Binding and degredation of insulin by plasma membranes from bovine liver isolated by a large scale preparation

With the large-scale preparation described, as much as 1 kg of bovine liver can be processed, giving a yield of more than 1 g plasma membrane protein. From analytical and morphological criteria the plasma membrane fraction isolated mainly derives from bile-canalicular and contiguous areas of the hepatocytes.The insulin binding activity is quite similar to insulin receptors in otherr cell systems and membrane preparations. Insulin-degrading activity is very low in the isolated plasma fraction. Most of degrading activity is located in a microsomal membrane fraction. Neverthless the K_m and the pH dependence of the insulin-degrading activity in both fractions are nearly identical.From these studies we conclude that binding and degradation of insulin are two independent processes located on different cell organelles.     

Internalization of insulin and its receptor in the isolated rat adipose cell. Time-course and insulin concentration dependency

The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37 degrees C. The internalization of insulin was assessed by examining the subcellular distribution of cell-associated [125I]insulin among plasma membrane, and high-density (endoplasmic reticulum-enriched) and low-density (Golgi-enriched) microsomal membrane fractions prepared by differential ultracentrifugation. The distribution of receptors was measured by the steady-state exchange binding of fresh [125I]insulin to these same membrane fractions. At 37 degrees C, insulin binding to intact cells is accompanied initially by the rapid appearance of intact insulin in the plasma membrane fraction, and subsequently, by its rapid appearance in both the high-density and low-density microsomal membrane fractions. An apparent steady-state distribution of insulin per mg of membrane protein among these subcellular fractions is achieved within 30 min in a ratio of 1:1.54:0.80, respectively. Concomitantly, insulin binding to intact cells is associated with the rapid disappearance of approx. 30% of the insulin receptors initially present in the plasma membrane fraction and appearance of 20-30% of those lost in the low-density microsomal membrane fraction. However, the number of receptors in the high-density microsomal membrane fraction does not change. This redistribution of receptors also appears to reach a steady-state within 30 min. Both processes are insulin concentration-dependent, correlating with receptor occupancy in the intact cell, and are partially inhibited at 16 degrees C. While the steady-state subcellular distributions of insulin and its receptor do not correlate with that of acid phosphatase, chloroquine markedly increases the levels of insulin associated with all three membrane fractions in apparent proportion to the distribution of this lysosomal marker enzyme activity, without more than marginally potentiating insulin's effects on the distribution of receptors. These results demonstrate that insulin, initially bound to the plasma membrane of the isolated rat adipose cell, is rapidly translocated by a receptor-mediated process into at least two intracellular compartments associated with the cell's high- and low-density microsomes. Furthermore, insulin simultaneously induces the translocation of its own receptor from the plasma membrane into the latter compartment. These translocations appear to represent the internalization and partial dissociation of the insulin-receptor complex through insulin-induced receptor cycling.     

Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to the plasma membrane

Insulin stimulates the accumulation of iron by isolated fat cells by increasing the uptake of diferric transferrin. Analysis of the cell-surface binding of diferric 125I-transferrin indicated that insulin caused a 3-fold increase in the cell surface number of transferrin receptors. This result was confirmed by the demonstration that insulin increases the binding of an anti-rat transferrin receptor monoclonal antibody (OX-26) to the surface of fat cells. The basis of this effect of insulin was examined by investigating the number of transferrin receptors in membrane fractions isolated from disrupted fat cells. Two methods were employed. First the binding isotherm of diferric 125I-transferrin to the isolated membranes was studied. Second, the membranes were solubilized with detergent, and the number of transferrin receptors was measured by immunoblotting using the monoclonal antibody OX-26. It was observed that insulin treatment of intact fat cells resulted in an increase in the number of transferrin receptors located in the isolated plasma membrane fraction of the disrupted fat cells. Furthermore, the increase in the number of plasma membrane transferrin receptors was associated with a concomitant decrease in the transferrin receptor number in a low density microsome fraction previously shown to consist of intracellular membranes. This redistribution of transferrin receptors between cellular membrane fractions in response to insulin is remarkably similar to the regulation by insulin of glucose transporters and type II insulin-like growth factor receptors. We conclude that insulin stimulates fat cell iron uptake by a mechanism that may involve the redistribution of transferrin receptors from an internal membrane compartment (low density microsomes) to the cell surface (plasma membrane).     

Internalization of insulin and its receptor in the isolated rat adipose cell: Time-course and insulin concentration dependency

The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37°C. The internalization of insulin was assessed by examining the subcellular distribution of cell-associated [¹²⁵I]insulin among plasma membrane, and high-density (endoplasmic reticulum-enriched) and low-density (Golgi-enriched) microsomal membrane fractions prepared by differential ultracentrifugation. The distribution of receptors was measured by the steady-state exchange binding of fresh [¹²⁵I]insulin to these same membrane fractions. At 37°C, insulin binding to intact cells is accompanied initially by the rapid appearance of intact insulin in the plasma membrane fraction, and subsequently, by its rapid appearance in both the high-density and low-density microsomal membrane fractions. An apparent steady-state distribution of insulin per mg of membrane protein among these subcellular fractions is achieved within 30 min in a ratio of 1:1.54:0.80, respectively. Concomitantly, insulin binding to intact cells is associated with the rapid disappearance of approx. 30% of the insulin receptors initially present in the plasma membrane fraction and appearance of 20–30% of those lost in the low-density microsomal membrane fraction. However, the number of receptors in the high-density microsomal membrane fraction does not change. This redistribution of receptors also appears to reach a steady-state within 30 min. Both processes are insulin concentration-dependent, correlating with receptor occupancy in the intact cell, and are partially inhibited at 16°C. While the steady-state subcellular distributions of insulin and its receptor do not correlate with that of acid phosphatase, chloroquine markedly increases the levels of insulin associated with all three membrane fractions in apparent proportion to the distribution of this lysosomal marker enzyme activity, without more than marginally potentiating insulin's effects on the distribution of receptors. These results demonstrate that insulin, initially bound to the plasma membrane of the isolated rat adipose cell, is rapidly translocated by a receptor-mediated process into at least two intracellular compartments associated with the cell's high- and low-density microsomes. Furthermore, insulin simultaneously induces the translocation of its own receptor from the plasma membrane into the latter compartment. These translocations appear to represent the internalization and partial dissociation of the insulin-receptor complex through insulin-induced receptor cycling.     

Insulin receptor kinase following internalization in isolated rat adipocytes

We have studied how insulin-mediated internalization of insulin receptors and insulin activation of the insulin receptor kinase might be inter-related. Isolated rat adipocytes were exposed to 0, 6, or 500 ng/ml insulin for 40 min at 37 degrees C. Subsequently, plasma membrane, low-density microsomal membrane and high-density microsomal membrane subcellular fractions were prepared. Measurement of insulin binding to insulin receptors isolated from the membrane fractions revealed that exposure of cells to insulin resulted in a loss of binding activity (13% at 6 ng/ml, 27% at 500 ng/ml insulin) from the plasma membranes which was completely accounted for by the appearance of receptors in the low-density and high-density microsomal membrane fractions, indicating that insulin had induced translocation of insulin receptors from the surface to the cell interior. Measurement of kinase activity of the isolated receptors revealed that exposure of intact cells to 500 ng/ml insulin resulted in as much as a 35-fold increase in the intrinsic kinase activity of receptors from subcellular fractions. The kinase activity per receptor was equal in all fractions at 3-4 min but by 20 min the activity of the internalized receptors fell approximately 40% to a steady state; plasma membrane receptors, on the other hand, remained fully active over time. This indicates that newly internalized receptors retain their kinase activity but undergo subsequent deactivation. Following exposure of cells to 6 ng/ml insulin, the degree of activation of the insulin receptor kinase was lower in the plasma membrane fraction (24% of the insulin effect at 500 ng/ml) than in the low-density and high-density microsomal membrane fractions (54 and 77%, respectively, of the insulin effect at 500 ng/ml). These results suggest that receptors with an activated kinase are preferentially internalized. We conclude that exposure of adipocytes to insulin causes endocytosis of insulin receptors and activation of insulin receptor kinase, newly internalized receptors are fully active tyrosine kinases but are deactivated as they traverse the intracellular organelles represented by low-density and high-density microsomal membranes, and insulin receptor occupancy, possibly by stimulating phosphorylation and activating the insulin receptor kinase, is important for targeting insulin receptors for internalization.     

Potential mechanism of insulin action on glucose transport in the isolated rat diaphragm. Apparent translocation of intracellular transport units to the plasma membrane

[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to quantitate the number of functional glucose transport units in plasma and microsomal membranes prepared from intact rat diaphragm. In a series of three experiments, plasma membranes prepared from diaphragms which have not been incubated with insulin bind approximately 16 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable binding site. If 280 nM (40,000 microunits/ml) insulin is present during the incubation, cytochalasin B binding to the plasma membranes is increased approximately 2-fold without alteration in the dissociation constant of this site. Membranes in the microsomal fraction prepared from diaphragms which have been incubated for 30 min in the absence of insulin contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. However, in the presence of insulin during the incubation period, the number of these sites in the microsomal fraction is decreased to 12 pmol/mg of membrane protein. These results suggest that insulin stimulates glucose transport in the isolated rat diaphragm primarily through a translocation of functional glucose transport units from an intracellular membrane pool to the plasma membrane. These results are similar to the results observed in rat adipose cells (Cushman, S. W., and Wardzala, L. J. (1980) J. Biol. Chem. 255, 4758-4762) and suggest that this mechanism of insulin-stimulated glucose transport activity may be general to other cell types.     

Demonstration that the insulin receptor undergoes an early structural modification following insulin binding

Processing of the insulin receptor by hepatocytes was studied using a 125I-labelled photoreactive insulin derivative which could be covalently attached to the receptor and facilitate the analysis of receptor structure in isolated subcellular fractions by SDS-polyacrylamide gel electrophoresis. Following binding at the cell surface, the label was rapidly internalised and located in a low-density subcellular fraction ('endosomes'). The intact receptor (350 000 molecular weight) and binding (alpha) subunit (135 000), produced by in vitro disulphide reduction of the samples, were found in the plasma membrane fraction but not in endosomes. In endosomes, the label was concentrated in a band at 140 000 (non-reduced) which on reduction generated species of 100 000 and 68 000 predominantly. The insulin receptor therefore undergoes an early structural change during endocytosis. This modification does not involve complete disulphide reduction and may be due to a proteolytic event.     

Changes in Insulin-Binding Capacity of the Plasma Membrane Fraction during Culture in vitro of Cells Derived from Micromeres of 16-Cell-Stage Sea Urchin Embryos

In cultured cells derived from isolated micromeres of 16-cell stage sea urchin embryos, which undergo insulin-induced pseudopodial cable growth, specific and reversible insulin binding by a 52-kDa protein, probably an insulin receptor in the plasma membrane, is augmented during 5 h of culture without any change in the dissociation constant (Kuno et al : 1994). The increase in insulin-binding capacity in micromere-derived cells was only minimally blocked by actinomycin D and cycloheximide, which inhibited [U-³H]uridine incorporation into RNA and [³⁵S]methionine incorporation into protein, respectively. Insulin binding capacity was found in the plasma membrane fraction and the microsome fraction of isolated micromeres. The capacity in the plasma membrane fraction increased, accompanied by its decrease in the microsome fraction, during 5 h of culture of micromere-derived cells. The insulin receptor is probably accumulated in microsomes of presumptive micromeres prior to the 16-cell stage and transferred to the plasma membrane, resulting in an increase in the insulin binding capacity of micromere-derived cells during 5 h of culture.     

Insulin degradation V. Unmasking of glutathione-insulin transhydrogenase in rat liver microsomal membrane

Glutathione-insulin transhydrogenase (glutathione:protein disulfide oxidoreductase, EC 1.8.4.2) inactivates insulin by cleaving its disulfide bonds. The distribution of GSH-insulin transhydrogenase in subcellular fractions of rat liver homogenates has been studied. From the distribution of insulin-degrading activity and marker enzymes (glucose-6-phosphatase and succinate-INT reductase) (INT, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) after cell fractionation by differential centrifugation, the immunological analysis of the isolated subcellular fractions with antibody to purified rat liver GSH-insulin transhydrogenase, and chromatographic analysis (on a column of Sephadex G-75 in 50% acetic acid) of the products formed from ¹²⁵I-labelled insulin after incubation with the isolated subcellular fractions, it is concluded that GSH-insulin transhydrogenase is located primarily in the microsomal fraction of rat liver homogenate. An enzyme(s) that further degrades insulin by proteolysis is located mainly in the soluble fraction; a significant amount of the protease(s) activity is also present in the mitochondrial fraction. The possibility has been discussed that the protease(s) acts upon the intermediate product of insulin degradation, A and B chains of insulin, rather than upon the intact insulin molecule itself.The GSH-insulin transhydrogenase in intact microsomes occurs in a latent state; it is readily released from the microsomal membrane and its activity is greatly increased by treatments which affect the lipoprotein membrane structure of microsomal vesicles. There include homogenization with a Polytron homogenizer, sonication, freezing and thawing, alkaline pH, the nonionic detergent Triton X-100, and phospholipases A and C.     

Distribution of insulin receptors among mouse liver endomembranes.

Specific binding of insulin to highly purified preparations of rough endoplasmic reticulum, Golgi apparatus, and plasma membrane of mouse liver was determined. 125I-labeled insulin bound maximally to the plasma membrane in radio-receptor assays. Golgi apparatus fractions exhibited binding 10--20% that of plasma membrane and rough endoplasmic reticulum exhibited only 1--2% of plasma membrane binding. Binding was proportional to membrane concentration and dose vs. response curves were very similar for the different fractions. Scatchard analysis of the insulin binding data for the plasma membrane and Golgi apparatus fractions showed curvilinear plots yielding similar apparent binding affinities (0.9 and 3.0-10(8) M-1, respectively). Purity of the isolated endomembranes was analyzed by morphometry and (Na+ + K+ + Mg2+)-ATPase and these preparations displayed less than 1% contamination by plasma membrane. These findings provide important confirmation of the presence of insulin receptors in Golgi apparatus membranes comparable to those located on the plasma membrane. Finally, the present study did not allow us to verify the existence of insulin receptors in the endoplasmic reticulum.     

Insulin stimulates the translocation of Na+/K(+)-dependent ATPase molecules from intracellular stores to the plasma membrane in frog skeletal muscle.

The mechanism of the stimulation of Na+/K+ transport by insulin in frog skeletal muscle was studied. The ouabain-binding capacity in detergent-treated plasma membranes of insulin-exposed muscles was increased 1.9-fold compared with that of controls. Na+/K(+)-ATPase activity was found in an intracellular 'light fraction' (fraction II) prepared by using anion-exchange chromatography. Marker enzyme activities for plasma and Golgi membranes were not detected in this fraction. The specific activity of Na+/K(+)-ATPase in fraction II from insulin-exposed muscles was 58% of that in an identical fraction from control muscles. No significant difference in the protein yield of the plasma membrane preparation was observed between these two groups. In parallel with the decrease in the Na+/K(+)-ATPase activity in fraction II from insulin-exposed muscles, the ouabain-binding capacity in this fraction was also decreased. The addition of saponin to fraction II increased both Na+/K(+)-ATPase activity and ouabain binding, indicating that some of the Na+/K(+)-ATPase is located in sealed vesicles. These findings support the view that insulin stimulates the translocation of Na+/K(+)-ATPase molecules from fraction II to the plasma membrane.     

Distribution of insulin receptors among mouse liver endomembranes

Specific binding of insulin to highly purified preparations of rough endoplasmic reticulum, Golgi apparatus, and plasma membrane of mouse liver was determined. ¹²⁵I-labeled insulin bound maximally to the plasma membrane in radio-receptor assays. Golgi apparatus fractions exhibited binding 10–20% that of plasma membrane and rough endoplasmic reticulum exhibited only 1–2% of plasma membrane binding. Binding was proportional to membrane concentration and dose vs. response curves were very similar for the different fractions. Scatchard analysis of the insulin binding data for the plasma membrane and Golgi apparatus fractions showed curvilinear plots yielding similar apparent binding affinities (0.9 and 3.0 · 10⁸ M^?1, respectively). Purity of the isolated endomembranes was analyzed by morphometry and (Na⁺ + K⁺ + Mg²⁺)-ATPase and these preparations displayed less than 1% contamination by plasma membrane. These findings provide important confirmation of the presence of insulin receptors in Golgi apparatus membranes comparable to those located on the plasma membrane. Finally, the present study did not allow us to verify the existence of insulin receptors in the endoplasmic reticulum.     

Stimulation of prostaglandin E1 binding to human blood platelet membrane by insulin and the activation of adenylate cyclase

Incubation of human platelet-rich plasma with physiological amounts of insulin resulted in the increase of the binding of prostaglandin E1 by more than 2-fold when compared to the control platelets. Scatchard analysis of the binding of the prostaglandin to the hormone-treated platelets showed that the increased binding was due to the increase of receptor numbers rather than the increase of affinity of the binding sites. The membranes prepared from the insulin-treated platelets also showed similar enhanced binding of the prostaglandin. However, addition of insulin directly to the membranes isolated from the platelets which had not been previously incubated with the hormone failed to show any effect. This increased binding of the prostanoid to the membranes prepared from the insulin-treated platelets resulted in the stimulation of adenylate cyclase by more than 2-fold when compared with the control of membrane preparation by the prostaglandin alone. In contrast, treatment of platelets with the hormone which increased the prostanoid binding to these cells did not influence the cyclic AMP phosphodiesterase activity of either the membrane or cytosolic fraction. The increase in the cellular level of cyclic AMP by prostaglandin E1 was 2-fold greater in the hormone-treated cells than in the case of untreated platelets stimulated by the agonist alone. The incubation of platelet-rich plasma with insulin, as expected, decreased the amount of prostaglandin E1 needed to inhibit platelet aggregation by more than 50% when compared to the control platelets.     

CMP-N-acetylneuraminic acid hydrolase, an ectoenzyme distributed unevenly over the hepatocyte surface.

The regional localization of CMP-N-acetylneuramic acid hydrolase at the hepatocyte surface was studied by using plasma membranes and hepatocytes isolated from rat liver. 1. By homogenization of the rat liver plasma membrane preparations and subsequent discontinuous sucrose gradient centrifugation, one light and two heavy membrane fractions were obtained. The origin of these three subfractions is discussed based on the specific activities in the three fractions of 5'-nucleotidase, alakaline phosphatase and Mg2+-ATPase and on electron microscopic examination of the fractions. Evidence is given suggesting that the light fraction is derived from the bile canalicular surface of the plasma membrane, and that the heavy fractions are derived predominantly from the sinusoidal and lateral surfaces of the liver cell membrane. CMP-AcNeu hydrolase was present at highest specific activity in one of the heavy subfractions. Therefore it is concluded that CMP-AcNeu hdyrolase is located preferentially in the sinusoidal and/or lateral plasma membrane parts of the liver cell. 2. Experiments with intact and disintegrated hepatocytes isolated from rat liver indicated that CMP-AcNeu hydrolase is located at the surface of the cell membrane, with its functional group directed to the outside.     

Characterization of the Escherichia coli membrane domain responsible for binding oriC DNA.

It has previously been shown that hemimethylated DNA from the Escherichia coli replication origin (oriC) binds with high specificity to membrane fractions isolated from disrupted cells. In this article, the membrane localization of oriC-binding activity was studied by subjecting crude membrane preparations to successive cycles of sedimentation and flotation gradient analysis. This revealed that approximately two-thirds of the membrane-associated oriC-binding activity of the cell was not associated with the outer membrane fraction as previously suggested but was recovered instead in a unique membrane fraction (OCB1) whose buoyant density and protein profile differed from those of both inner and outer membranes. The specific activity of oriC binding in OCB1 was approximately fivefold higher than the activity of the isolated outer membrane peak. It is likely that membrane fraction OCB1 includes the membrane domain responsible for the binding of hemimethylated oriC to the cell envelope in intact cells.     

Perturbation of insulin-receptor interactions by intramolecular hormone cross-linking. Analysis of relative movement among residues A1, B1, and B29

We have evaluated, by use of isolated canine hepatocytes, the importance of intramolecular hormone cross-linking (and of concomitant changes in molecular flexibility) to the interaction of insulin with its plasma membrane receptor. Cross-linked hormone analogs were prepared by reacting porcine insulin, N alpha A1-t-butyloxycarbonyl insulin or N alpha A1-t-butyloxycarbonyl [D-LysA1]insulin with various dicarboxylic acid active esters to obtain alpha-GlyA1/epsilon-LysB29-, alpha-PheB1/epsilon-LysB29-, and epsilon-D-LysA1/epsilon-LysB29-cross-linked insulins, respectively. In the aggregate, insulin analogs cross-linked by groups containing 2-12 atoms retained 1.4-35% of the receptor binding potency of native insulin. Analysis of our results suggests that: (a) loss of chemical functionality, steric interference, and restriction of potential intramolecular movement can all play roles in determining the receptor binding potencies of cross-linked insulin analogs; (b) restriction of intramolecular movement between residues A1 and B29 affects negatively the binding of insulin to its receptor (but accounts for only a fraction of the conformational change which insulin must undergo to achieve a high affinity state of ligand-receptor interaction); and (c) introduction of a cross-link between residues B1 and B29 (residues that are in fact in proximity in one crystalline form of the hormone) decreases markedly the receptor binding potencies of the corresponding analogs. The importance of these findings is discussed in relation to the potential structure of insulin when it is bound to its plasma membrane receptor.     

Isolation by preparative free-flow electrophoresis and aqueous two-phase partition from rat adipocytes of an insulin-responsive small vesicle fraction with glucose transport activity

Preparative free-flow electrophoresis and aqueous two-phase polymer partition were used to obtain a plasma membrane-enriched fraction of adipocytes isolated from epididymal fat pads of the rat together with a fraction enriched in small vesicles with plasma membrane characteristics (thick membranes, clear dark-light-dark pattern). The electrophoretic mobility of the small vesicles was much less than that of the plasma membrane consistent with an inside-out orientation whereby charged molecules normally directed to the cell surface were on the inside. When plasma membranes and the small vesicle fraction were isolated from fat cells treated or not treated with 100 μU/ml insulin and the resident proteins of the two fractions analyzed by SDS-PAGE, the two fractions exhibited characteristics responses involving specific protein bands. Insulin treatment for 2 min resulted in the loss of a 90 kDa band from the plasma membrane. At the same time, a ca. 55-kDa peptide band that was enhanced in the plasma membrane was lost from the small vesicle fraction. The latter corresponded on Western blots to the GLUT-4 glucose transporter. Thus, we suggest that the small vesicle fraction with characteristics of inside-out plasma membrane vesicles may represent the internal vesicular pool of plasma membrane subject to modulation by treatment of adipocytes with insulin.     

Evidence for an early degradative event to the insulin molecule following binding to hepatocyte receptors

We have used photoreactive insulin analogues to investigate as related processes, early structural modification of the receptor-bound insulin molecule and internalisation of the insulin-receptor complex. In isolated rat hepatocytes an initial modification of bound insulin leads to the generation of a molecular species unchanged in molecular weight but with reduced receptor and antibody binding affinities and altered electrophoretic mobility. Using photoreactive insulin analogues and density gradient cell fractionation the insulin receptor complex has been shown to undergo internalisation from the plasma membrane to a low density vesicular fraction, the endosome. No labelled material was found in lysosomal fractions after up to 10 min incubation at 37 degrees C. The degree of labelling of the endosome fraction depended on the position of the photoreactive group within the insulin molecule. The data suggest that before or during endocytosis, a small peptide is proteolytically cleaved from the C terminus of the insulin B chain.     

Characterization of the stimulatory action of insulin on insulin-like growth factor II binding to rat adipose cells. Differences in the mechanism of insulin action on insulin-like growth factor II receptors and glucose transporters

Insulin is known to increase the number of cell surface insulin-like growth factor II (IGF-II) receptors in isolated rat adipose cells through a subcellular redistribution mechanism similar to that for the glucose transporter. The effects of insulin on these two processes, therefore, have now been directly compared in the same cell preparations. 1) Insulin increases the steady state number of cell surface IGF-II receptors by 7-13-fold without affecting receptor affinity; however, insulin stimulates glucose transport activity by 25-40-fold. 2) The insulin concentration required for half-maximal stimulation of cell surface IGF-II receptor number is approximately 30% lower than that for the stimulation of glucose transport activity. 3) The half-time for the achievement of insulin's maximal effect at 37 degrees C is much shorter for IGF-II receptor number (approximately 0.8 min) than for glucose transport activity (approximately 2.6 min). 4) Reversal of insulin's action at 37 degrees C occurs more rapidly for cell surface IGF-II receptors (t1/2 congruent to 2.9 min) than for glucose transport activity (t1/2 congruent to 4.9 min). 5) When the relative subcellular distribution of IGF-II receptors is examined in basal cells, less than 10% of the receptors are localized to the plasma membrane fraction indicating that most of the receptors, like glucose transporters, are localized to an intracellular compartment. However, in response to insulin, the number of plasma membrane IGF-II receptors increases only approximately 1.4-fold while the number of glucose transporters increases approximately 4.5-fold. Thus, while the stimulatory actions of insulin on cell surface IGF-II receptors and glucose transport activity are qualitatively similar, marked quantitative differences suggest that the subcellular cycling of these two integral membrane proteins occurs by distinct processes.