Insulin Receptor Activation with Transmembrane Domain Ligands

Complementary surfaces are buried when peptide hormones, growth factors, or cytokines bind and activate cellular receptors. Although these extended surfaces provide high affinity and specificity to the interactions, they also present great challenges to the design of small molecules that might either mimic or antagonize the process. We show that the insulin receptor (IR) and downstream signals can be activated by targeting a site outside of its ligand-binding domain. A 24-residue peptide having the IR transmembrane (TM) domain sequence activates IR, but not related growth factor receptors, through specific interactions with the receptor TM domain. Like insulin-dependent activation, IR-TM requires that IR have a competent ATP-binding site and kinase activation loop. IR-TM also activates mutated receptors from patients with severe insulin resistance, which do not respond to insulin. These results show that IR can be activated through a pathway that bypasses its canonical ligand-binding domain.     

Peptide binding to intestinal epithelium: distinct sites for insulin, EGF and VIP

Several peptides, including insulin, epidermal growth factor and vasoactive intestinal polypeptide bind to intestinal epithelial cells. However, it is unclear whether one binding site binds several peptides or whether separate sites exist for each peptide. These studies were designed to examine the specificity of peptide binding sites on intestinal epithelial cells. Peptide binding was measured directly with [125I]radiolabelled peptides to isolated enterocytes prepared from rabbit ileum. The characteristics of insulin and epidermal growth factor binding were similar. Both insulin and epidermal growth factor specific binding was saturable, directly correlated to cell concentration and temperature and pH dependent. The total number of insulin binding sites per cell was 4500, that for epidermal growth factor was 2280. Scatchard analysis for both peptides produced curvilinear plots. Dissociation of both peptides from the binding site was increased in the presence of their respective unlabelled peptide. However, insulin specific binding was not altered by epidermal growth factor, and epidermal growth factor specific binding was unaffected by insulin. Further, both insulin and epidermal growth factor failed to inhibit the specific binding of vasoactive intestinal polypeptide to ileal enterocytes, and vasoactive intestinal polypeptide did not inhibit insulin or epidermal growth factor specific binding. These studies demonstrate that insulin, epidermal growth factor and vasoactive intestinal polypeptide interact with three distinct membrane binding sites on the enterocyte.     

Basal autophosphorylation of insulin receptor occurs preferentially on the receptor conformation exhibiting high affinity for insulin and stabilizes this conformation

Insulin signal transmission through the plasma membrane was studied in terms of relationship between basal autophosphorylation of the β-subunit and the ability by bind insulin by the -subunit of the insulin receptor. In a cell free system, receptors phosphorylated on tyrosine residues in the absence of insulin were separated from non-phosphorylated receptors using antiphosphotyrosine antibodies. Insulin binding assays were then performed on basally autophosphorylated and on non-phosphorylated receptors. We found that the tyrosine phosphorylated receptors, which corresponded to 25% of the total number of receptors, were accountable for 60–80% of insulin binding. Scatchard representation of binding data has shown that the plot corresponding to tyrosine phosphorylated receptors was localized above, and was steeper than the plot corresponding to non-phosphorylated receptors. These data make it likely that the conformation of -subunit which favours ligand binding is connected to the conformation of β-subunit which favours phosphate reception on tyrosine residues. Reciprocally, the high-affinity conformation of insulin receptor seems to become stabilized by basal autophosphorylation.     

Carbohydrate inhibitors of concanavalin A that inhibit binding of insulin-sepharose to fat cells and antagonize and mimic insulin's bioactivity. A possible role for membrane carbohydrate in insulin's action.

A consistent pattern of insulin-like properties is expressed by a variety of glycoside inhibitors of concanavalin A (Con A), and is suggestive of a common mechanism of action to explain these effects. Various exogenously added glycoside derivatives inhibit the binding of insulin-Sepharose beads to insulin receptors on isolated intact rat fat cells with a specificity resembling that for Con A-Sepharose binding to these cells. A more limited number of glycosides tested were also found to inhibit the binding of 125I-insulin, although some enhancement of binding that preceded the inhibition was observed for some of these saccharides. The glycosides also antagonize insulin-stimulated glucose utilization by the cells, but in some cases also mimic the hormone by stimulating glucose utilization. A few glycosides mimic insulin without appearing to antagonize its bioactivity. Radiolabeled glycoside inhibitors fail to bind to insulin in equilibrium dialysis experiments although they readily bind to Con A, indicating that the glycosides act directly on the cell rather than on the insulin molecule. The latter observation is consistent with the ability of those glycosides that act like insulin to do so independent of the hormone. In view of the known insulin-like properties of Con A, the effects of the glycosides seen in the present study suggest roles for a membrane carbohydrate and a carbohydrate binding site in the mechanisms of action of both insulin and Con A. In addition to various alternative explanations, a working hypothesis is presented to rationalize the present observations. It proposes that the effects of the exogenously added glycosides (and Con A) may reflect the presence on the membrane of a native carbohydrate moiety by either mimicking or competitively inhibiting its ability to interact reversibly with a lectin-like carbohydrate binding site associated with the function of the insulin receptor.20     

Specific Binding of Insulin to Membranes from Dendrodendritic Synaptosomes of Rat Olfactory Bulb

The external plexiform layer of the olfactory bulb is among the brain regions where insulin receptors are most abundant. In vitro binding of porcine 125I-insulin to membranes of dendrodendritic synaptosomes isolated from adult rat olfactory bulbs was studied to test the hypothesis that dendrodendritic synapses are major insulin-receptive sites in the external plexiform layer of olfactory bulbs. Of the specific insulin binding sites present in a total particulate fraction from the olfactory bulbs, approximately half were recovered in the dendrodendritic synaptosome fraction. The only other subcellular fraction to which substantial insulin binding was observed was the conventional (axodendritic/axosomatic) synaptosome fraction. Analysis of equilibrium binding of insulin to dendrodendritic synaptosomal membranes, at total insulin concentrations of 0.5-1,000 nM, revealed binding site heterogeneity consistent with a two-site model for insulin binding to a high-affinity (KD = 6 nM), low-capacity (Bmax = 110 fmol/mg of protein) site and a low-affinity (KD = 190 nM), high-capacity (Bmax = 570 fmol/mg of protein) site. The results indicate that the intense labeling of the external plexiform layer of the olfactory bulb in autoradiographic studies of insulin binding can be attributed to insulin receptors on dendrodendritic synaptic membranes in this region.     

Effect of proteolytic enzymes on masked insulin receptors in rat submaxillary gland microsomes

Trypsin and alpha-chymotrypsin effects on masked insulin receptors were studied. Phospholipase C treatment, incubation in a high ionic strength buffer or solubilization were used as alternative procedures for the unmasking of insulin receptors. These three methods expose receptor structures which are inaccessible to insulin in the current experimental conditions of binding assays without any significant change in binding affinity. Both exposed and masked receptors proved to be equally sensitive to trypsin and alpha-chymotrypsin degradation. At 25 degrees C, about 5 micrograms trypsin/ml for 50 min or 80 micrograms alpha-chymotrypsin/ml for 200 min were necessary in each case to cause a 50% inhibition of the binding of 125I-iodo insulin to microsomes. The results suggest that masked receptors are only nonfunctional to bind insulin but they are not located in compartments inaccessible to molecules present in the medium.     

Immunocytochemical verification of the insulin receptor's specificity in the nuclear envelope of Tetrahymena. Comparison with receptors of the plasma membrane

The unicellular Tetrahymena possess hormone receptors in the nuclear envelope similarly to higher rank animals. These receptors bind insulin and their specificity is detectable by monoclonal antibodies developed to insulin. The hormonal (insulin) pretreatment (imprinting) of the cell did not alter the binding capacity of the nuclear membrane, demonstrated by antibody-technique. The specific binding characteristics of the plasma membrane was demonstrated and this was significantly increased following imprinting. In the nucleus of Tetrahymena presence of insulin was not detected by immunocytochemical method.     

Characterization of a second ligand binding site of the insulin receptor

Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the alpha subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K(d) of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site.     

Interaction of decay-accelerating factor with coxsackievirus B3

Many entero-, parecho-, and rhinoviruses use immunoglobulin (Ig)-like receptors that bind into the viral canyon and are required to initiate viral uncoating during infection. However, some of these viruses use an alternative or additional receptor that binds outside the canyon. Both the coxsackievirus-adenovirus receptor (CAR), an Ig-like molecule that binds into the viral canyon, and decay-accelerating factor (DAF) have been identified as cellular receptors for coxsackievirus B3 (CVB3). A cryoelectron microscopy reconstruction of a variant of CVB3 complexed with DAF shows full occupancy of the DAF receptor in each of 60 binding sites. The DAF molecule bridges the canyon, blocking the CAR binding site and causing the two receptors to compete with one another. The binding site of DAF on CVB3 differs from the binding site of DAF on the surface of echoviruses, suggesting independent evolutionary processes.     

Characterization of an Insulin Receptor in Human Y79 Retinoblastoma Cells

Cultured human Y79 retinoblastoma cells bind [125I]iodoinsulin in a manner similar to that of other CNS and peripheral tissues. The only difference noted between the insulin binding properties of the Y79 cells and other CNS preparations is that insulin binding to Y79 cells is down-regulated by prolonged exposure of the cells to insulin. By contrast, studies with the various brain preparations indicate that the brain insulin receptor is not down-regulated by circulating levels of insulin. Insulin binding to Y79 cells exhibits negative cooperativity, has a pH optimum of 7.8, is responsive to cations, and gives a curvilinear Scatchard plot. Y79 cell insulin binding capacity is 26 fmol/100 micrograms of cell protein, corresponding to about 125,000 binding sites per cell. These findings are the first to report insulin binding in a human cell line of retinal origin. The characterization of the insulin binding in this cell line may facilitate an understanding of the relationship between insulin and its specific functions in the human retina.     

Structural and Functional Characteristics of Insulin Receptors in Rat Neuroblastoma Cells

Insulin receptors were detected in a variety of rat neuroblastoma and glioma cell lines. The binding of 125I-insulin to B103 neuroblastoma cells had characteristics typical of insulin receptors in other tissues, including high affinity for insulin, low affinity for insulin-like growth factor I (IGF-I), and curvilinear Scatchard plots. Using photoaffinity labeling procedures and sodium dodecyl sulfate (SDS) gel electrophoresis to analyze the subunit structure of insulin receptors in B103 cells, the predominantly labeled protein had an apparent molecular weight of 125K and the mobility of this protein was shifted after removal of sialic acid residues. On the basis of size and susceptibility to neuraminidase, the insulin binding subunit in neuroblastoma cells was identical to the alpha-subunit of insulin receptors in adipocytes and different from the 115K subunit found in brain. The presence of an "adipocyte" form of the insulin receptor in clonal cells derived from brain is probably a consequence of transformation and results from more extensive oligosaccharide processing of the 115K receptor expressed in normal brain cells. The fully glycosylated receptors in neuroblastoma cells were capable of exerting functions typical of insulin receptors in adipocytes such as internalization of insulin and stimulation of glucose transport.     

The insulin receptor and type I IGF receptor: Comparison of structure and function

The insulin receptor and type I IGF receptor are closely related in structure and function. The receptors are heterotetrameric glycoproteins, of structure αββα, which are widely distributed in mammalian tissues. A third member of this receptor family has been described, the insulin receptor-related receptor, for which a ligand has still to be identified. It has also been demonstrated that the insulin receptor and IGF receptor form αββ′α′ hybrids in cells expressing both receptors.The key elements in the function of any receptor are recognition of ligand and transmission of an intracellular signal. In the insulin and IGF receptors, determinants of binding specificity are contained within amino-terminal and cysteine-rich domains of the extracellular α-subunit. Intracellular signalling is dependent on ligand activated tyrosine kinase activity in the transmembrane β-subunit, which phosphorylates both the receptor itself and the specific substrate insulin receptor substrate-1 (IRS-1). Phosphorylated IRS-1 binds the enzyme phosphatidylinositol 3-kinase and may act as a multivalent docking site for SH2 domains of other proteins involved in signalling. The possibility that some signalling molecules interact directly with the receptors has not been ruled out.The specificity of action of insulin and IGFs in vivo depends on differences between the respective receptors in tissue distribution, ligand binding specificity and intrinsic signalling capacity. However, the detailed aspects of gene and receptor structure which underly these functional differences are still poorly understood. Moreover, the issue of specificity is complicated by the existence of hybrid and atypical receptors, which in principle could bind and respond to both insulin and IGF-I, although the physiological significance of these receptor subtypes is at present unclear.     

Role of ATP in specific binding of 125I-insulin to cytoplasmic receptors of the liver and muscle membranes of controls and diabetic rats

A study was made of the action of various concentrations of ATP on insulin ability to bind to the receptors of the liver and muscle membranes in control and streptozocin-induced diabetes animals. Specific binding of 125I-insulin to the receptors of the liver and muscle membranes was shown to rise in animals with streptozocin-induced diabetes as compared to control. This effect was most pronounced in the muscle membranes. Preincubation of the membranes with ATP did not affect insulin binding to the liver and muscle receptors of control animals. However, hormone binding to the liver receptors of diabetic rats was drastically suppressed by ATP (10(-3) M). Less ATP concentrations (10(12) M) produced an additional inhibitory action which was not marked. ATP led to decreased insulin binding to the muscle receptors of diabetic rats only at extremely low concentrations (10(-12) M). The data obtained may be of importance for regulation of membrane phosphorylation in the states characteristic of insulin resistance.     

Effects of trypsin on binding of insulin and concanavalin A and on glucose and proline transport in the R3230AC mammary adenocarcinoma

Effects of trypsin treatment on insulin and concanavalin A binding to, and glucose and proline transport in, dissociated R3230AC mammary adenocarcinoma cells were examined. Reduction of binding of ¹²⁵I-labelled insulin was dependent on the amount of trypsin used, the temperature and the time of the incubation period. Under conditions that reduced insulin binding by greater than 75%, transport of glucose and proline was reduced by less than 15%. Scatchard analysis of insulin binding after trypsin treatment yielded slopes similar to those from cells not exposed to trypsin, assuming either two classes of receptors or an average affinity, $\text{K}\text{?}{}_{\mathrm{<mtext>e</mtext>}}$. Dissociation of bound insulin from untreated or trypsin-treated cells was enhanced by addition of excess unlabelled ligand. Insulin added in vitro, which decreased glucose transport in untreated cells, produced a decrease in glucose transport in cells treated with trypsin for 5 min (insulin binding was decreased 35%), but not in cells treated for 45 min (insulin binding was decreased 90%). Binding of the plant lectin concanavalin A was also reduced by trypsin treatment, but to a lesser extent and with a different time-course than for insulin. Scatchard analysis of the binding of concanavalin A in untreated and trypsin-treated cells yielded comparable values for K_d. The insulinomimetic actions of concanavalin A on glucose transport were abolished after brief exposure to trypsin. Pre-treatment of cells with concanavalin A reduced insulin binding and partially protected insulin receptors from trypsin digestion, but the inability to remove all of the concanavalin A precluded its use as a method to protect insulin receptors. Thus, in this rat mammary tumor, the number, but not the affinity or functional activity, of insulin receptors can be reduced by trypsin treatment without significant effects on glucose or A system amino acid transport.     

The effect of placement of tryptophan residues in selected A-chain positions on the biological profile of insulin

In continuation of our efforts to study the solution structure and conformational dynamics of insulin by time-resolved fluorescence spectroscopy, we have synthesized and examined the biological activity of five insulin analogues in which selected naturally occurring residues in the A-chain have been replaced with the strongly fluorescent tryptophan residue. The potency of these analogues was evaluated in lipogenesis assays in isolated rat adipocytes, in receptor binding assays using rat liver plasma membranes, and in two cases, in receptor binding assays using adipocytes. [A3 Trp]insulin displays a potency of 3% in receptor binding assays in both liver membranes and in adipocytes, but only 0.06% in lipogenesis assays as compared to porcine insulin. [A10 Trp] insulin displays a potency ofca. 40% andca. 25% in rat liver receptor binding and lipogenesis assays, respectively. [A13 Trp]insulin displays a potency ofca. 39% in rat liver receptor binding assays, but onlyca. 9% in receptor binding in adipocytes; in lipogenesis assays, [A13 Trp] insulin displays a potency ofca. 12%, comparable to its potency in adipocyte receptor binding assays. [A15 Trp]insulin exhibits a potency of 18% and 9% in rat liver receptor binding and lipogenesis assays, respectively. The doubly substituted analogue, [A14 Trp, A19 Trp] insulin, displays a potency ofca. 0.7% in both rat liver receptor binding assays and lipogenesis assays. These data suggest two major conclusions: (1) the A3 and A15 residues lie in sensitive regions in the insulin molecule, and structural modifications at these positions have deleterious effects on biological activity of the hormone; and (2) [A13 Trp]insulin appears to be a unique case in which an insulin analogue exhibits a higher potency when assayed in liver tissue than when assayed in fat cells.     

The N-terminal hexapeptide fragment of IGF II stimulates thymidine incorporation into fibroblasts.

We synthesized the N-terminal hexapeptide fragment of IGF II to study potential binding to NMDA receptors in analogy to the N-terminal tripeptide of IGF I. The amino acid sequence of the hexapeptide is furthermore identical with the C-terminal sequence of the casiragua insulin B chain. The hexapeptide did not bind to the NMDA receptors, but was found to promote [3H]-thymidine incorporation into fibroblasts at concentrations of 10(-8) - 10(-5) M in a dose-dependent manner. Since [125I]-hexapeptide did not bind to IGF receptors, indirect competition studies using either labelled IGFs or insulin had to be used. The competition of hexapeptide at a concentration of 10(-5) M with labelled IGF I or II was about equal to that of 10(-9) M IGF I or II. IGF receptors were apparently up-regulated by the hexapeptide, as has also been described for insulin. When using casiragua insulin as labelled ligand, IGF II and casiragua insulin competed with equal potency, whereas the hexapeptide at 10(-7) M caused an apparent up-regulation of the casiragua insulin binding sites. Our results that the hexapeptide stimulates [3H]-thymidine incorporation and up-regulates IGF II and casiragua insulin binding sites may be connected to one or several of the following findings: the hystricomorph insulins--of which the casiragua insulin is a member--stimulate DNA synthesis to a greater extent than other insulins; the insulin and type 1 IGF receptor binding regions are localized predominantly in the C-terminal region of the insulin B chain; and the "cooperative" site regulating the affinity of the insulin receptor is also located in the C-terminal region of the insulin B chain. Further experiments will be needed to clarify the exact mechanism.     

Phage display screening against a set of targets to establish peptide-based sugar mimetics and molecular docking to predict binding site

A novel selection approach is presented to screen phage display peptide libraries against sets of receptors that share specificity for the same ligand. This strategy was applied to the discovery of glycomimetic peptides. Through these screens, a number of peptide clones were discovered that bind the lectins used in the screen, in a sugar competitive manner. In addition, the majority of the selected peptides demonstrate sugar type mimicry consistent with lectin specificity. Docking studies were conducted to establish whether the mimetic peptides bind to the lectin ConA at the sugar binding site or to a nearby, alternative site shown to bind to YPY-containing peptides previously discovered from single-target screens. Of the three cyclic peptides subjected to computational docking, CNTPLTSRC had the highest predicted affinity and CSRILTAAC demonstrated specificity for the sugar binding site comparable to the natural ligand itself.     

An extracellular domain of the insulin receptor beta-subunit with regulatory function on protein-tyrosine kinase

Anti-insulin receptor monoclonal antibody MA-10 inhibits insulin receptor autophosphorylation of purified rat liver insulin receptors without affecting insulin binding (Cordera, R., Andraghetti, G., Gherzi, R., Adezati, L., Montemurro, A., Lauro, R., Goldfine, I. D., and De Pirro, R. (1987) Endocrinology 121, 2007-2010). The effect of MA-10 on insulin receptor autophosphorylation and on two insulin actions (thymidine incorporation into DNA and receptor down-regulation) was investigated in rat hepatoma Fao cells. MA-10 inhibits insulin-stimulated receptor autophosphorylation, thymidine incorporation into DNA, and insulin-induced receptor down-regulation without affecting insulin receptor binding. We show that MA-10 binds to a site of rat insulin receptors different from the insulin binding site in intact Fao cells. Insulin does not inhibit MA-10 binding, and MA-10 does not inhibit insulin binding to rat Fao cells. Moreover, MA-10 binding to down-regulated cells is reduced to the same extent as insulin binding. In rat insulin receptors the MA-10 binding site has been tentatively localized in the extracellular part of the insulin receptor beta-subunit based on the following evidence: (i) MA-10 binds to insulin receptor in intact rat cells; (ii) MA-10 immunoprecipitates isolated insulin receptor beta-subunits labeled with both [35S]methionine and 32P; (iii) MA-10 reacts with rat insulin receptor beta-subunits by the method of immunoblotting, similar to an antipeptide antibody directed against the carboxyl terminus of the insulin receptor beta-subunit. Moreover, MA-10 inhibits autophosphorylation and protein-tyrosine kinase activity of reduced and purified insulin receptor beta-subunits. The finding that MA-10 inhibits insulin-stimulated receptor autophosphorylation and reduces insulin-stimulated thymidine incorporation into DNA and receptor down-regulation suggests that the extracellular part of the insulin receptor beta-subunit plays a role in the regulation of insulin receptor protein-tyrosine kinase activity.     

Insulin receptor desensitization correlates with attenuation of tyrosine kinase activity, but not of receptor endocytosis.

A model of insulin-receptor down-regulation and desensitization has been developed and described. In this model, both insulin-receptor down-regulation and functional desensitization are induced in the human HepG2 cell line by a 16 h exposure of the cells to 0.1 microM-insulin. Insulin-receptor affinity is unchanged, but receptor number is decreased by 50%, as determined both by 125I-insulin binding and by protein immunoblotting with an antibody to the beta-subunit of the receptor. This down-regulation is accompanied by a disproportionate loss of insulin-stimulated glycogen synthesis, yielding a population of cell-surface insulin receptors which bind insulin normally but which are unable to mediate insulin-stimulated glycogen synthesis within the cell. Upon binding of insulin, the desensitized receptors are internalized rapidly, with characteristics indistinguishable from those of control cells. In contrast, this desensitization is accompanied by a loss of the insulin-sensitive tyrosine kinase activity of insulin receptors isolated from these cells. Receptors isolated from control cells show a 5-25-fold enhancement of autophosphorylation of the beta-subunit by insulin; this insulin-responsive autophosphorylation is severely attenuated after desensitization to a maximum of 0-2-fold stimulation by insulin. Likewise, the receptor-mediated phosphorylation of exogenous angiotensin II, which is stimulated 2-10-fold by insulin in receptors from control cells, is completely unresponsive to insulin in desensitized cells. These data provide evidence that the insulin-receptor tyrosine kinase activity correlates with insulin stimulation of an intracellular metabolic event. The data suggest that receptor endocytosis is not sufficient to mediate insulin's effects, and thereby argue for a role of the receptor tyrosine kinase activity in the mediation of insulin action.     

Different ligand affinities of the two human insulin receptor splice variants are reflected in parallel changes in sensitivity for insulin action

The human insulin receptor (hIR) is expressed in two variant forms that are generated by tissue-specific alternative splicing of the 11th exon of the IR gene. This leads to receptors that differ in their affinities for insulin based on the absence (hIR-A) or presence (hIR-B) of a 12-amino acid insert near the C-terminus of the alpha-subunit. To explore further the functional significance of the difference in these receptor subtypes, the properties of hIR-A(exon 11-) and hIR-B(exon 11+) receptors have been compared in parallel. Despite their different affinities for insulin, the receptor variants retain equivalent acid sensitivity for insulin binding and bind proinsulin with the same relative affinity. Both hIR-A and hIR-B are able to signal a variety of insulin's actions, but the insulin dose-response curves for receptor autophosphorylation and for mitogenesis and glycogen synthase stimulation in cells are shifted to the right for hIR-B receptors compared to hIR-A receptors. The magnitude of these rightward shifts, 1.5- to 3-fold in the assays listed above, are similar to and presumably accounted for by the 2-fold difference in insulin affinity exhibited by the receptor variants. Occupied hIR-A and hIR-B receptors undergo indistinguishable endocytotic itineraries after insulin binding. Both lead to insulin degradation that is quantitatively and kinetically similar, and both down-regulate when exposed to saturating insulin for 24 h. Thus, the functional consequences of the alternative splicing of IRs are limited to those related to the variants' differing affinities for insulin.