Similar to many cell surface receptors, the insulin receptor is internalized into intracellular vesicular compartments following ligand binding and tyrosine kinase activation (3
). Recent studies have begun to examine the structural determinants required for insulin receptor endocytosis (2
). It is generally accepted that insulin receptor kinase tyrosine autophosphorylation in conjunction with the dileucine motif located in the juxtamembrane domain of the β subunit is essential for insulin-mediated endocytosis (5
). However, the identification of the functional tyrosine autophosphorylation sites involved in this process has been enigmatic. Multiple studies expressing various insulin receptor mutants in the juxtamembrane domain have provided evidence both for and against the involvement of consensus GPLY and NPEY tyrosine-based internalization motifs (2
). Similarly, several studies have reported evidence both for and against clathrin-mediated endocytosis of the insulin receptor (40
To address these issues, we examined the role of dynamin in insulin receptor internalization. Previous studies have demonstrated that dynamin plays a critical role in clathrin-mediated endocytosis. Dynamin appears to associate with clathrin-coated pits through its association with the adapter protein amphiphysin and the AP2 adapter complex (59
). Once localized to coated pits, dynamin forms a ring structure or collar which is thought to pinch off coated vesicles (61
). Although it is not clear whether dynamin itself is the so-called pinchase, dynamin GTPase activity is essential for the subsequent formation of clathrin-coated vesicles (60
). Thus, expression of a GTPase-defective dynamin mutant results in a dominant-interfering phenotype by competing with endogenous dynamin and thereby preventing formation of the endocytic coated vesicles (57
Using a dominant-interfering mutant of dynamin I (K44A/dynamin), we have observed an inhibition of both insulin and transferrin receptor endocytosis. This finding is consistent with previously published results which demonstrate that the transferrin receptor is internalized through a dynamin- and clathrin-dependent mechanism (16
). Our observation that K44A/dynamin inhibits insulin receptor internalization in the same manner as the transferrin receptor is consistent with both receptors utilizing a dynamin- and clathrin-dependent pathway.
In addition to the sequestration of the insulin receptor into clathrin-coated pits, the physiological role of ligand-mediated endocytosis has long been debated in the field of insulin receptor signaling. Controversy has centered around whether the endocytic process is primarily a means for inactivating the insulin receptor and/or for localizing the kinase-activated insulin receptor to appropriate signaling molecules (12
). Therefore we examined the ability of the cell surface insulin receptors to interact with a variety of established downstream effectors. Our data demonstrate that inhibition of insulin receptor endocytosis had no significant effect on the overall extent of β-subunit autophosphorylation or IRS1 tyrosine phosphorylation. These results are consistent with those of two previous studies in which inhibition of insulin receptor internalization inhibited by reduced temperature had no effect on either receptor autophosphorylation or IRS1 tyrosine phosphorylation (9
In contrast, we did observe a small reduction in Shc tyrosine phosphorylation which correlated with a reduction in the extent of ERK1 and ERK2 activation. There was an approximate 50% diminution in the insulin-stimulated association of PI 3-kinase with IRS1 which directly correlated with a 50% reduction in both IRS1- and phosphotyrosine-immunoprecipitated PI 3-kinase activity. It is possible that this was a result of differences in specific sites of IRS1 tyrosine phosphorylation, as there was no significant change in the total levels of either IRS1 or p85 expression. In any case, these data suggest that endocytic compartmentalization of the insulin receptor, and possibly IRS1, is a prerequisite for maximal PI 3-kinase activation.
Nevertheless, insulin was still capable of inducing a substantial activation of the PI 3-kinase in the absence of insulin receptor endocytosis. In this regard, several studies have indicated that Akt activation is mediated through a PI 3-kinase-dependent pathway (10
). The ability of insulin to fully activate the Akt kinase in the K44A/dynamin-expressing cells further suggests that the inhibition of insulin-stimulated PI 3-kinase activity was a functionally minor event. Although less likely, it remains possible that a specific subcellular pool of PI 3-kinase is directly responsible for Akt activation, and it is this pool of PI 3-kinase that is not affected by K44A/dynamin expression.
Ultimately, insulin signaling leads to increases in the cellular storage of energy in the form of protein, lipid, and carbohydrate. Since the proximal insulin signaling events were only marginally altered by the inhibition of insulin receptor endocytosis, we anticipated that these endpoint biological responses would also be essentially unaffected. As predicted, insulin was fully capable of stimulating amino acid uptake and DNA synthesis in the K44A/dynamin adenovirus-infected H4IIE cells. Unexpectedly, inhibition of dynamin-dependent endocytosis resulted in an enhanced basal uptake of AIB, suggesting an increased number of AIB transporters at the cell surface under these conditions. To determine if this was a more general phenomenon, we attempted to determine the effect of K44A/dynamin on insulin-stimulated glycogen synthesis and lipogenesis. However, in our hands the H4IIE cells displayed a negligible insulin stimulation of these activities. Therefore, we used the differentiated 3T3L1 adipocytes which are markedly responsive to insulin and also are quantitatively infected by adenovirus (reference 21
and data not shown). In addition, essentially identical effects of K44A/dynamin on insulin receptor β-subunit autophosphorylation, IRS1 and Shc tyrosine phosphorylation, and PI 3-kinase and Akt kinase activation were observed (data not shown). In any case, insulin was also fully effective in the stimulation of glucose transport, glycogen synthesis, and lipogenesis. Expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis. The increases in glycogen synthesis and lipogenesis are most likely due to the increased glucose uptake, which is the rate-limiting step in the basal state. Consistent with this hypothesis, we have also observed that expression of K44A/dynamin resulted in the cell surface accumulation of the GLUT4 glucose transporter (unpublished data).
In summary, the data presented in this report demonstrate that insulin-stimulated insulin receptor internalization occurs through a dynamin-dependent, and hence clathrin-mediated, endocytic pathway. Although the insulin-stimulated kinase-activated insulin receptor was unable to localize to endosomes, there was only minor effects on substrate tyrosine phosphorylation and association/activation of several downstream effectors. Our results demonstrate that neither amino acid transport, DNA synthesis, glucose transport, glycogen synthesis, nor lipogenesis is dependent on insulin receptor endocytosis and that each is fully responsive when the insulin receptor remains localized to the plasma membrane.