Characteristics of various synthetic peptide calpain inhibitors and their application for the analysis of platelet reaction

Calpeptin (a cell permeable synthetic peptide calpain inhibitor) inhibited the generation of thromboxane B2 (TxB2) by the direct inhibition on Tx synthetase in platelets at the concentrations more than 30 microM. Calpeptin, its analogues and E-64d (EST) were further examined with regard to cell permiability and inhibitory spectra. Among all compounds, only calpeptin inhibited the degradation of substrate proteins of calpain with negligible effect on TxB2 generation in intact platelets at the concentrations less than 30 microM. These concentrations of calpeptin did not inhibit the platelet aggregation, the elevation of [Ca2+], nor the formation of inositol 1,4,5-trisphosphate (IP3) in thrombin or collagen activated platelets. These results indicate that calpain dose not participate in the process of platelet activation induced by thrombin or collagen.     

SNARE protein degradation upon platelet activation: calpain cleaves SNAP-23

In order to better understand the molecular mechanisms of platelet granule secretion, we evaluated the effect of activation-induced degranulation on three functional platelet SNARE proteins, SNAP-23, VAMP-3, and syntaxin 4. Initial studies showed that SNAP-23 is lost upon SFLLRN-induced platelet activation. Experiments with permeabilized platelets demonstrated that proteolysis of SNAP-23 was Ca(2+)-dependent. Ca(2+)-dependent proteolysis of SNAP-23 was inhibited by the cell-permeable calpain inhibitors, calpeptin and E-64d, as well as by the naturally occurring calpain inhibitor, calpastatin. In addition, purified calpain cleaved SNAP-23 in permeabilized platelets in a dose-dependent manner. In intact platelets, calpeptin prevented SFLLRN-induced degradation of SNAP-23. In contrast, calpeptin did not prevent SFLLRN-induced degradation of VAMP-3 and syntaxin 4 did not undergo substantial proteolysis following platelet activation. Calpain-induced cleavage of SNAP-23 was a late event occurring between 2.5 and 5 min following exposure of permeabilized platelets to Ca(2+). Experiments evaluating platelet alpha-granule secretion demonstrated that incubation of permeabilized platelets with 10 microM Ca(2+) prior to exposure to ATP inhibited ATP-dependent alpha-granule secretion from permeabilized platelets. SNAP-23 was cleaved under these conditions. Incubation of permeabilized platelets with either calpeptin or calpastatin prevented Ca(2+)-mediated degradation of SNAP-23 and reversed Ca(2+)-mediated inhibition of ATP-dependent alpha-granule secretion. Thus, activation of calpain prior to secretion results in loss of SNAP-23 and inhibits alpha-granule secretion. These studies suggest a mechanism whereby calpain activation serves to localize platelet secretion to areas of thrombus formation.     

Possible participation of calpain in myosin light chain phosphorylation of human platelets

We previously demonstrated that myosin light chain kinase (MLCK) of gizzard is proteolyzed by platelet calpain. It has been also reported that partially cleaved MLCK may phosphorylate myosin light chain (20K) in the absence of calmodulin. Therefore, a possible participation of calpain in 20K phosphorylation was studied in human platelets, utilizing various inhibitors. An epoxy succinate derivative (E-64) or N-ethylmaleimide (NEM), used as calpain antagonist, inhibited 20K phosphorylation of Ca2+-stimulated lysed platelets. A synergistic effect between these calpain antagonists and calmodulin antagonist W-7 was observed. Also, the similar results were obtained in 20K phosphorylation of intact platelets. From these observations, it was suggested that 20K phosphorylation in platelets is mediated by two separate pathways, namely calmodulin and calpain dependent pathways, provided that calpain activity is specifically inhibited by the antagonists used.     

Evidence that agonist-induced activation of calpain causes the shedding of procoagulant-containing microvesicles from the membrane of aggregating platelets

One of the responses of platelets to stimulation is activation of intracellular calpain (the Ca(2+)-dependent protease). Previously, we have shown that activation of calpain in platelets is involved in the generation of platelet procoagulant activity. Because procoagulant activity is present on the microvesicles that are shed from activated platelets, in this study we examined whether calpain is involved in the shedding of microvesicles. Platelets were incubated with the physiological agonists collagen or thrombin. The extent of activation of calpain correlated positively with the amount of procoagulant-containing microvesicles that formed, and the shedding of procoagulant-containing microvesicles was inhibited by calpeptin, MDL, and EST (E-64-d), three membrane-penetrating inhibitors of calpain. The protein composition of the microvesicles shed from aggregating platelets was similar to that of microvesicles shed by platelets in which the association of the membrane skeleton with the plasma membrane had been disrupted by incubation of platelets with dibucaine or ionophore A23187. Furthermore, like microvesicles shed from dibucaine- or ionophore A23187-treated platelets, those shed from the aggregating platelets possessed procoagulant activity. These results are consistent with the possibility that activation of calpain in aggregating platelets causes the shedding of procoagulant-containing microvesicles. We suggest that the shedding of microvesicles results from the calpain-induced hydrolysis of the platelet membrane skeleton.     

Superior prevention of calcium ionophore cataract by E64d.

The purposes of this experiment were: (1), to compare effect of three E64 derivatives, E64, E64c and E64d in preventing nuclear opacity and proteolysis in calcium ionophore-induced cataract and (2), to measure the accumulation of E64 derivatives in the cultured lenses. In vitro E64 and E64c strongly inhibited purified calpain II from porcine heart, while E64d showed weaker inhibition than E64 and E64c. In cultured lenses, all three E64 derivatives reduced nuclear opacity by calcium ionophore A23187 in a concentration-dependent manner, and E64d, the ethyl-ester of E64c, was the most effective. When lenses were cultured in E64d for 2 h, the resulting concentration of E64 derivative in the lens was markedly higher than during culture in E64 or E64c. All three E64 derivatives prevented proteolysis of crystallins seen in A23187 cataract. The stronger effect of E64d against A23187 cataract was likely due to an earlier penetration into the lens, conversion to E64c and inhibition of activated calpain.     

Elucidation of calpain dependent phosphorylation of myosin light chain in human platelets

Newly synthetized calpain inhibitors (CI-I approximately III) were used to prove potential participation of calpain in protein phosphorylation. CIs were about 1,000 times more potent against platelet calpain I than N-ethyl-maleimide (NEM) and an epoxy succinate derivative (E-64). CI-II inhibited 20K (myosin light chain) and 47K phosphorylation of Ca2+-stimulated lysed platelets as well as protein degradation (actin binding protein, P235). Both myosin light chain kinase (MLCK) and C-kinase dependent phosphorylation of 20K were inhibited by CI-II as demonstrated in phosphopeptides mapping. Electropermeabilized platelets (EP) were employed to examine the effects of CI-II on Ca2+ mediated reactions in non-lysed platelets. Phosphorylation of 20K and 47K induced by Ca2+ addition to EP was inhibited by CI-II, though secretory response was not modified. Only MLCK dependent phosphorylation of 20K was observed in Ca2+-activated EP, which was inhibited by CI-II. Collectively, the data indicated that calpain may activate both MLCK and C-kinase to phosphorylate 20K by partial proteolysis.     

Possible role of calpain in normal processing of beta-amyloid precursor protein in human platelets

Abnormal proteolytic processing of beta-amyloid precursor protein (APP) underlies the formation of amyloid plaques in aging and Alzheimer's disease. The proteases involved in the process have not been identified. Here we found that spontaneous proteolysis of intact APP in detergent-lysed human platelets generated a N-terminal fragment that was immunologically indistinguishable from secreted APP, reminiscent of the action of a putative alpha-secretase. This proteolysis of APP was inhibited by EDTA, suggesting that a metal-dependent protease was involved. Among the several metals tested, calcium was the only one that enhanced APP proteolysis and the reaction was blocked by EGTA as well as by several calpain inhibitors. The APP fragments generated by spontaneous proteolysis in platelet lysates were identical to those produced by exposure of partially purified APP to exogenous calpain. Finally, the secretion of APP from intact platelets was inhibited by cell-permeable calpain inhibitors. Taken together, these results suggest that normal processing of APP in human platelets is mediated by a calcium-dependent protease that exhibits calpain-like properties.     

Inhibition of calpain expression by E-64d in the rat retina subjected to ischemia/reperfusion injury

To investigate the effect of E-64d, a selective inhibitor of calpain, on the expression of calpain and calpastatin in rat retina subject to ischemia/reperfusion injury (IRI). An animal model of retinal IRI was set up by increasing the intraocular pressure (110 mmHg) of a rat eye for 1 h. The retinal thickness and morphologic changes were detected by histology. The protein expression of m-calpain (a calpain isoform) in the retina was assessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain as well as calpastatin (an endogenous protein inhibitor of calpain) in the retina was assessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated. To evaluate the effect of E-64d on the expression of calpain, the drug (5 microl of 100 microM) was injected intravitreously immediately after IRI. There were retinal edematous changes, particularly in the inner plexiform layer after IRI. The protein expression of m-calpain in the retina was increased 24h after IRI, an effect that was inhibited by E-64d (P < 0.05). The mRNA expression of m-calpain and calpastatin was also increased 24 h and 3 h after IRI, respectively. Neither m-calpain nor calpastatin mRNA expression was influenced by E-64d (P > 0.05). The mRNA ratio of m-calpain to calpastatin was increased at the 6 h, 24 h and 72 h after IRI, and only at 24 h the increase of the ratio of m-calpain to calpastatin was inhibited by E-64d (P < 0.05). In the rat retina of IRI, E-64d inhibits the increase of m-calpain protein expression, as well as the mRNA ratio increase of m-calpain to calpastatin. E-64d also inhibited the retinal damage induced by IRI, suggesting a role for E-64d in the protection of the retinal apoptosis induced by IRI.     

Superior prevention of calcium ionophore cataract by E64d

The purposes of this experiment were: (1), to compare effect of three E64 derivatives, E64, E64c and E64d in preventing nuclear opacity and proteolysis in calcium ionophore-induced cataract and (2), to measure the accumulation of E64 derivatives in the cultured lenses. In vitro E64 and E64c strongly inhibited purified calpain II from porcine heart, while E64d showed weaker inhibition than E64 and E64c. In cultured lenses, all three E64 derivatives reduced nuclear opacity by calcium ionophore A23187 in a concentration-dependent manner, and E64D, the ethyl-ester of E64c, was the most effective. When lenses were cultured in E64d for 2 h, the resulting concentration of E64 derivative in the lens was markedly higher than during culture in E64 of E64c. All three E64 derivatives prevented proteolysis of crystallins seen in A23187 cataract. The stronger effect of E64d against A23187 cataract was likely due to an earlier penetration into the lens, conversion to E64c and inhibition of activated calpain.     

Inhibition of calpain expression by E-64d in the rat retina subjected to ischemia/reperfusion injury

To investigate the effect of E-64d, a selective inhibitor of calpain, on the expression of calpain and calpastatin in rat retina was subjected to ischemia/reperfusion injury (IRI). An animal model of retinal IRI was set up by increasing the intraocular pressure (110 mm Hg) of a rat eye for 1 h. The retinal thickness and morphologic changes were detected by histology. The protein expression of m-calpain (a calpain isoform) in the retina was assessed by immunohistochemistry and Western blot assay. The mRNA of m-calpain, as well as calpastatin (an endogenous protein inhibitor of calpain), in the retina was assessed by RT-PCR, and the ratio of m-calpain/calpastatin was then calculated. To evaluate the effect of E-64d on the expression of calpain, the drug (5 μl of 100 μM) was injected intravitreously immediately after IRI. There were retinal edematous changes, particularly in the inner plexiform layer after IRI. The protein expression of m-calpain in the retina was increased 24 h after IRI, an effect that was inhibited by E-64d (P < 0.05). The mRNA expression of m-calpain and calpastatin was also increased 24 h and 3 h after IRI, respectively. Neither m-calpain nor calpastatin mRNA expression was influenced by E-64d (P > 0.05). The mRNA ratio of m-calpain to calpastatin was increased at the 6 h, 24 h and 72 h after IRI, and only at 24 h the increase of the ratio of m-calpain to calpastatin was inhibited by E-64d (P < 0.05). In the rat retina of IRI, E-64d inhibits the increase of m-calpain protein expression, as well as the mRNA ratio increase of m-calpain to calpastatin. E-64d also inhibited the retinal damage induced by IRI, suggesting a role for E-64d in the protection of the retinal apoptosis induced by IRI. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 2, pp. 258–264. The text was submitted by the authors in English.     

Vinculin is proteolyzed by calpain during platelet aggregation: 95 kDa cleavage fragment associates with the platelet cytoskeleton

The focal adhesion protein vinculin contributes to cell attachment and spreading through strengthening of mechanical interactions between cell cytoskeletal proteins and surface membrane glycoproteins. To investigate whether vinculin proteolysis plays a role in the influence vinculin exerts on the cytoskeleton, we studied the fate of vinculin in activated and aggregating platelets by Western blot analysis of the platelet lysate and the cytoskeletal fractions of differentially activated platelets. Vinculin was proteolyzed into at least three fragments (the major one being approximately 95 kDa) within 5 min of platelet activation with thrombin or calcium ionophore. The 95 kDa vinculin fragment shifted cellular compartments from the membrane skeletal fraction to the cortical cytoskeletal fraction of lysed platelets in a platelet aggregation-dependent manner. Vinculin cleavage was inhibited by calpeptin and E64d, indicating that the enzyme responsible for vinculin proteolysis is calpain. These calpain inhibitors also inhibited the translocation of full-length vinculin to the cytoskeleton. We conclude that cleavage of vinculin and association of vinculin cleavage fragment(s) with the platelet cytoskeleton is an activation response that may be important in the cytoskeletal remodeling of aggregating platelets.     

Calpain,calpastatin activities and ratios during myocardial ischemia-reperfusion

The purpose of this study was to test the hypothesis that myocardial ischemia-reperfusion (I/R) is accompanied by an early burst in calpain activity, resulting in decreased calpastatin activity and an increased calpain/calpastatin ratio, thereby promoting increased protein release. To determine the possibility of a calpain burst impacting cardiac calpastatin inhibitory activity, rat hearts were subjected (Langendorff) to either 45 or 60 min of ischemia followed by 30 min of reperfusion with and without pre-administration (s.c.) of a cysteine protease inhibitor (E-64c). Myocardial function, calpain activities (casein release assay), calpastatin inhibitory activity and release of CK, LDH, cTnI and cTnT were determined (n = 8 for all groups). As expected no detectable changes in calpain activities were observed following I/R with and without E-64c (p > 0.05). Both I/R conditions reduced calpastatin activity (p < 0.05) while E-64c pre-treatment was without affect, implicating a non-proteolytic event underlying the calpastatin changes. A similar result was noted for calpain–calpastatin ratios and the release of all marker proteins (p < 0.05). In regard to cardiac function, E-64c resulted in transient improvements (15 min) for left ventricular developed pressure (LVDP) and rate of pressure development (p < 0.05). E-64c had no effect on end diastolic pressure (LVEDP) or coronary pressure (CP) during I/R. These findings demonstrate that restricting the putative early burst in calpain activity, suggested for I/R, by pre-treatment of rats with E-64c does not prevent downegulation of calpastatin inhibitory activity and/or protein release despite a transient improvement in cardiac function. It is concluded that increases in calpain isoform activities are not a primary feature of I/R changes, although the role of calpastatin downregulation remains to be elucidated.     

Thiol Protease Inhibitor,E-64-d,Prevents Spindle Formation during Mouse Oocyte Maturation1

E-64-d, a membrane permeant derivative of E-64, the thiol protease inhibitor, was found to prevent meiotic maturation of mouse oocytes in a dose dependent manner. When immature mouse oocytes were incubated with E-64-d for up to 14 hr, first polar body emission was blocked to 36% at 200 μg/ml and 6% at 400 μg/ml, but germinal vesicle breakdown occurred normally. Cytological analysis revealed that meiotic spindles were not formed, while chromosome condensation occurred. Thus, E-64-d prevents oocytes from progressing to the first meiotic metaphase. When exposed to E-64-d after 8 hr of incubation without E-64-d, one-fourth of oocytes completed the first meiotic division but never progressed to the second metaphase. In three-fourth of the oocytes inhibited to emit the first polar body, spindles disappeared after incubation with E-64-d. The results suggest that E-64-d promotes disassembly of meiotic spindles resulting in inhibition of meiotic maturation. We propose that thiol protease is involved in spindle formation in mouse meiotic maturation.     

Impact of cysteine proteinase inhibition in midgut fluid and oral secretion on fecundity and pollen consumption of western corn rootworm (Diabrotica virgifera virgifera)

Cysteine proteinases predominate in the midgut fluid (MF) and oral secretion (OS) of adult western corn rootworm (WCR) based on their mild acidic pH optima (pH 6.0), enhanced activities after treatment with thiol reducing agents, and inhibition by selective cysteine proteinase inhibitors (PIs). Four cysteine PIs including E-64, calpeptin, calpain inhibitor II, and leupeptin (also a serine PI) strongly inhibited azocaseinolytic activity in a dose-dependent manner in both the MF and OS. The most significant effect on adult female WCR of cysteine PI consumption with corn pollen was the reduction in fecundity, but female survival was not apparently affected. Mean fresh weights for all PI-fed females were also lower than control groups. All PI-fed groups [E-64, calpain inhibitor I (Cal I) and leupeptin] had a significantly lower daily egg production than respective corn pollen-fed controls. E-64 was more potent than leupeptin and Cal I on inhibiting fecundity, which correlates with their relative anti-proteinase potency in vitro. E-64, Cal I, and leupeptin at 1.5-2 nmol/beetle/day reduced fecundity down to 25-45% of control values. Reduced egg production by PI-fed beetles results from a combination of the direct inhibition of protein digestion and a post-ingestive negative feedback mechanism, which reduces food intake. The supplement of ten essential amino acids into the E-64-treated pollen enhanced up to 3.7-fold the number of eggs laid compared to the E-64-fed group without these amino acids, suggesting that egg production is dependent on the supply of essential amino acids from corn pollen proteolysis.     

SNAP-23 is a target for calpain cleavage in activated platelets

The role of calpain in platelet function is generally associated with aggregation and clot retraction. In this report, data are presented to show that one component of the platelet secretory machinery, SNAP-23, is specifically cleaved by calpain in activated cells. Other proteins of the membrane fusion machinery, e.g. syntaxins 2 and 4 and alpha-SNAP, are not affected. In vitro studies, using permeabilized platelets, demonstrate that cleavage is time- and calcium-dependent. Analysis of SNAP-23 cleavage products suggests that the calpain cleavage site(s) is in the C-terminal third of the molecule potentially between the cysteine-rich acyl attachment sites and the C-terminal coiled-coil domain. The time course of cleavage is most consistent with late calpain-mediated events such as pp60(c-src) cleavage, but not early events such as protein-tyrosine phosphatase-1B activation. SNAP-23 cleavage is inhibited by calpeptin, calpastatin, calpain inhibitor IV, and E-64d, but not by caspase 3 inhibitor III or cathepsin inhibitor I. When tested for their effect on secretion, none of the calpain-specific inhibitors significantly affected release of soluble components from any of the three platelet granule storage pools. These results indicate that SNAP-23 cleavage occurs after granule release and therefore may play a role in affecting granule membrane exteriorization. This is consistent with the ultrastructural morphology of calpeptin-treated platelets after activation.     

Inhibition of thrombin-induced platelet activation by leupeptin. Implications for the participation of calpain in the initiation of platelet activation

Inhibitors of calcium-dependent proteases (calpains) such as leupeptin and antipain have been shown to selectively inhibit platelet activation by thrombin. Based upon this observation, it has been proposed that calpains play a role in the initiation of platelet activation. In the present studies, we have examined the effect of leupeptin on the earliest known event in thrombin-induced platelet activation: the interaction between the agonist, its receptors, and the guanine nucleotide-binding proteins which stimulate phospholipase C (Gp) and inhibit adenylyl cyclase (Gi). We found that leupeptin inhibited thrombin's ability to stimulate phosphoinositide hydrolysis, suppress cAMP formation, and dissociate Gp and Gi into subunits. Leupeptin had no effect, however, on the same responses to other agonists or on thrombin binding to platelets. Although these observations might suggest, as others have concluded, that calpain is involved in the initiation of platelet activation by thrombin, we also found that: 1) substituting platelet membranes for intact platelets and decreasing the free Ca2+ concentration below the threshold required for calpain activation did not diminish the effects of leupeptin on phosphoinositide hydrolysis and cAMP formation, 2) washing the platelets after incubation with leupeptin reversed the effects of the inhibitor, 3) permeabilizing the platelets with saponin did not enhance the inhibitory effects of leupeptin, and 4) leupeptin inhibited the proteolysis of fibrinogen and the hydrolysis of S2238 by thrombin. Similar results in these assays were obtained with antipain. Therefore, our observations suggest that the inhibition of platelet activation by leupeptin is due to a direct interaction with thrombin and need not reflect a role for calpain in the initiation of platelet activation.     

Synthesis of a new cell penetrating calpain inhibitor (calpeptin)

N-terminal of Leu-norleucinal or Leu-methioninal was modified to obtain a cell penetrative peptide inhibitor against calpain. Benzyloxycarbonyl (Z) derivatives had less active against papain than phenylbutyryl derivatives and leupeptin. Z-Leu-nLeu-H (calpeptin) was more sensitive to calpain I than Z-Leu-Met-H and leupeptin. Calpeptin was most potent among synthesized inhibitors in terms of preventing the Ca2+-ionophore induced degradation of actin binding protein and P235 in intact platelets. After 30 min incubation with intact platelets, calpeptin completely abolished calpain activity in platelets but no effect was observed in case of leupeptin. Calpeptin also inhibited 20K phosphorylation in platelets stimulated by thrombin, ionomycin or collagen. Thus calpeptin was found to be a useful cell-penetrative calpain inhibitor.     

Calpain abolishes the effect of filamin on the actomyosin system in platelets

Platelet filamin was shown to cross-link F-actin and inhibit actomyosin ATPase activity. Filamin was also shown to be degraded by calpain (calcium-activated neutral proteinase; CANP) when the platelet was activated. The consequences of the proteolysis of filamin on the actomyosin system have been investigated. When degraded by calpain in the presence of Ca2+, filamin loses its ability to cross-link F-actin. Under the same conditions, its inhibitory effects on the superprecipitation and ATPase activity of actomyosin are abolished. The result suggests that the degradation of filamin is favorable for contraction of the activated platelets.     

Inhibition of calpain blocks platelet secretion, aggregation, and spreading

Previous studies have indicated that the Ca(2+)-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced alpha-granule secretion (IC(50) = 20 microM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC(50) = 50 microM), and platelet spreading on glass surfaces (IC(50) = 34 microM). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.     

Proteolytic processing of the insulin receptor beta subunit is associated with insulin-induced receptor down-regulation

This report describes the use of an antibody directed against the carboxyl terminus of the insulin receptor beta subunit to assess the fate of the insulin receptor protein over the time course of insulin-induced receptor down-regulation. The insulin receptor beta subunit is lost from the cellular membranes of insulin-treated 3T3-C2 fibroblasts with a time course superimposable with the insulin-induced loss of cellular insulin binding activity. Concomitant with the time-dependent loss of the intact beta subunit from the membranes, a 61,000-Da fragment of the insulin receptor beta subunit accumulates in the cytosol of the cells in a time-dependent manner. The insulin-induced loss of the intact beta subunit from the cellular membranes is inhibited by cycloheximide. Chloroquine and the thiol protease inhibitors leupeptin and E-64 inhibit the insulin-induced loss of the intact beta subunit from the membranes and induce an accumulation of the intact subunit in the membranes. However, in the presence of leupeptin, E-64, or chloroquine, the insulin-induced loss of insulin binding activity occurs normally. These data indicate that down-regulation results in the loss of the intact beta subunit from the cellular membranes with the production of a fragment of the beta subunit in the cytosol. The protease responsible for the generation of the fragment is a thiol protease which requires acidic conditions. Since the insulin-induced proteolysis of the beta subunit can be totally inhibited under conditions where the insulin-induced loss of insulin binding activity proceeds normally, the proteolysis of the beta subunit is a process which is separate and distinguishable from the insulin-induced loss of insulin binding activity.