Inhibition of Clathrin-Mediated Endocytosis Selectively Attenuates Specific Insulin Receptor Signal Transduction Pathways

Abstract

To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor.Receptor-mediated endocytosis is an essential mechanism for several important physiological processes. These include down regulation of cell surface receptors, degradation of the receptor and/or ligand, absorption and retention of essential nutrients, transcellular transport of specific ligands, and the activation of intracellular signal transduction cascades (42,49,52). It is well established that the insulin receptor undergoes endocytosis upon insulin stimulation (3,28,35,37); however, the exact physiological significance of insulin receptor internalization is poorly understood. Several studies have demonstrated that following insulin stimulation, the endosome-localized insulin receptor exhibits increased autophosphorylating and exogenous substrate tyrosine kinase activity compared to plasma membrane-associated insulin receptors (3,31,32). Based on the time course of endosome association, kinase activation, and compartmentalization of effector substrates, it has been hypothesized that the internalized endosome-associated insulin receptor population is responsible for the activation of intracellular signaling pathways (12,18,31). However, other studies have reported that low-temperature (4°C) blockade of insulin receptor internalization does not impair either insulin receptor autophosphorylation or tyrosine phosphorylation of the major insulin receptor substrate, IRS1 (9, 26). Thus, in contrast, these data suggest that activa...

An arginine to cysteine252 mutation in insulin receptors from a patient with severe insulin resistance inhibits receptor internalisation but preserves signalling events

“…In contrast, the decrease of insulin-IR complexes endocytosis is not accompanied by a loss of IRS 1/IRS 2 phosphorylation. These observations are in agreement with previous studies showing that IRS 1 phosphorylation is preserved at 4°C (a temperature at which IR endocytosis is prevented) [16,17] as well as in cells expressing a dominant interfering dynamin which prevents clathrin-coated pits mediated endocytosis [15]. Along the same line, we have recently described a mutant IR (IR C860S ) capable of phosphorylating IRS 1 in spite of its defective internalisation.…”

“…The recent observation that inhibition of endocytosis by expression of a dominant interfering dynamin mutant has no effect on any insulin signalling pathways tested, including the Shc pathway, would support the first possibility [15]. On the other hand, present data would lend support to the observations that in the case of the EGF receptor, a subset of (but not all) signal transducers require the normal endocytic trafficking of the activated receptor for full activation [43].…”

“…So far, the role of IR internalisation on insulin signal transduction has remained controversial. On the one hand, several mutant IR have been shown to keep some of their functional properties although they failed to undergo insulin-induced internalisation [12,13,14,15]. Likewise, under conditions where IR internalisation is inhibited (incubation at 4°C or expression of a dominant negative mutant of dynamin), IRS 1 is still phosphorylated [15,16,17], suggesting that IR internalisation is not required for the transduction of its biological signal.…”

“…On the one hand, several mutant IR have been shown to keep some of their functional properties although they failed to undergo insulin-induced internalisation [12,13,14,15]. Likewise, under conditions where IR internalisation is inhibited (incubation at 4°C or expression of a dominant negative mutant of dynamin), IRS 1 is still phosphorylated [15,16,17], suggesting that IR internalisation is not required for the transduction of its biological signal. On the other hand, subcellular fractionation experiments (on 3T3-L1 adipocytes and rat hepatocytes) have shown that the pool of IR present on internal membranes undergoes a higher autophosphorylation in response to insulin than the IR present at the cell surface [18,19], and that IRS 1 is mainly distributed in intracellular compartments, probably endosomes, where it could be tyrosine phosphorylated in the presence of insulin [16,18,19,20].…”

EGF receptor (EGFR) signaling promoting invasion is disrupted in androgen‐sensitive prostate cancer cells by an interaction between EGFR and androgen receptor (AR)

“…46 Furthermore, blocking of EGFR endocytosis by using a dynamin mutant results in downregulation of ERK and PI3K activation in response to EGF 47 and insulin. 48 Whether EGFR-AR interaction may be responsible for decreased EGFR signaling and internalization remains to be addressed; however, it is possible that such interaction disrupts the ability of the receptor to autophosphorylate by attenuating its intrinsic tyrosine kinase activity, determining as a consequence a reduction of internalization and PI3K activation.…”