(Received for publication, April 10, 1995; and in revised form, July 31, 1995)
From the
pp120/HA4 is a hepatocyte membrane glycoprotein phosphorylated by the insulin receptor tyrosine kinase. In this study, we have investigated the role of pp120/HA4 in insulin action. Transfection of antisense pp120/HA4 cDNA in H35 hepatoma cells resulted in inhibition of pp120/HA4 expression and was associated with a 2-3-fold decrease in the rate of insulin internalization. Furthermore, insulin internalization in NIH 3T3 fibroblasts co-transfected with insulin receptors and pp120/HA4 was increased 2-fold compared with cells expressing insulin receptors alone. In contrast, no effect on internalization was observed in cells overexpressing a naturally occurring splice variant of pp120/HA4 that lacks the phosphorylation sites in the intracellular domain. Insulin internalization was also unaffected in cells expressing three site-directed mutants of pp120/HA4 in which the sites of phosphorylation by the insulin receptor kinase had been removed (Y488F, Y488F/Y513F, and S503A). Our data suggest that pp120/HA4 is part of a complex of proteins required for receptor-mediated internalization of insulin. It is possible that this function is regulated by insulin-induced phosphorylation of the intracellular domain of pp120/HA4.
Insulin binding to its receptor triggers the rapid endocytosis of the ligand-receptor complex(1, 2) . Internalization of the insulin-insulin receptor complex constitutes the major mechanism of insulin degradation and down-regulation of cell surface receptors (3, 4, 5) . The molecular events involved in the internalization process, however, are yet to be well defined. Specific sequences in the submembranous portion of the receptor are required for internalization to occur. Furthermore, activation of the receptor kinase is required for ligand-induced receptor internalization(6, 7, 8, 9, 10, 11, 12, 13, 14, 15) . Following insulin-induced receptor autophosphorylation, several intracellular substrates are phosphorylated, the best characterized of which is insulin receptor substrate-1 (IRS-1)(16) . However, the role of substrate phosphorylation in receptor internalization is not established.
In the present study, we have investigated the role
of pp120/HA4, a substrate of the insulin receptor kinase that is
predominantly expressed in liver, in the internalization process.
pp120/HA4 is a transmembrane glycoprotein that is phosphorylated by the
insulin receptor tyrosine kinase in intact cells (17, 18, 19, 20) and in cell-free
systems(21) . It is composed of a large extracellular domain
containing 16 sites of potential N-linked glycosylation and a
71-amino acid cytoplasmic domain(22) . Studies of site-directed
mutants indicate that the intracellular domain contains a site of
constitutive serine phosphorylation (Ser) as well as one
tyrosine residue that is phosphorylated in response to insulin
(Tyr
)(20, 23) . Alternative splicing of
pp120/HA4 mRNA generates two isoforms, one of which lacks 61 amino
acids at the C terminus of the cytoplasmic domain, including all the
potential phosphorylation sites(24) . The function of pp120/HA4
is still unknown. It has been proposed to serve as a
Ca
/Mg
-dependent ecto-ATPase (22) and a bile acid transporter(25, 26) .
We report that insulin-induced receptor internalization is inhibited
2-3-fold in H35 hepatoma cells transfected with an antisense
pp120/HA4 cDNA. Inhibition of internalization paralleled the reduction
of expression of pp120/HA4. Moreover, expression of pp120/HA4 in NIH
3T3 cells transfected with insulin receptors increased the rate of
internalization of the insulin-insulin receptor complex. In contrast,
internalization rates were unaffected in NIH 3T3 cells expressing the
short isoform of pp120/HA4 or by expression of mutant pp120/HA4
molecules in which the potential phosphorylation sites of the
intracellular domain (Tyr and Ser
) had been
replaced by site-directed mutagenesis with nonphosphorylatable amino
acids.
NIH 3T3 cells co-expressing human insulin receptors and wild-type (WT) pp120/HA4 (full-length and truncated isoforms) or phosphorylation-defective pp120/HA4 mutants (Y488F, Y513F, Y488F/Y513F, and S503A) were described previously(20) .
Figure 1:
Expression of antisense pp120/HA4 RNA
in H35 hepatoma cells. Cell extracts were prepared from untransfected
H35 hepatoma cells (H35), H35 cells transfected with Neo vector, and clones transfected with antisense pp120/HA4 cDNA
(AS1-AS6) and analyzed by immunoblotting with
-295 (anti-pp120/HA4
polyclonal antibody; upperpanel). Quantitation was
done by phosphorImager analysis of the radioactive blot (lowerpanel). This figure shows one of two different
representative experiments.
Figure 2: Insulin internalization and degradation in H35 cells. Insulin internalization (A) was assayed as described under ``Experimental Procedures.'' Acid-resistant radioactivity was measured in H35 cells (open circles), Neo cells (solidcircles), and AS cells (opensquares) at the indicated times. Insulin degradation (B) was evaluated as trichloroacetic acid-soluble radioactivity in the extracellular medium after 20 min at 37 °C. These data represent mean ± S.D. of four experiments in duplicates performed with at least two different clones of each cell type.
Following receptor-mediated internalization, insulin molecules are released from the receptor by the acidic pH of the endocytic vesicles, and insulin is subjected to intracellular degradation. Insulin degradation was measured as trichloroacetic acid-soluble radioactivity in clones expressing antisense pp120/HA4 RNA (Fig. 2B). Consistent with the decreased internalization rate, insulin degradation was also decreased 2-fold in AS cells (15% of bound insulin, following 20 min incubation at 37 °C) compared with both untransfected and Neo-transfected cells (35 and 42% of bound insulin, respectively). After 30 min at 37 °C, 78% of bound insulin was degraded in H35, 82% in Neo cells, and 28% in AS cells, indicating that insulin degradation occurred at a slower rate in cells expressing antisense pp120/HA4 RNA. Similar results were also obtained when insulin degradation was measured in cell lysates (data not shown).
Insulin-induced receptor down-regulation was analyzed in AS and control cells following incubation with 100 nM insulin for 24 h at 37 °C. At the end of the incubation period, the receptor number on the surface of control cells (H35 and Neo) was decreased by 15-20%. In contrast, AS cells did not down-regulate the number of cell surface receptors in response to insulin (data not shown).
Figure 3:
Insulin receptor internalization in NIH
3T3 cells expressing pp120/HA4. Insulin internalization in NIH 3T3
cells expressing hIR alone (opencircles),
co-expressing hIR and WTpp120 (opensquares), or
co-expressing hIR and 448 (solidcircles) was
measured as described in the legend to Fig. 2. The data
represent mean ± S.D. from three different experiments performed
in triplicate on at least two different clones of each cell
type.
Figure 4:
PDGF
receptor internalization in NIH 3T3 cells expressing pp120/HA4. Binding
of I-PDGF to 75% confluent monolayers was carried out as
indicated in (28) . Thereafter, PDGF internalization in NIH 3T3
cells (opensquares) or cells expressing WTpp120 (filledsquares) was measured by the acid wash
technique as described. Experiments were performed in
triplicate.
Insulin receptor internalization was also studied using biotin
labeling of cell surface proteins followed by immunoprecipitation with
anti-insulin receptor antibody and immunodetection with
[I]streptavidin. Cell surface proteins were
biotinylated at 4 °C and then transferred to a 37 °C incubator
for 15 min in the absence or presence of 100 nM insulin (Fig. 5). Thereafter, monolayers were treated with Pronase to
remove residual cell surface proteins, lysed with Triton X-100,
immunoprecipitated with anti-insulin receptor antibody (Ab50) and
probed with [
I]streptavidin. In the absence of
Pronase treatment, two major bands were detected at approximately 130
and 95 kDa, corresponding to
- and
-subunits of the insulin
receptor(29) . Pronase treatment resulted in a 90-95%
decrease of cell surface receptors (Fig. 5, solid
bars). Preincubation with insulin rendered a larger fraction of
insulin receptors expressed in NIH 3T3 cells resistant to digestion
with Pronase as a result of having undergone internalization (Fig. 5, hatchedbars). Consistent with
insulin internalization data, the amount of internalized receptors was
2-fold higher in WTpp120 cells (33 ± 5% of the total
biotinylated receptors were internalized) than in hIR (16 ± 4%)
and
448 cells (12 ± 3%).
Figure 5: Internalization of biotin-labeled insulin receptors. After labeling of cell surface protein with biotin, monolayers were incubated at 37 °C for 20 min in the presence (fullbars) or absence (hatchedbars) of 100 nM insulin. After incubation with Pronase, cells were lysed and immunoprecipitated with anti-insulin receptor antibodies (Ab50). These data were obtained by PhosphorImager quantitation of blots from three separate experiments.
Thus, expression of full-length pp120/HA4, but not of the truncated isoform, was associated with increased internalization of insulin-receptor complexes in NIH 3T3 cells.
In NIH
3T3 cells co-transfected with hIR and phosphorylation-defective mutants
of pp120/HA4 (Y488F, Y488F/Y513F, or S503A), insulin internalization
rates were similar to those observed in cells expressing hIR alone and
cells co-expressing hIR and 448 (Fig. 6A). In
contrast, insulin internalization rates in cells transfected with Y513F
mutant were similar to those observed in cells expressing WTpp120 (Fig. 6A). Thus, we observed a correlation between
increased rates of insulin endocytosis and expression of
phosphorylation-competent forms of pp120/HA4.
Figure 6: Insulin internalization and degradation in cells expressing pp120/HA4 phosphorylation-defective mutants. Insulin internalization (upperpanel) and degradation (lowerpanel) were measured as in the legend to Fig. 2. Insulin internalization rates (upperpanel) were determined according to Lund et al.(27) . Insulin degradation (lowerpanel) represents the fraction of trichloroacetic acid-soluble radioactivity detected in the extracellular medium after 20 min at 37 °C. All results are expressed as the mean ± S.D. of three triplicate experiments with at least two different clones of each cell type.
Receptor down-regulation was evaluated in clones
transfected with hIR and different forms of pp120/HA4. In hIR cells,
insulin binding decreased by 37% after 24 h of insulin treatment. In
contrast, at the same time point, the number of surface receptors in
WTpp120 cells decreased by 50% (Fig. 7). In cells transfected
with the truncated isoform of pp120/HA4 (448), insulin receptor
down-regulation was similar to hIR cells (34%). Insulin-induced
receptor down-regulation of Y488F, Y488F/Y513F, and S503A cells was
comparable with that observed in
448 cells. In cells expressing
Y513F, receptor down-regulation displayed a pattern similar to that of
WTpp120 cells, with 47% of receptors being removed from the cell
surface by 24 h (Fig. 7).
Figure 7:
Insulin-induced receptor down-regulation
in NIH 3T3 transfected cells. Down-regulation was measured in hIR (opencircles), WTpp120 (opensquares), 448 (opentriangles),
Y488F (solidsquares), Y513F (solidcircles), Y488F/Y513F (solidtriangles), and S503A (opendiamond)
cells as described under ``Experimental Procedures.'' Data
are means ± S.D. of three triplicate experiments with at least
two clones of each cell type.
pp120/HA4 is a transmembrane glycoprotein that is phosphorylated on tyrosine residues by the insulin receptor kinase(17, 18, 19, 20, 21) . In the present study we have shown that inhibition of the expression of pp120/HA4 is associated with decreased endocytosis of insulin. We have also shown, using transfected cells, that overexpression of pp120/HA4 is associated with increased insulin endocytosis. Thus, manipulation of pp120/HA4 expression correlates with changes in insulin receptor-mediated internalization and degradation of insulin. Moreover, this effect of pp120/HA4 appears to require phosphorylation by the insulin receptor or other kinases, inasmuch as expression of phosphorylation-defective mutants of pp120/HA4 (20) is not associated with changes in internalization of the insulin-insulin receptor complex. The effect is specific for insulin endocytosis, because internalization of PDGF, a ligand that is also internalized via a receptor tyrosine kinase, is not affected by expression of pp120/HA4, nor is internalization of the unrelated molecule transferrin. Our data do not indicate whether there is a causal relation between expression of pp120/HA4 and endocytosis of insulin. It is, however, interesting to note that pp120/HA4 is predominantly expressed on the plasma membrane of the hepatocyte, a major site of insulin clearance from the circulation. The mechanism by which pp120/HA4 might affect insulin receptor internalization is not clear. Our data suggest that phosphorylation of pp120/HA4 by the insulin receptor kinase may play a role in this process. The lack of a dominant negative effect by phosphorylation-defective mutants of pp120/HA4 on insulin internalization is consistent with the notion that there might exist pp120-dependent and pp120-independent internalization pathways. The tissue distribution of pp120/HA4, which is predominantly found in liver, is also consistent with the idea that pp120 may play a specific role in hepatic insulin clearance. pp120/HA4 may be part of a complex of proteins contributing to the interaction of insulin receptors with clathrin-coated pits(30, 31, 32) . In support of the latter hypothesis, it is of interest to note that the amino acid sequence of pp120/HA4 shares homology with tyrosine-containing sequences thought to be important recognition elements for AP-2 adaptors binding(33, 34) . The sequences are found at positions 488-491 (Tyr-Ser-Val-Leu) and 513-516 (Tyr-Ser-Val-Val) of pp120/HA4.
Insulin's ability to stimulate thymidine incorporation is increased in H35 cells transfected with antisense pp120/HA4 cDNA and decreased in NIH 3T3 cells overexpressing the full-length isoform of pp120/HA4. This finding is in agreement with a recent report showing that increased endocytosis of a splice variant of the IGF-1 receptor is associated with a decreased response to the mitogenic action of IGF-1 (35) . It remains to be seen whether this effect is due to down-regulation, ligand degradation, or sequestration of substrates.
The process of ligand-induced endocytosis of insulin and growth factor receptors can be summarized in the following steps: 1) binding of the ligand; 2) receptor autophosphorylation and kinase activation; 3) surface redistribution of receptors from microvilli to the nonvillous surface of the cell; and 4) interaction with clathrin and various components of the coated pits, such as adaptins (AP-2) and perhaps other proteins(11, 30) . Carpentier and co-workers (11) have recently shown that insulin-induced activation of receptor autophosphorylation releases a constraint maintaining the receptor on microvilli. Whether a phosphorylated intermediate such as pp120/HA4 is involved at this level cannot be determined from our experiments. Further, it is not clear whether the insulin receptor binds directly to adaptor proteins or clathrin. On the other hand, it has also been demonstrated by co-immunoprecipitation experiments that epidermal growth factor receptor associates with AP-2 complex upon epidermal growth factor stimulation of cells(31, 32) .
In conclusion, our data are compatible with the hypothesis that pp120/HA4 is involved in insulin internalization and degradation. Further studies are needed to understand the mechanism by which this effect is elicited. Differential expression of the two isoforms of pp120/HA4 in liver may be important to modulate insulin clearance and to regulate receptor number at the cell surface.