, 17, 18) ,
since all mutants examined had normal kinase activation and an intact
juxtamembrane domain. Regulation of internalization independent of
kinase activation has also been suggested by Trischitta et
al.(34) , who showed that insulin receptor antibodies that
do not activate receptor tyrosine kinase can also induce receptor
internalization, and by Androlewicz et al.(35) , who
reported an insulin-resistant melanoma cell line that exhibited
decreased insulin-induced receptor internalization despite normal
ligand-dependent kinase activation. Along with present results, these
observations indicate that the mechanisms by which ligand-dependent
internalization can be regulated are complex and that the structure of
the TM domain and its flanking charged amino acids are major components
in the regulation of this system. The uptake of insulin-receptor
complexes inside target cells is preceded by surface events that can be
subdivided in three steps: 1) freeing of the receptor from microvilli,
where they preferentially localize in the absence of bound ligand; 2)
surface redistribution from microvilli to the nonvillous domain of the
cell surface; and 3) anchoring of the receptor in clathrin-coated
pits(15, 16) . The first of these events is
ligand-specific and depends on receptor kinase activation and
autophosphorylation, while the third requires specific signal sequences
present in the juxtamembrane domain of the receptor (15, 16, 17, 18) . Neither of these
two events seems affected by the mutations used in the present study
since all TM domain receptor mutants analyzed have a normally activable
kinase, intact autophosphorylation sites, and preserved juxtamembrane
domains. Moreover, as demonstrated by the morphological analysis, the
three TM domain receptor mutants that exhibit decreased or increased
internalization can leave microvilli and show a normal propensity to
associate with clathrin-coated pits on the nonvillous domain of the
cell surface. This is in contrast to what has been previously observed
in the case of kinase-inactive or juxtamembrane mutated
receptors(15, 16) . Thus, the altered surface
redistribution of these three receptor mutants (TM-AA/GP,
++++exo/cyt, and TM/c-neu) most probably reflects
altered surface mobility of these receptors. Indeed, using fluorescence
photobleaching, the TM-AA/GP receptors have been shown to exhibit
increased lateral mobility within the plane of the plasma
membrane(30) . This suggests that the structure of the
transmembrane domain may play an important role in the interaction with
the phospholipid bilayer of the plasma membrane. It is interesting to
note that internalization and intracellular processing of insulin
receptors are reduced in type II diabetes and obesity (36, 37, 38, 39, 40) ,
disorders in which there is a change in lipid composition of the
membrane (41, 42, 43) as well as decreased
receptor kinase activation. The exact role of internalized receptors
in insulin action remains
uncertain(44, 45, 46, 47) . The fact
that double alanine substitution for Gly Another important
finding in this study is a tight correlation between insulin-dependent
receptor internalization and insulin-induced receptor down-regulation
in each mutation. Internalization-defective mutations, which include
TM/c-neu, +exo/cyt, and ++++exo/cyt mutants,
all exhibited decreased levels of insulin receptor down-regulation and
insulin degradation, whereas the TM-AA/GP mutant, which had enhanced
receptor internalization, showed accelerated receptor down-regulation
and increased insulin degradation. Correlations among internalization,
receptor down-regulation, and insulin degradation have also been
observed with chimeric receptor constructs and other
mutants(48, 49, 50) . Moreover, in studies
analogous to the present one, we (15) and others (10, 51) reported similar observations in the case of
various mutations of the insulin receptor accompanied by changes in
receptor internalization rate and magnitude. Although insulin-induced
receptor down-regulation is thought to be a complicated phenomenon that
includes not only the endocytosis, but also degradation, recycling, and
biosynthesis of the insulin receptors(45, 46) , we
conclude that insulin-dependent receptor internalization is the major
factor in insulin-induced down-regulation and intracellular insulin
degradation.
Volume 270,
Number 7,
Issue of February 17, 1995 pp. 3115-3122
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
or of Ala for either Gly
or Pro
had
no effect on internalization. Replacement of the TM domain with that of
c-neu or conversion of the charged amino acids on the
cytoplasmic flank to uncharged amino acids, on the other hand, resulted
in a 40-60% decrease in insulin-dependent internalization rate
constants. By contrast, substitution of Ala for both Gly
and Pro increases lateral diffusion mobility and
accelerates internalization rate. These changes in internalization were
due to decreased or increased rates of redistribution of receptors from
microvilli to the nonvillous cell surface. In all cases, receptor
down-regulation and receptor-mediated insulin degradation paralleled
the changes in internalization. Thus, the structure of the
transmembrane domain of the insulin receptor and flanking amino acids
are major determinants of receptor internalization, insulin
degradation, and receptor down-regulation.
- and two 
-subunits in
an 
-heterotetrameric
form(1, 2) . The -subunits are located outside
the cell and contain the insulin-binding site. The -subunits are
transmembrane proteins; each has a 194-amino acid external domain, a
single transmembrane (TM) (
)domain of 23 amino acids, and a
large intracellular domain containing the receptor tyrosine
kinase(3, 4) . Binding of insulin to the -subunit
on the cell-surface receptor results in activation of the -subunit
kinase, which in turn phosphorylates intracellular substrates such as
insulin receptor substrate-1 (IRS-1), initiating the intracellular
events that lead to the final biological effects of
insulin(5, 6, 7) . At the same time, the
insulin-receptor complex undergoes receptor-mediated internalization (8, 9) . Recent studies have demonstrated that
insulin-dependent internalization of the receptor requires both an
active -subunit kinase and an intact intracellular juxtamembrane
region(10, 11, 12, 13, 14, 15, 16) .
The latter contains sequence motifs, which like those involved in
internalization of other receptors, form a tight type I -turn
exposing at least one aromatic
residue(17, 18, 19, 20, 21, 22, 23, 24) .
Materials
I-Insulin was prepared
to a specific activity of 3000 Ci/mol at the Peptide Synthesis Core,
Joslin Diabetes Center. NaI was purchased from Amersham
Corp. Fetal bovine serum and tissue culture medium were from Life
Technologies, Inc. Reagents for SDS-polyacrylamide gel electrophoresis
were from Bio-Rad. L-1-Tosylamido-2-phenylethyl chloromethyl
ketone-treated trypsin was from Worthington. All other reagents
including lactoperoxidase, glucose oxidase, soybean trypsin inhibitor,
and monensin were purchased from Sigma. Human insulin receptor-specific
monoclonal antibody 83-14 directed against the -subunit of the
human insulin receptor was kindly supplied by Dr. Kenneth Siddle
(Cambridge University, London) and iodinated with IODO-GEN (Pierce)
when needed.In Vitro Mutagenesis and Establishment of CHO Cell Lines
Which Overexpress Mutant Insulin Receptors
cDNA sequences
encoding human insulin receptors were prepared by
oligonucleotide-directed mutagenesis as described
previously(18, 25) . Positive candidates were chosen
by DNA sequencing the area of interest; selected clones were sequenced
over the mutated region plus >50 bases upstream and downstream to
confirm the absence of unexpected mutations. Monolayer CHO cells were
grown in Ham's F-12 medium containing 10% fetal bovine serum.
Cells were cotransfected with a neomycin resistance plasmid and the
mutated insulin receptor expression plasmids by calcium phosphate
precipitation (25) and selected by growth in the presence of
450 mg/ml Geneticin (Life Technologies, Inc.). Transfected cells were
enriched for expression by two or three passages of
fluorescence-activated cell sorting using human insulin receptor
monclonal antibody 83-14, and clonal cell lines were obtained by
limiting dilution. Clones expressing equivalent receptor numbers were
identified by insulin binding and Scatchard
analyses(25, 26) .
Confluent CHO cells in
24-well plates were incubated with I-Insulin Uptake in CHO Cells
Overexpressing Mutant Insulin ReceptorsI-insulin (100,000
cpm/well) in internalization medium (serum-free Ham's F-12 medium
supplemented with 50 mM Hepes, pH 7.6, and 0.3% bovine serum
albumin). After incubation at 37 °C for the indicated times, cells
were washed with either PBS at pH 7.4 or PBS at pH 3.5 three times
using a 3-min incubation for each wash. Cells were lysed in 0.1 N NaOH, 0.1% SDS, and the radioactivity was counted to determine
total cell-associated insulin, i.e. the counts/minutes
remaining after the neutral wash, and internalized insulin, i.e. the I-insulin remaining after the acidic
wash(13) .
) was calculated based on the internalization
data as described by Lund et al.(27) and Backer et al.(28) . Briefly, the instantaneous velocity of
ligand internalization was expressed as dL
/dt = K[LR
], where
L is the amount of internalized ligand and
[LR] is the concentration of cell-surface
ligand-receptor complex. Integrating both sides of the equation,
L = K[LR]dt. Thus, the K is the slope of the line when L is
plotted versus [LR] as a function of
time. Integrals were approximated by the trapezoidal rule taking an
interval of dt = 2 min.Quantitative Electron Microscopy
Autoradiography
Fixed cells from two different experiences were
dehydrated, processed for electron microscopy autoradiography, and
quantitated as described
previously(8, 16, 29) . For each incubation
time, three Epon blocks were prepared, and three sections were cut from
each block. Thus, for each time point studied for each cell line, 18
separate grids were examined, out of which 1000-2000 grains were
analyzed from all cells judged to be morphologically intact. Grains
within a distance of 250 nm from the plasma membrane were considered
associated with the cell surface, whereas grains overlying the
cytoplasm and >250 nm from the plasma membrane were considered
internalized. Grains associated with the plasma membrane were divided
into the following classes: 1) microvilli, 2) coated pits, 3)
nonvillous noncoated pits segments, and 4) uninterpretable. Grains were
considered associated with microvilli or coated pits if their center
was <250 nm from these surface domains; they were categorized as
uninterpretable when the structures underlying the grain could not be
unequivocally identified.Degradation of Insulin by CHO Cells
Confluent CHO
cells in 24-well plates were incubated with I-insulin
(100,000 cpm/well) for 18 h at 4 °C. Cells were washed twice with
chilled PBS and quickly warmed to 37 °C by addition of 0.5 ml of
internalization medium to each well. At the indicated times, aliquots
of medium were taken and mixed with an equal volume of 15%
trichloroacetic acid to give a final trichloroacetic acid concentration
of 7.5%. The intact insulin was precipitated by centrifugation in a
microcentrifuge, and the radioactivity in the supernatant was counted
to determine the amount of degraded insulin. Data are expressed as the
ratio of degraded insulin to that initially bound to the cells.Insulin-induced Down-regulation of Mutant Insulin
Receptors
Confluent CHO cells in 24-well plates were incubated
with or without 10
M insulin at 37 °C
for the indicated times in 0.5 ml of internalization medium/well. After
incubation, the medium was aspirated, and the cells were washed twice
with chilled PBS, pH 3.5, supplemented with 0.3% bovine serum albumin
and then twice with PBS, 0.3% bovine serum albumin, pH 7.4, to remove
all surface-bound insulin. Each wash included a 3-min incubation. To
quantify cell-surface insulin receptors, cells were incubated with
either 0.1 ng/ml I-insulin and unlabeled insulin or
iodinated monoclonal antibody 83-14 (200,000 cpm/well) in 0.5 ml of
internalization medium/well at 4 °C. After 18 h of incubation,
cells were washed twice with chilled PBS, pH 7.4, and digested in 0.1 N NaOH, 0.1% SDS, and the associated radioactivity was counted
in an automated 
-counter. In some experiments, cells were
preincubated with insulin in the presence of 50 mM monensin,
followed by cell-surface receptor quantification.
Insulin Binding and Autophosphorylation of Mutant and
Wild-type Receptors
The mutants used in this study include
entire replacements of the TM domain by TM domains of the
proto-oncoprotein c-neu (TM/c-neu) or the platelet-derived
growth factor receptor (TM/PDGFR), an aspartic acid substitution for
Val (TM-D/V) analogous to the point mutation in the neu oncoprotein, an insertion of the 3 amino acids Val-Phe-Leu
between Leu
and Phe
, double alanine
substitutions for Gly and Pro, single
alanine substitutions for either Gly or
Pro, and replacements of 3 flanking charged amino acids
at the intracellular face of the TM domain
(Arg
-Lys
-Arg
) with the
uncharged amino acids Ala-Asn-Ala with or without insertion of
Arg-Lys-Arg into the exofacial surface of the TM domain
(++++exo/cyt and +exo/cyt, respectively). The
sequences of all of these mutant TM domains are shown in Fig. 1.
10
and 3.6
10
binding sites/cell (Table 1). This is
20-150 times the number of endogenous hamster insulin receptors,
estimated to be 1-2 10
/cell. As described
previously (25, 30) , each of these receptors was
normally processed to give - and -subunits, and the affinity
for insulin binding in all cases was normal (Table 1).
Autophosphorylation of these TM domain mutant insulin receptors in
intact cells was also normal as assessed by anti-phosphotyrosine
immunoblots of cell extracts after insulin stimulation in vivo (Fig. 2). As a negative control, we also included the
Ala
receptor mutant, which has an alanine for lysine
substitution in the ATP-binding site and is known to be a
kinase-defective mutant (19, 31, 32) (data
not shown).
M insulin in Ham's F-12 medium at
37 °C for 5 min. Cells were then solubilized as described under
``Experimental Procedures,'' and insulin receptors were
immunoprecipitated with antibody 83-14. Tyrosine phosphorylation of the
insulin receptor was detected by immunoblotting with
anti-phosphotyrosine antibody. In all cases, the odd-numbered lanes represent basal phosphorylation, and the even-numbered lanes represent insulin-stimulated samples. Representative experiments
are show for the wild-type receptor (A, lanes 1 and 2; B, lanes 1 and 2; and C, lanes 1 and 2), +exo/cyt (A, lanes 3 and 4),
++++exo/cyt (A, lanes 5 and 6), TM/c-neu (B, lanes 3 and 4),
TM/PDGFR (B, lanes 5 and 6), TM-D/V (B, lanes 7 and 8), TM+3 (B, lanes 9 and 10), TM-AA/GP (C, lanes 3 and 4), TM-A/P (C, lanes 5 and 6), and TM-A/G (C, lanes 7 and 8).
Insulin Uptake in CHO Cells
As with most
cell-surface receptors, following insulin binding at physiological
temperature (37 °C), some of the occupied insulin receptors are
internalized along with the bound insulin. This pool of internalized
bound insulin can be distinguished from the cell-surface pool by
resistance to elution with an acidic wash as described under
``Experimental Procedures.'' The time course of
internalization at 37 °C for all the TM domain mutants, the
wild-type receptor, and the Ala mutant is shown in Fig. 3A. As described
previously(10, 11, 12, 15, 16) ,
the kinase-defective Ala mutant showed little or no
insulin-stimulated internalization over the time course of study. Among
the TM domain mutants studied, TM/c-neu, +exo/cyt and
++++exo/cyt also showed reduced levels of I-insulin uptake when compared with the wild-type
receptors. By contrast, internalization of insulin receptors was
increased by 10-100% for the TM-AA/GP receptor mutant (Fig. 3A, right panel). Internalization was
also studied in the CHO cells expressing normal insulin receptors and
selected TM domain mutants using quantitative autoradiography at the
electron microscopic level (Fig. 3B). Again, by this
method, internalization was impaired for both the TM/c-neu and
++++exo/cyt mutants and increased in the TM-AA/GP
mutant (Fig. 3B). Morphological and biochemical
analysis also indicated that the inhibition of I-insulin
internalization observed in TM/c-neu and
++++exo/cyt CHO cells was not as complete as that
observed in cells expressing the Ala insulin receptor
mutant (Fig. 3, A and B).
I-insulin. CHO cells expressing mutant and wild-type (WT) receptors were incubated with I-insulin
(100,000 cpm/well) at 37 °C for the indicated times. Cells were
washed with either PBS at pH 7.4 or PBS at pH 3.5, after which the
cell-associated radioactivity was determined. In A, the
acid-resistant radioactivity was considered as internalized. In B, the percentage of the total number of autoradiographic
grains seen associated with the cells at the electron microscopic level
that are centered >250 nm from the plasma membrane represents the
percentage I-insulin internalized. In these experiments,
cells were continuously incubated in the presence of I-insulin for the indicated periods of time at 4 or 37
°C. Results concerning Ala mutant insulin receptor
are derived from (16) .
values were calculated from the acid wash data
as described previously (28) (Fig. 4). The K values for TM domain mutants TM/c-neu,
+exo/cyt, and ++++exo/cyt were decreased by
40-60% as compared with that of the wild-type receptor (Fig. 4). The Ala mutant, which has abolished
tyrosine kinase activation, showed the lowest level internalization
rate (K 
15% of the wild-type K). On the other hand, the TM-AA/GP insulin
receptor mutant, in which 2 naturally occurring helix-breaking amino
acids (Gly and Pro) were replaced with 2
helix-favoring alanines, was found to have an accelerated insulin
uptake rate corresponding to 70% above that of the wild-type
receptor (Fig. 4, A, rightpanel, opensquares; and B). All other mutant
receptors showed a normal insulin uptake rate ( Fig. 3and Fig. 4).
values for each
mutant and wild-type (WT) receptor were determined as
described under ``Experimental Procedures.'' Data represent
the means ± S.E. of three to six separate experiments. LR, concentration of cell-surface ligand-receptor
complex
Surface Localization of the Insulin Receptor
Two
surface steps precede the entry of insulin receptors inside the cells:
the first is ligand-specific and consists of the insulin-induced
redistribution of the receptor from microvilli to the nonvillous domain
of the cell surface; the second is ligand-independent and common to
many receptors and consists of the anchoring of receptors to
clathrin-coated pits, which represent the internalization
gate(16) . To distinguish whether the modified rates of
internalization observed with the various insulin receptor TM domain
mutants were related to altered surface events, we analyzed
morphologically the surface localization and redistribution of these
receptors. With respect to the first step, both the TM/c-neu and
++++exo/cyt mutants exhibited defects in
internalization and redistributed from microvilli to the nonvillous
region of the cell surface in response to I-insulin
binding more slowly and to a lesser extent than wild-type receptors,
but faster than the Ala mutant receptors (Fig. 5). By contrast, the TM-AA/GP mutant receptor, which
exhibited an increased internalization rate, was more rapidly
redistributed than the normal human insulin receptor (Fig. 5).
I-insulin in CHO cells expressing mutant and wild-type
receptors. Results presented are the means ± S.E. of the
analysis of three different Epon blocks from two different experiments (n = 6). For each time point and each cell line, 2000
autoradiographic grains were quantitated. Cells were continuously
incubated in the presence of I-insulin for the indicated
periods of time at 4 or 37 °C. Results concerning the Ala mutant insulin receptor are derived from (16) . WT, wild-type receptor.
I-insulin were considered (Fig. 6A).
However, if we considered only labeled material present on the
nonvillous cell surface, these receptor mutants showed the same
propensity to associate with clathrin-coated pits (Fig. 6B). Thus, these mutants are able to anchor to
clathrin-coated pits, but are not concentrated in these surface
invaginations because they do not have access to the surface domain of
the cell surface where these structures are located. In the case of the
TM-AA/GP receptors, an increased association with clathrin-coated pits
was observed whichever mode of calculation was used (Fig. 6, A and B). This suggests that the increased mobility
of the TM-AA/GP receptor also increased its capacity to anchor to
clathrin-coated pits.
I-insulin
present on the total cell surface (A) or on the nonvillous
surface (B) with clathrin-coated pits in CHO cells expressing
mutant and wild-type receptors. Results presented are the means
± S.E. of the analysis of three different Epon blocks from two
different experiments (n = 6). For each time point and
each cell line, 2000 autoradiographic grains were quantitated. Cells
were continuously incubated in the presence of I-insulin
for the indicated periods of time at 4 or 37 °C. Results concerning
the Ala mutant insulin receptor are derived from (16) . WT, wild-type
receptor.
Insulin Degradation
Fig. 7shows the
degradation of I-insulin by CHO cells expressing the four
mutant receptors that exhibited decreased or accelerated insulin uptake
as well as the wild-type receptor and the Ala mutant.
For the wild-type receptor, there was relatively little insulin
degradation over the first 10 min of incubation and then an increase in
rate such that 24% of the cell-associated insulin was degraded after 30
min. The TM/c-neu, +exo/cyt, and ++++exo/cyt
receptors showed a reduced level of insulin degradation (ranging from
12 to 18% at 30 min), whereas the TM-AA/GP mutant had increased insulin
degradation compared with the wild-type receptor (36% at 30 min). The
kinase-defective Ala mutant showed the lowest level of
insulin degradation (11% at 30 min). Thus, the amount of degraded
insulin changed in proportion to the level of insulin uptake.
I-insulin at 4 °C for 18 h. Cells were
washed with chilled PBS and warmed up to 37 °C in Ham's F-12
medium. After incubation for the indicated times at 37 °C, the
amount of degraded insulin was determined by trichloroacetic acid
precipitation. Data are expressed as a percentage of initial bound
insulin.
Down-regulation of Mutant Insulin
Receptors
Preincubation of CHO cells with high concentrations of
insulin (10M) causes a loss of
cell-surface insulin receptor, i.e. down-regulation in a
time-dependent manner. Fig. 8shows a representative time course
for down-regulation of I-insulin binding with CHO cells
that overexpress wild-type insulin receptors. After 16 h of incubation,
the bound/free ratio for I-insulin decreased by 35% as
compared with that in cells incubated with buffer but without insulin
for 16 h. Each mutant receptor also showed reduced surface binding of
insulin after 16 h of preincubation with insulin; however, the degree
of down-regulation was only 22, 18, and 15% for +exo/cyt,
++++exo/cyt, and TM/c-neu, respectively, all of
which showed reduced internalization. As with other parameters, the
TM-AA/GP mutant receptor showed an increased extent of down-regulation,
which was 50% greater than that of the wild-type receptor. These
changes in binding were due to changes in cell-surface receptor
concentration, as demonstrated by Scatchard analysis (data not shown).
When assessed by insulin binding, the kinase-defective mutant receptor
Ala showed an unexplained, but reproducible, small
increase in ligand binding following prolonged insulin pretreatment.
M insulin for the indicated
times at 37 °C. Cells were washed twice with PBS at pH 3.5 and
twice with PBS at pH 7.4 to remove surface-bound insulin. Cells were
then incubated with I-insulin for 18 h at 4 °C. After
washing, cells were digested, and the associated radioactivity was
counted. Data are expressed as the bound/free ratio of
``down-regulated'' to control. Each point represents the mean
± S.E. of three separate
experiments.
M insulin for 18 h,
binding of iodinated monoclonal antibody 83-14 was decreased by 30% for
the wild-type receptor. The +exo/cyt,
++++exo/cyt, and TM/c-neu mutations showed reduced
antibody binding by 19, 20, and 24%, respectively, whereas the TM-AA/GP
mutant had a 41% decrease in antibody binding with insulin
preincubation. These data confirm the fact that the loss of insulin
binding is due to a loss of immunoreactive receptor protein, rather
than some modification of the insulin-binding site. Furthermore, the
degree of insulin-induced receptor down-regulation varied in proportion
to the change in receptor internalization in each receptor mutant. In
contrast to the studies with labeled insulin, however, no change in
receptor was found in cells expressing the Ala mutant
when assessed by antibody binding.
M insulin and washed as
described in the legend to Fig. 8. Cells were incubated with I-labeled monoclonal antibody 83-14 to the insulin
receptor (2 10 cpm/well) at 4 °C for 18 h. Cells were
washed with PBS and digested, and the associated radioactivity was
counted. WT, wild-type receptor.
Effect of Monensin on Insulin-induced Down-regulation of
Mutant Receptors
Insulin-induced receptor down-regulation is a
reflection not only of receptor endocytosis, but also of synthesis,
recycling, and degradation. Monensin, a calcium ionophore that inhibits
receptor recycling without affecting internalization(33) , was
used to further investigate the relationship between internalization
and down-regulation of mutant insulin receptors. In this experiment,
preincubation of CHO cells with 10M insulin for 3 h at 37 °C caused receptor down-regulation by
27% for the wild-type receptor and between 6 and 39% for the various
mutants (Table 2). When cells were incubated with insulin in the
presence of 50 mM monensin for 3 h, in each case, loss of
cell-surface receptors became even greater than that without monensin.
For example, with monensin, the wild-type receptor was down-regulated
by 36%. One can obtain an estimate of the ``recycled
fraction'' by calculating the percent increase in down-regulation
in the presence versus the absence of monensin, and for the
wild-type receptor, this value was 28%. Based on similar calculations,
the recycled fraction of internalized receptors was estimated to be
between 23 and 36% for all cell lines. Thus, there was no significant
difference in recycling of the internalized receptor to the cell
surface among mutant and wild-type receptors, suggesting that the
differences in receptor down-regulation can be accounted for mainly by
the differences in internalization among these receptors.
I-insulin internalization both biochemically and
morphologically and by performing insulin degradation and insulin
receptor down-regulation studies. and Pro (TM-AA/GP) exhibited an accelerated ligand-dependent
internalization suggests that the native structure of the transmembrane
domain does not necessarily provide the best conformation for
ligand-dependent internalization. This further suggests that
internalization of the insulin receptor may be regulated in some way
that provides the best efficiency for transmission of the extracellular
signal to intracellular receptor kinase domain.
))
We thank C. Maeder and G. Berthet for skilled
technical assistance and Terri-Lyn Bellman and Nanda E. Barker for
excellent secretarial assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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