(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 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.
The insulin receptor is a member of the family of receptor
tyrosine kinases and consists of two - 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) .
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) 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.
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 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).
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
10M 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).
Figure 3:
Internalization of 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) .
The K 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).
Figure 4:
Rate constant for insulin
internalization. The K 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
Figure 5:
Surface redistribution of 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.
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 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.
Figure 6:
Association of 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.
Figure 7:
Insulin degradation in CHO cells. CHO
cells expressing mutant and wild-type (WT) receptors were
incubated with 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.
Figure 8:
Down-regulation of mutant receptors
(preincubation time course). CHO cells were preincubated with or
without 10M 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.
Loss of cell-surface receptor was also estimated by changes in
binding of labeled anti-receptor antibody (Fig. 9). After
preincubation with 10M 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.
Figure 9:
Down-regulation as measured by
anti-receptor antibody binding. CHO cells were preincubated with or
without 10M 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.
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 I-insulin internalization both biochemically and
morphologically and by performing insulin degradation and insulin
receptor down-regulation studies.
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 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.
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.