Insulin-stimulated GLUT4 Glucose Transporter Recycling

THE JOURNAL OF BIOWGICAL CHEMISTRY

Vol. 269, No. 26, Issue of July 1, pp. 1751617524, 1994 Printed in U.S.A.

Insulin-stimulated GLUT4 Glucose Transporter Recycling A PROBLEM IN MEMBRANE PROTEIN SUBCELLULAR TRAFFICKING THROUGH MULTIPLE POOLS* (Received for publication, March 28, 1994) Geoffrey D. Holmanl, Leila Lo Leggio, and Samuel W. CushmanO From the Department of Biochemistry, The University of Bath, Bath BA2 7AE: United Kingdom and the §Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

The subcellular trafficking of GLUT4 in isolated rat adipose cellsand 3T3-Ll adipocytes exhibits many of the properties observed in regulated secretory processes and neurosecretion. GLUT4 is sorted and sequestered from endosomes into a specialized secretory compartment in thebasal state and the initial stimulation of its exocytosis by insulin is more rapid than its recycling through the endosomes and secretory compartment during the steady-state response to insulin. We present a mathematical analysis which showsthat thisbehavior is inconsistent with a simple 2-pool model with one plasma membrane and one intracellular compartment, but that a 3-pool model, with two intracellular compartments, can simulate these properties. We extend this model to include the presence of occluded pools in the plasma membrane.Our analysis compares the behaviorexpected when these occluded pools are precursors in stimulation and/or clathrin-associated-likeintermediates in endocytosis. The presence of a precursor occluded pool can account for a lag between the appearance ofGLUT4 in the membrane and before the full stimulation of glucose transport activity. The analysis alsoshows that since the poolsize of the occluded GLUT4 is relatively small, the formation of endocytic occluded intermediates such as GLUT4 in clathrincoated pits is likely to be slow compared with the rateof endocytosis of the coated vesicles.

tosed, retrieved from the plasma membrane via clathrin-coated vesicles into endosomes, and sorted into thespecialized secretory pool which is then available for further stimulated exocytosis. The retrieval of membrane receptors from the plasma membrane into the endosome system also occurs via clathrincoated vesicles. However, recycling of receptors does not always occur through a single intracellular pool. Receptors can be rea constitutive recycling turned to the plasma membrane in process or enter endosome vesicles that undergo a maturation process t o produce a more specialized compartment in which receptors are sorted from other vesicle proteins before being returned to the surface (12-16). We present a mathematical analysis of membrane protein trafficking through multiple pools and show how this type of processing can account for observations made on the stimulation of glucose transporter subcellular trafficking by insulin. The analysis identifies kinetic differences that would be expected to occur between a simple recycling system in which only a single intracellular pool of endosomes is involved and the more complex schemes applicable to retrieval of membrane proteins into secretory vesicles. In 1980, Suzuki and Kono (17) and Cushman and Wardzala (18) reported that glucose transporters in nonstimulated rat adipose cells are located in a large intracellularpool and canbe recruited to the plasma membrane inresponse to insulin. Immunocytochemical studies carried out by Slot et al. (19,20) using brown adipose tissue localize glucose transporters in 11 separate cellular locations. Clearly, the kinetic consequences of Two membrane trafficking processes that are the focus of an insulin-stimulated redistribution of glucose transporters much current interest in cell biology are secretory processes among these locations will be complex. On the other hand, a involving regulated exocytosis and the retrieval of membrane simple analysis based on the assumption of only one intracelproteins by endocytosis. Many important concepts have lular pool, while being analyticallyof use, may oversimplify the emerged in both these areas and new proteins are being dis- physiological situation. covered which directly participate in these subcellular traffickRecently, photoaffinity labeling reagents have become availing mechanisms (1-4). Cain et al. (5)have recently made the able for studying glucose transporter subcellular trafficking important discovery that theV-SNAREprotein, synaptobrevin, (21-27). In ratadipose cells, we have tracer-tagged the GLUT4 involved in subcellular trafficking of synaptic vesicles in brain glucose transporter isoform with the impermeant bismannose is also associated with glucose transporter-containing vesicles photolabel ATB-BMPA1 in fully insulin-stimulated cells and in adipose cells. Laurie et al. (6) and Thoidis et al. ( 7 ) have have then followed the redistribution of these labeled glucose found SCAMPS (secretory carrier membraneproteins) in these transporters between the plasma membrane and thelow denglucose transporter-containing vesicles. These findings suggest sity microsomes (23, 24). Jhun et al. (25) have used animpera common vesicle processing mechanism for glucose transport- meable bisglucose photolabel B3GL to compare GLUT4 trafers and secretory systems. In the secretory systems found in ficking in rat adipose cells in the basal and insulin-stimulated chromaffin cells (8),pancreatic acinar cells (9), mastcells (101, states. Inaddition, we have followed the subcellular trafficking and nerve synapses (ll),vesicle membrane proteins areexocy- of both GLUT4 and GLUT1 in the basal and insulin-stimulated states in the insulin responsive adipocyte cell line, 3T3-L1(26, * The costs of publication of this article were defrayed in part by the 27). These kinetic data, together with the GLUT4 subcellular localization data described by Slot et al. (191, provide conpayment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section1734solely to straints on the modeling of the GLUT4 trafficking process and indicate this fact. .$ To whom correspondence should be addressed: Dept. of BiochemisThe abbreviation used is: ATB-BMPA, 2-N-4-(1-azi-2,2,2-trifluorotry, The University of Bath, Bath BA2 7AY, UK. Tel.: 44-225-826874; ethyl)benzoyl-l,3-bis~~-mannos-4-yloxy~-2-propylamine. Fax: 44-225-826449.

17516

17517

Glucose Dansporter Subcellular Daficking 5-Pool Model

2-Pool Model

ken

( TP )

kex

Q

Model

ek

Tee

Tee’ Q

‘Q

4a-Pool

Tpc

4b-Pool Model ken

\

1

kex

Tp -Tpo hen

(

ken

[

kex

FIG.2. A multiple pool membrane protein recycling model. In this model, two occluded plasma membrane pools are included occurFIG.1. Membrane protein recycling models. In the2-pool model, ring both before (T,J and after (T ) the fully functional plasma membrane form of the protein (T ).Two%racellular pools are also included, the fully functional plasma membrane protein (T,) is in equilibrium one associated with the earfy endosomes (Tee)and the other associated with only one intracellular pool (Tee).In the 3-pool model, two distinct intracellular pools are designated, the early endosome pool (Tee) and the with a tubulovesicular system (TJ tubulovesicular compartment (TtJ In the4-pool models, occluded forms are added to the plasma membrane occurring either before (T,,,, the la-pool model) or after (Tw, the 4b-pool model) the fully functional exponential equation. plasma membrane pool. t y , = In 2/(k, + ken) (Eq. 3)

lead us to suggest that the subcellular trafficking can only be adequately described by a minimum of four intermediate pools, with two localized insidethe cell and two localized to the plasma membrane. The properties of a 5-pool model with an additional plasma membrane pool occurring as an intermediate in endocytosis is also examined.

Quon (29) has analyzed this 2-pool model. His equations suggest that consideration must be given to the influence of nonlabeled protein on the trafficking of tracer-tagged protein. However, this is unnecessary, since such interactionsonly require consideration whenthe trafficking pathway contains saturable steps. Equations1-3 are applicable to the movements of both labeled and unlabeled membrane protein in this simple 2-pool model. EXPERIMENTAL PROCEDURES The 3-POOl Model-In this consecutive intracellularpool model (Fig. l), the total cellularpool of membrane proteins is distributed between Development of Models a plasma membrane form (T,) and two intracellular pools, the early A simplerecycling system involvingjust one plasma membranepool endosomes (Tee)and the tubulovesicular system (TJ; the latter is a and one intracellular pool (a 2-pool model) is initially compared with a specialized “secretory” compartment. The rate constants in thismodel more complicated system involving two intracellular pools (a %pool are k, for exocytosis from the secretory compartment,k,, for endocytomodel). Later, we develop a series of extensions of the latter model by sis from the plasma membrane, and k, for sequestratiodsortlng from considering the possibility that the plasma membrane contains octhe endosomes to the secretory compartment. The fractional concentracluded pools as well as a fully functionalpool. These occluded pools are of King and assumed to be inactive in catalysis and may be intermediates in exo- tions of the intermediates are calculated using the approach cytosis or endocytosis (the4-pOOl models) or both (a 5-pool model). We Altman (301. These fractional concentrations, together with the differentialequationsdeterminingthechangesintheseintermediates, show that the main features which distinguish the consecutive intracellular pool recycling schemes from a model with one intracellular pool are shown in Table I. Only two differential equations are necessary and numerical integration is required to predict changes in the interare exemplified in the 3-pool model but that the occluded pool models (the 4-pool and B-pool models) can account for the apparent presence in mediates. The 4-Pool Model-In the 4a-pool consecutive intracellular pool rethe plasma membrane of inactive proteins. In the 3-, 4-, and 5-pool cycling model, the total cellular pool of membrane proteins is distribmodels, numerical integration of the differential equations describing are active (T,) or are the models is required. For this we have used a numerical integration uted between plasma membrane forms which software package called ISIM (Simulation Science, Manchester, United occluded from participating in function(TJ and the two intracellular Kingdom) employing a RungeKutta numerical integration algorithm. pools (Teeand TtJ. In this model, the process of fusion and opening of Figs. 1 and 2 illustrate the intermediates and rate constants consid- occluded vesicles (T,) is determined by the rate constant k,. Three differential equations are required for the 4a-pool model (Fig. 1, Table ered in thesemodels as follows. (e.g. glucose transport activity) The 2-Pool Model-Here the plasma membrane form (T,) and thelow I). The changes in the functional activity density microsome form present in the endosomes (Tee)are intercon- are assumed to be associated with changes in the level of Tp, whereas verted by just two rate constants, one determining the ofrate exocytosis the total plasma membrane pool of proteins, as detected experimentally (k,) and one determining the rate of endocytosis (ken) (28). Only a single by Western blotting, will reflect the combined levels of the T, and T,, differential equation is required in describing the redistributionprocess pools. A variation of this model, the 4b-pool model, is also considered in because the increase inT, is associated with a corresponding decrease which an occluded pool (T,) occurs as an intermediate in endocytosis. in Tee, The formation of this intermediate isgiven by the rate constant kc. The 8-Pool Model-In this model, the kinetic consequences ofocdT dtP = k, . (1 - TP) - ken.T, (Eq. 1) cluded pools occurring both before (TpJ and after (TpJ the fully functional form T, are examined. Four differential equations are required for this model (Table I). We assume that impermeantphotoprobes (e.g. where T, is the plasma membrane pool expressed as a fraction of the total cellularpool. This can be integrated give to the following equation. ATB-BMPA photolabel) combine mainly with the T, pool, but also label a precursor or partially occluded pool. The partitioning of such photoprobes between fully occluded proteins and partlyoccluded proteins is suggested to be a rapid equilibrium process. A fractional equilibrium constant K, then describes the extent of interaction of photoprobes with Tpo.This assumption avoids the separate analysis of a model in which a This equation shows that thet , of recycling in thecontinuous presence separate intermediate exists between the fully occluded and the fully of insulin and thet, values for the transitionsbetween the basal state active pools. The intermediate is still assumed to be present but is and the insulin-stimulated state are all determined by the following accounted for by simply multiplyingK, by T,.

17518

Glucose Dansporter Subcellular Dafticking

Constraints on the Models In applying these models to the recycling of glucose transporters in isolated rat adipose cells and 3T3-Ll adipocytes in culture, the following constraints are imposed. 1)A constant amount of glucose transporter in the cell is assumed. This assumption is supported by observations on the rate of degradation of photolabeled GLUT4 whichoccurs with at, of -36 h (31),much slower than any of the observed rates of recycling ( t , = 2-12 min) (23-27). Under steady-state conditions, in either the basal or insulinstimulated state, the constant amount of GLUT4 is assumed to be at equilibrium among all the subcellular pools. Evidence that this equilibration takes place has been obtained in the rat adipose cell system where the photolabel ATB-BMPA has been used to show that plasmamembrane-tagged glucose transporters fully redistribute to an equilibrium level which is the same as thatobtained by Western blotting, i.e. -55% are located inside the cell in the insulin-stimulated state. The steady-state assumption implies that each glucose transporter pool remains constant at the steady-state; the efflux from that pool must, therefore, be equal to the influx into it. 2) Constraints for the sizes of the glucose transporter pools in the basal and insulin-stimulated states aresummarized in Table I1 according to the data available in the literature. The studies appear compatible with each other for the most part and aretherefore used to estimate ranges of acceptable values for the pool sizes. In the Western blotting study examined, the size of the plasma membrane pool (the sum of the total in all its associated pools) in the basal state is 6.4% of the total GLUT4. Becauseof the inevitable contamination of plasma membrane fractions with low density microsomal protein, this isprobably an overestimate of the true value. In the rat adipose cell system, insulin has been shown to increase glucose transport activity by -30-fold or greater (22, 24, 32). The increase in GLUT4 at the cell surface available for photolabeling is only -20-fold (22, 24). These data and immunocytochemical studies suggest that in the basal state, the fraction of plasma membrane GLUT4 in an inactive pool is larger than in the insulinstimulated state. According to the immunocytochemical study, 1.2%of the total GLUT4pool in the basal state is in uncoatedcoated pits/ vesicles in the plasma membrane. For the insulin-stimulated state, the size of the total plasma membrane pool is -55% of the total and the immunocytochemicallydetermined uncoatedcoated pitlvesicle plasma membrane pool is 4.8%. The estimated size of the intracellular pool as determined by Western blotting is -95% in the basal state and 55% in the insulin-stimulated state. According to the immunocytochemical study by Slot et al. (19), 90% of the total GLUT4 is associated with tubulovesicular elements, whereas 6.4% is associated with early endosomes in thebasal state. In the insulin-stimulated state, the tubulovesicular and early endosome pools account for 42 and 14%, respectively,of total GLUT4. 3) Table I11 summarizes several different experimental protocols which have given t, values for the translocation of GLUT4 at 37 "C. The t , values measured for similar experimental protocols are somewhat variable between different studies and therefore an average t , for these experiments is used as thebasis for modeling.In simulating the t , values for transition from onesteady-state to another, it is assumed that the rateconstants are instantaneously changed. As well as evaluating models on the basis of their ability to closely approximate these different t , values, the ratios of the t , values observed in different experimental protocols placeconstraints on the models and exclude some simple models. These ratios are fairly consistent between the collected studies summarized. For example, the equilibration of tracer-tagged GLUT4 in the insulin-stimulated steady-state and the t , for stimulation of the appearance of GLUT4 in the plasma membrane as measured by Western blotting and by the reappearance of internalized tracer-tagged GLUT4 have been determined. Contrary to the expected result from a simple model (see below), these t,, values are unequal. Experimentally this ratio has been observedto be -2-3. Similarly, a comparison of the rateof stimulation of glucose transport activity with the rateof stimulation of the appearance of glucose transporters as detected by photolabeling and Western blotting provides another constraint. This ratio is -2.Althoughsome of the observations on glucose transporter translocation, when viewedseparately, do not place severe constraints on the possible models considered,these constraints collectively appear to rule out simple models. Choice of Rate Constants The one situation in which the equations for the 2-, 3-, and 4a-pool models reduce to similar equations is that in which the insulin effect on GLUT4 distribution is reversed and exocytosis is negligible. Here the equations for loss of the T, form approximate to the following equation.

(Eq. 4) Since the t , for this internalization of GLUT4 is -8 min (Table III), we can assume that ken is approximately 0.06-0.09 rnin". Under the steady-state conditions in which the net flux through each intermediate in thecycle is the same, the concentrations of the intermediates (Table 11)can be used to give approximate values for the additional rate constants. For example, in the 3-poolmodel, k and k,, can be approxi9 mated respectively from the following equations.

and

However, the utility of these equations relies too heavily on the relative values of T,, Tee,and T,. These values have been determined in brown adipose tissue and therefore may not be typical of white adipose cells where most of the kinetic data on GLUT4 t r a c k i n g have been obtained. Consequently, refinement of the choiceof rate constants requires simulations of the time course experiments. RESULTS

The %Pool Model-Insulin could influence the level of cell surface glucose transporters by either increasing exocytosis, decreasing endocytosis, or both. Although presently available data onGLUT4 trafficking make it difficult to rule out the possibility of insulin effects on both exocytosis and endocytosis, simple consideration of the 2-pool model (Fig. 1, Table I) suggests that inhibition of endocytosis alone is unlikely to account for insulin's action. Because the number of glucose transporters in the plasma membrane in the basal state is small, insulin would have t o induce a largedecrease in theendocytic process in order to give the necessary increase in steady-state glucose transporters. However, we knowfrom Equation 3 that the stimulation t,, for the 2-pool modelis dependent on the sum of the endocytosis (ken)and exocytosis (hex)rate constants. Fig. 3 compares the expected stimulation time courses if the endocytosis rate constant is markedly reduced or the exocytosis rate constant is markedly increased. It can be seen that theeffect of a 40-fold decrease in ken has a much slower effect than a proportional increase in &, although the same steady-state plasma membrane level of glucose transporters is achieved in both conditions (a 20-fold stimulation above the basal level). The t , of stimulation is 138 min in the first case and 3.5 min in the second. An additional characteristic of this simple model is that the tu, for stimulation and the t , for equilibration are the same (Equation 3). However, the ratio of these values observed experimentally is on the order of 2-fold (Table 111). The .%pool Model-This model can simulate many of the t , values observed experimentally and also the discrepancy between the t , values for the insulin stimulation of GLUT4 appearance and the equilibration of tracer-tagged GLUT4 in the presence of insulin. In thismodel (Fig. 1,Table I), the involvement of a second intracellular compartment (e.g. the tubulovesicular compartment) allows a rapid initial stimulation of translocation to the plasma membrane without endocytosis contributing significantly to thestimulation t,, (as itdoes with only one intracellular pool, Equations 1 and 2).With two consecutive intracellular pools, the recycling in the continuous presence of insulin would depend on endosome processingand recycling which would markedly slow the turnover t,, that maintains the distribution of glucose transporters at steady state. Several different combinations of rate constants were examined so that we could determine those which best accounted for

17519

Glucose Dansporter Subcellular Daficking TABLEI Equations used for modeling Model equations

Differential

Fractional pool size

T,' = kex41- T,) - k,;T,

T, = k,J(k,, + hex) Tee= keml(ken+ k,) T, = k,;k,dden Tee= k,;k,Jden T,, = k,;k,,,Iden Den = sum of all numerator terms T, = k,.k, .k,,,lden T, = k;k,;lkJden Tee= k,;k,;kJden T, = k,;k,;kJden Den = sum of all numerator terms T, = k,;k,,~k,,Iden T = k;k,;k$den (e = k,;k;k,Jden T,, = k,;k,;kJden Den = sum of all numerator terms T,, = k,;k, .k,;kJden T, = k;k;~,;k,,,Iden T e = k;k,;k;k,Jden = k,;k;k;k,Jden T, = k,;k,;k;kJden Den = sum of all numerator terms

T,' = kex.(l- T - T,) - k,;T, Tee'= k,;T, - $;T, T,,,' = k,.(l - T, - T, - Tee)- k,.T, T, = k;T,, - k,;T Tee'= k,;T, - k,,.$, T,' = kex.(l- T,, - T, - Tee)- k;T, Tpc'= k;T - k,;T e T,' = ken.lfpe- ksq.!hee

- Westernblotting W

1.o

z

2rn

0.4

I

H

--.........

r

Tracer-taggedGLUT4 (+ insulin) Tracer-tagged GLUT4 (- insulin)

\ \

Insulin stimulation of exocytosis

0.8

-\ 1

3

0.6

0.4 0

20

40

60

80

TIME h i d

FIG.3. Simulation of the effects of insulin on GLUT4 subcellular distribution using the 2-pool membrane protein recycling model. In fully stimulated cells, halfof the glucose transporters are at the cell surface. When insulin is removed, the level of glucose transporters at thecell surface declines to the basal level (1120 of the insulin level) at a rate determined by the endocytosis rate constant (ken = 0.1 rnin") and the exocytosis rate constant (kex= 0.0025 min") in thebasal state. At 45 min, the effect of an insulin-dependent increase in exocytosis (k, = 0.1 rnin") or decrease in endocytosis (ken = 0.0025 min") is simulated. The former givesa stimulation with a tl,z of 3.5 min, whereas the latter gives the same final level of stimulation but with a t, of 138 min.

0.2

0

0

10

20 30 40

50

60 70 80 90

TIME (min) FIG.4. Simulation of glucose transporter subcellular trafficking using the %pool membraneprotein recycling model. A nonreversible cycling between the three pools is assumed with ken = 0.06 rnin", k,, = 0.15 rnin", and k, = 0.001 min" (basal) and 0.12 min" (insulin). The fractional steady-state pool sizes of T,, Tee, and T,, in the basal and insulin-stimulated states are0.016,0.006, and 0.97, and 0.53, 0.21, and 0.26, respectively. Fromthese parameters, a t,, of 4.7 min is obtained for the equilibration of tracer-tagged GLUT4 in the continuous presence of insulin, and t , values of 11 and 2.5 min are obtained, respectively, for the reversal of insulin stimulation of GLUT4 translocation and for the stimulation itself by insulin of GLUT4 translocation as assessed by either Western blotting or photolabeling. The subcellular distributions of immunodetectable GLUT4 (solidline) and tracertagged GLUT4 either when insulin is removed and readded (dotted line) or in the continuous presence of insulin (broken line) are shown. The tracer-tagged GLUT4 that is internalized following insulin removal is returned to the cell surface following insulin restimulation (60 min) with the same tl,z as that observed for immunodetectable GLUT4 and reaches the same steady-state subcellular distribution level as observed with tracer-tagged GLUT4 that recycles in the continuous presence of insulin.

the observed t, values within the constraints listed under "Experimental Procedures." Using the set of parameters for the simulation shownin Fig. 4 (optimized with a starting valuefor k,, of 0.06 rnin") produces a ratio of the t,,, for GLUT4 recycling in the continuouspresence of insulin to thet , for stimulation of GLUT4 translocation in response to insulinof 1.9 (4.7 m i d 2.5 rnin). This value is close to the ratio of 2.7 obtained by averaging the literature values shown Table in I11 (6.0 mid2.2 min). The inclusion of reversible reactiondprocesses confers only minor improvements in the fitting, and into simplify order the modeling, is not considered in detail here. A feature of all of the more complex models examined, including the 4- and 5-pool models, is their production of overshoots in the stimulation time courses such as those illustrated endosome comin Fig. 4 for the 3-pool model. These overshoots are due to a ment andbefore they equilibrate with the early transient increase inglucose transporters in the plasma mem- partment. These overshoots are lesspronounced when the rate introduced into the brane as they rapidly exit from the tubulovesicular compart- constant k,, is highor if reversible steps are

Glucose llansporter Subcellular n a f i c k i n g

17520

0.6

0.4

Western blotting Glucose transport activity

0

5

10

15

20

TIME (mid

0.2

Wastern blotting Glucose transport activity

Q I

0

5

10

15

20

TIME (rnin)

FIG.5. Simulation of glucose transporter subcellular trafficking using the 4-pool membrane protein recycling models. In A (the 4a-pool model),k,, and k,, are 0.095and 0.15 min", respectively.K, and k, are increased by insulin from 0.001to 0.11min" and from 0.13 to 0.35 rnin", respectively. The fractional steady-state pool sizes of T,, Tp, T,, and T, in the basal and insulin-stimulated states are0.010, 0.008,0.007, and 0.975 and 0.361,0.098,0.229, and 0.311,respectively. The t , values to reach the steady-state stimulation level are 2.5min (Western blotting) and 4.7 min (glucosetransport activity). These rate constants produce a t , for recycling in the continuous presence of insulin of 4.0 min and for reversal of insulin stimulation of 10 min (glucosetransport activity), 8 min (tracer-tagged GLUT4 equilibration), and 9 min (Western blotting). In B (the 4b-pool model),ken, kc, and k, are 0.15,0.08, and 0.15rnin", respectively.k,, is increased by insulin from 0.002 to 0.08min". The fractional steady-state pool sizes of T,, T,,, Tee,and T,, in the basal and insulin-stimulated states are 0.023, 0.013, and 0.013, 0.95and 0.326,0.174, 0.174, and 0.326,respectively. The tu, values to reach the steady-state stimulation level of GLUT4 as detected by glucose transport activity and Western blotting are 3.0and 5.0min, respectively. These rate constants produce a t , for recyclingin the continuous presence of insulin of 9.5min and t , values for reversal of insulin stimulation of 9 min (glucosetransport activity), 15 min (tracer-tagged GLUT4 equilibration), and 11.5min (Western blotting). In A, the increase in glucose transport activity shows a lag phase and reaches a maximum after the Western blotting signal reaches a maximum. In B, the increase in glucose transport activity reaches a maximum before the Western blotting signal. In A and B overshoots of the steady-state equilibrium level of GLUT4 occur in the stimulation time courses.

exocytosis step (kJ. Although the %pool model can account for many of the observations on glucosetransporter recycling, it only involves one plasma membrane poolof glucose transporters. This model therefore cannot account for the differences observed between the t,,2 values for stimulation of glucose transport activity and the appearance of GLUT4 in theplasma membrane as detected by Western blotting. The 4-Pool Model-Occluded plasma membrane glucose transporters may occur before (T,) or after (TPJ the pool in which the glucose transporters are fully participating in glucose transport (TJ. T,, represents apool in which vesicleshave docked with the plasma membrane but have not yet fused with it, whereas the T,, pool represents the vesicle internalization assembly state (e.g. clathrin-coated pit) in the plasma membrane. These two 4-pool models are called the 4a-pool and the 4b-pool models, respectively (Fig. 1, Table I). Fig. 5 shows that the4a-pool modelis much more likelythan the 4b-pool model to account for the separation during stimulation of the appearance of GLUT4 in the plasma membrane and its participation in glucose transport. In this figure, the calculated increases in theWestern blotting signal and glucose transport activity are normalized to the same final level. When this is done, it is seen that the rise in the Western blotting signal is complete before the increase in glucose transport activity reaches a maximum in the 4a-pool model, whereas the Western blotting signal continues to rise after the increase in glucose transport activity reaches a maximum in the 4b-pool model. The latter effect occurs because with insulin stimulation, the occluded form only fullyequilibrates when the pool of glucose transporters participating in glucose transport is large and this only occurs toward the end of the stimulation time course. Although none of the rate constants other than k,, require modification to account for most of insulin's effect on translo-

cation, we note that if insulin also modifies the rate constant determining the rate of fusion of dockedvesicles with the plasma membrane, k,, then the fraction of the plasma membrane glucose transporters in occluded vesicles can be reduced by insulin action. That thisfraction is greater in the basal state than theinsulin-treated state is still a matterof some dispute because of the experimental difficulty in estimating the proportion of inactive or occludedglucose transporters when the plasma membrane fraction is generally highly contaminated by glucose transporters present in thelow density microsome pool. However, the available immunocytochemical studies (Ref. 19 and cited in Table 11) suggest that insulin may reduce the proportion of plasma membrane GLUT4 present in occluded/ clathrin-coateduncoated vesicles from 55 to 11% of the total plasma membrane pool in the basal and insulin-stimulated states, respectively. Although the 4a-pool model givesan adequate fit to most of the datasummarized in Tables I1 and 111, subcellular trafficking mustoccur at least in part through clathrin-coated vesicles, because GLUT4 association with clathrin has been observed experimentally (19,34). In order to retain the featuresof both an occluded precursor step and a vesicle internalization assembly step inthe trafficking process, we examined the properties of a 5-pool model (Fig. 2, Table I). The 5-Pool Model-As expected from consideration of the simpler 3- and 4-pool models,the 5-pool model accounts for the steady-state pool sizes and kinetic parameters of GLUT4 trafficking that have been reported to date and summarized in Tables I1 and 111. A set of parameters has been chosen that retains many of the features suggested to be important through consideration of the simpler models. Simulations of experiments inwhich basal cells are stimulated by insulin reveal that if the rise in the Western blotting signal reaches a maximum before the rise in glucose transport activity reaches a maxiT, mum, then the T, pool has to be at least as abundant as the

Glucose Subcellular Transporter

Daficking

17521

TABLE I1 Experimental data used as constraints in modeling pool sizes Insulin

Basal

% total cell pool

48

Plasma membrane pools (PM) Total PM 45 Western blotting Cell surface photolabeling PM surface 39 PM noncoated invaginations + coated pits and vesicles

4.8

6.4 2.5 1 1.2

% total

55

93.6 97.5 6.4 91.4

% total

Ref.

PM pool

50

% total cell pool

Intracellular, low density microsome pools (LDM) Total LDM Western blotting 50 Cell surface photolabeling 14.2 Early endosome vesicles 42 Trans-Golgi reticulum and tubulovesicular structures

Insulin

Basal

89 11

24 24 19 19

75

24 24 19 19

LDM pool

55 25

6.6 93.4

TABLE I11 t , , , values for translocationof GLUT4 at 37 "C Reported

t , values

Ref.

min

min f S.E.

Basal steady-state recycling t , values Adipose cells 3T3-Ll cells 10-37 "C perturbation of glucose transport activity in basal state Adipose cells ATB-BMPA-tagged GLUT4 recycling t, following insulin removal Adipose cells ATB-BMPA labeling of GLUT4 remaining after insulin removal Adipose cells 3T3-Ll cells Continuous insulin steady-state recycling t , values Adipose cells Adipose cells 3T3-Ll cells Insulin stimulation of glucose transport activity Adipose cells Adipose cells 3T3-Ll cells Insulin stimulation of translocation of cytochalasin B binding sites Adipose cells Insulin stimulation of GLUT4 appearance determined by photolabeling Adipose cells 3T3-Ll cells Insulin restimulation of GLUT4 appearance from internalized photolabel Adipose cells Insulin stimulation of GLUT4 appearance determined by Western blotting Adipose cells

1.7 f 0.4 5.6 f 0.3

Mean value

for simulation

25 27

10.8 9.4 f 0.8

24

12.3 3.0 6.8 f 1.7

23 26

7.8

10.6 f 1.5 3.2 f 0.5 4.2 f 0.3

24 25 27

6.0

3.2 f 0.2 4.0 -3.8

23 33

2.5

2.2

b

33

2.2 & 0.2 -2.4

23

2.7 2 0.3

24

-1.5

3.7

b

24

A. E. Clark, G. D. Holman, L. Olsson, S. W. Cushman, and J. Stagsted, unpublished results (mean of two experiments). * J. Yang, and G. D. Holman, unpublished results (mean of two experiments).

pool. This constraint dictates in turn that the coated vesicle assembly be governed by a slow step; if this rateconstant, kc, is fast compared with the endocytosis rate constant, ken, then the T,, pool accumulates at theexpense of T, and Tpo.The insulin stimulatory steps occur mainly at k,, but with a smaller -3-fold effect on the fusion of docked vesicles with the plasma membrane, the k , step. By setting the proportion ofT,, that combines with the ATB-BMPAphotolabelat 0.3, a separationin the time courses for the increase in glucose transport activity, GLUT4 accessibilityto photolabel, and totalplasma membrane GLUT4 as detectable by Western blotting is adequately simulated as demonstrated in Fig. 6. DISCUSSION

Pool 'lkaffcking Models-Analysis of the simple 2-pool modelclearly shows that measuring internalization of surface-labeled proteins is not simply a measure of the endocytosis rate constant. Rather, the t , for the internalization process is highly dependent on the rate of recycling. If recycling is extensive as is thecase when Characteristics of SingleIntracellular

insulin stimulates exocytosis, then simply measuring the extent of internalization of tagged glucose transporters alone cannot measure the endocytosis rate constant; information on both the t,, and the steady-state distribution ratio is required. The combination of these parameters can then be used to either provide estimates of the endocytosis and exocytosis rate constants in a simple model or provide constraints on the more complex models considered here. Our analysis has thus used both t,, values and steady-statelevels to define an appropriate model of the glucose transporter subcellular trafficking process. The simulations obtained for a 2-poolmodel clearly show that changes in the ratefor GLUT4 exocytosis are more likely to be the main effectors of insulin-stimulated glucose transport than changes in the rate for GLUT4 endocytosis, simply because changes at the exocytotic step can affect translocation faster than changes at the endocytic step. However, much recent debate has centered around the issue of whether major effects of insulin can be attributed to inhibition of endocytosis (25, 35). The GLUT4 internalization study described by Czech

nafficking Subcellular Dansporter Glucose

17522

- -

”

.I.....

0.0

Glucose transport activity Western blottlng Tracer-tagged GLUT4 (- insulin) Tracer-tagged GLUT4 (+ insulin)

.../

-

........ ”_

Western blotting blotting Western ATE-BMPA ATE-BMPA photolabeling photolabeling Glucose transport actiiity

I

0

10

20

30

40

TIME (min)

0

5

10

15

TIME (min)

FIG.6. Simulation of glucose transporter subcellular trafficking usingthe &pool membrane protein recycling model. The rate constants kc, k,,, and k,, are 0.095, 0.35, and 0.15 min”, respectively. Ke, and k , are increased by insulin from 0.001 t o 0.11 min” and from 0.13 to 0.35 rnin”, respectively. The fractional steady-state pool sizes of T , T,,T,,, Tee, and T, in the basal and insulin-stimulated states are 0.010, 0.0075,0.028, 0.0065, and 0.948 and0.329,0.089,0.089,0.208, and 0.585, respectively.In A, these rate constants produce a t,, for recyclingin the continuous presence of insulin of 6.5 min and t,, values for reversal of insulin stimulation of 10 min (glucose transport activity), 10.5 min (tracer-tagged GLUT4 equilibration),and 10 min (Western blotting).In B, the t,,* values to reach the steady-state stimulation level are 2.3 min (Western blotting),3.2 min (accessibility toATB-BMPAphotolabel),and 4.5 min (glucose transport activity). The stimulation of glucose transport activity shows a lag phase and the GLUT4 in the plasma membrane overshoots the steady-state equilibrium level.

and Buxton (35) using trypsin cleavage of plasma membrane GLUT4 cannot be clearly taken as indicatingan inhibition of endocytosis by insulin, because the rate of recycling of GLUT4 i n t h econtinuous presenceof insulin (24-27) needs to be taken into account. The study of Jhun etal. (25) reportsa much faster is consistt,, for GLUT4 translocation in the basal state than ent with other studies on translocation in the basal (Table state 111). The experimental design in some of these studies, suchas those measuring changesin GLUT4 distribution following reversal of insulin treatment, could be criticized on the grounds that stimulation may still be ongoing during the treatments to remove insulin (36). In addition, the t,,2 for reversal may be slower than the t, for recycling in the basal state if, in the reversal experiment but not in the true basal state, the internalization process is saturated (possibly due to the limited availability of clathridadaptin molecules). However, the consistency of the reversalt,, values determined in severaldifferent procedures for removing insulin, the rapid decline in the signalingtoIRSl/phosphoinositol3-kinase intheseexperiments? and the similarity of these reversal t,,2 values to that obtained in experiments in which rat adipose cells (inthe complete absence of insulin) are subjected to a temperature transition from 10 to 37 “C (Table 111)’ suggest that internalization is not markedlyslowed by insulin andthat the resultsof J h u n et al. (25) are anomalous. Nishimura eta2. (37)have recentlyproposed a n occluded pool model for glucose transportertraffickingcomprisingthree plasma membrane pools and one intracellular pool of glucose transporters. In the plasma membrane, GLUT4is proposed to be distributed betweena pool where theseglucose transporters fully participate in transport andoccluded pools occurring before and after this active state. Their model is based in part on the observation that following insulin removal, glucose transport activity appears t o fall more rapidlythan the decrease in GLUT4 in the plasma membrane fraction detected by Western blotting. However, these results disagree with those of Satoh et al. (24) who observed that therates of loss of glucose transport J. Stagsted, M. J. Zarnowski, L. Olsson, G. D. Holman, and S. W. Cushman, unpublished data.

A. E. Clark et al., unpublished data.

activity and of immunodetectable and tracer-tagged GLUT4 occur with the same t,, values (Table 111). The reason for the discrepancy remains to beclarified. Nevertheless, simulations of this model suggest that a large proportion of GLUT4 is unlikely t o residein occluded endosomeintermediates.This model illustrates a fundamental problem of all models with just a single intracellular pool. Values for rate constants that are chosen t o fit the slow rate of glucose transporter internalization also predict that the rate of insulin stimulationof exocytosis is slow. Using the parameter valuesof Nishimura et al. (37), the predicted tUzfor the increase in glucose transport activity following insulin stimulation (-4.5 min) fits the experimentally observed results reasonably well (Table III), whereas the predicted t,, for the increase in plasma membrane GLUT4 as detected by Western blotting (-7 min) greatly contrasts to the experimentally observed time courses whichoccur with t , values of -2.5 min. Further development of this occluded pool model, with the inclusion of consecutive intracellular pools a s in the 5-pool model (see “Results”), greatly improves the fit to these experimental data. Characteristics of the Consecutive Intracellular Pool Daficking Models-Despite theusefulcluesthat a simple 2-pool model of GLUT4 translocation can give, it cannot describe the GLUT4 subcellular trafficking accurately, because it predicts that the t , values of recycling in the insulin-stimulated state and for the transition between the basal and insulin steady states should be the same (Equation 3). The most important characteristic of the multiple pool models is that they more fully account for t h e slow steady-state recycling of cell surface GLUT4 in the presenceof insulin, but fast initial insulin stimulation of the appearance of GLUT4 on the cell surface. This is because t h e tubulovesicular compartment acts as a reservoir which can rapidly empty to the plasma membrane during the initial transition but fill relatively slowly through the endosome recycling system. In our analysis of the %pool model we also considered t h e possibility that GLUT4, which is present the in early endosome pool, can be returned to the plasma membrane directly in a constitutive exocytosis process and/or move to the tubulovesicular pool for rapid exocytosis in response to acute insulin

Glucose Dansporter Subcellular n a f i c k i n g treatment. Such a model would be consistent with the known effects of insulin to stimulate both fluid phase endocytosis and exocytosis (38)but isdifficult to match with the early endosome pool size of GLUT4 (both in the absence and presence of insulin) asdetermined by immunocytochemistry(20).The inclusion of this constitutive exocytosis may be more important for simulating the trafflcking of GLUTl where exocytosis in the basal state ismore rapid than for GLUT4(27). It would beof interest to determine if the early endosome pool size of GLUTl is greater than GLUT4. We have also considered the possibility that a reversal process may occur in exocytosis. The recycling between the intracellular tubulovesicular pool of GLUT4 and the occluded precursor form in the plasma membrane is analogous to the recycling of synaptic vesicles which are thought to visit the plasma membrane many times before fusing with it (39). The inclusion of a reverse reactiodprocess in exocytosis allows a faster time course for stimulation in which k,, is increased to a greater extent than is possible without this reverse process. This occurs because the cycle between T, and T, allows a fast flux without building up a large steady-statelevel of the intermediate, T,. Further refinement of the present model may be required if it can be experimentally demonstrated that significant reverse reactions in GLUT4 exocytosis occur. An associated feature of the consecutive intracellular pool trafficking models is that they have the possibility of producing overshoots in the initial stimulation. Although glucose transport stimulation is considered to be analogous t o a secretory process and therefore may be expected t o show transient pulses of activity in response to the insulin stimulus, no overshoots have ever been reported in the insulin stimulation of glucose transport. This suggests that thepotential to produce an overshoot is dampened by a reversible step in the stimulation. Alternatively, the detection of the relatively small overshoots predicted by the models without reversible steps (Figs. 4-6) may be limited by experimental resolution when using only a limited number of time intervals in a time course experiment. However, reports of the stimulation of transferrin receptor appearance by insulin or epidermal growth factor have suggested that a transient overshoot is produced (28, 40). A transient overshoot also occurs in the insulin stimulation of the translocation of a,-macroglobulin receptors (41). Another property of the multiple pool recycling schemes described here is that they can account for the presence in the membrane of inactive glucose transporters which are occluded from participating in the glucose transport process. Similarly, occluded pools have been postulated to occur in the epidermal growth factor receptor recycling pathways (42). These occluded vesicles may account for the effects of adenosine and isoproterenol on insulin-stimulated glucose transport activity. In rat adipose cells, adenosine has been observed to produce an increase and isoproterenol a decrease in glucose transport activity and the accessibility of GLUT4 to the impermeable photolabel ATB-BMPA in fully insulin-stimulated cells, without a concurrent increase or decrease, respectively, in immunodetectable GLUT4 in theisolated plasma membrane fraction (43,44). However, it remains t o be determined whether the occluded state that appears to be modulated by adenosine and isoproterenol treatment is a precursor to the fully functional glucose transporter form or an intermediate in the endocytosis route involving the formation of endocytic vesicles. Evidence that we have presented here in our comparison of the 4a- and 4b-pOol models suggests that a distinction between these alternatives will be possible by comparing the time course forstimulation of glucose transport activity with that for the increase in immunodetectable GLUT4 in the plasma membrane following insu-

17523

lin stimulation of cells initially treated with various combinations of adenosine and isoproterenol. For example, if isoproterenol increases the proportion of GLUT4 in theprecursor pool, then the lag between the appearance of immunodetectable GLUT4 and the rise in glucose transport activity should be greater than with insulin alone. Conversely, if the isoproterenol-induced occluded pool occurs as an intermediate in endocytosis, then the stimulation time course of glucose transport activity should reach a maximum before the immunodetectable GLUT4 reaches a maximum. Limitations of the Modeling-The use of the more complex models of subcellular trafficking is associated with several problems. First, as the models become more complex and incorporate more rate constants, then more parameters must be considered as possible candidate sites of hormone action. In addition, the more complex the models become, the more difficulty is encountered in applying conventional least squares data fitting routines. Only the equation for the 2-pool model has an implicit integrated solution, whereas least squares fitting to the more complex models requires a fitting algorithm that simultaneously takes intoaccount the datafrom stimulation, reversal, and steady-state tracer flux experiments plus the steady-state pool sizes of intermediates and uses all these as constraints to fit the appropriate simultaneous differential equations. In view of these limitations, a major justification for consideration of the complex models is their applicability to currently available information on membrane components and compartments involved in the membrane protein recycling process. Relationships between the ConsecutiveIntracellular Pool Models and the Current Understanding of Intracellular Dafficking-A model with a single intracellular pool is oversimplified with respect t o current knowledge of subcellular trafficking, as well as being mathematically inadequate in describing the experimental findings. The translocated protein, GLUT4, has clearly been shownto be distributed between early endosome and tubulovesicular structures by Slot et al. (19) in their immunocytochemical study. Slot et al. (19) defined early endosomes containing GLUT4 as apparently empty vacuolar structures, 0.3-1 pm in diameter, which could take up extracellular albumin but did not contain cathepsin D, a lysosomal marker. Late endosomes were defined as vacuoles containing vesicular and amorphous material and were observed to be sparsely labeled with GLUT4 antibody even in the insulin-stimulated state. GLUT4 might therefore internalize via the same endosomal route followed by receptormediated endocytosis, occurring via a clathrin-coated pit! vesicle route (19, 34). The tubulovesicular structures containing GLUT4 are described by Slot et al. (19) as occurring in mitochondrion-free areas with very little apparent association with the Golgi cisternae and to be free from albumin immunostaining. These structures may therefore be distinct from the tubular multibranching structures described by Tooze and Hollingshead (16) which are thought to be involved in the latestages of receptormediated endocytosis. These latter structures are stained by fluid phase markers such as horseradish peroxidase, whereas the structures described by Slot et al. (19) do not appear t o be in equilibrium with endocytosed albumin. Although the tubulovesicular structures involved in GLUT4 recycling appear t o be distinct from the tubular endosomes involved in the recycling of receptors, the analysis of the models described here with consecutive intracellular pools may be generally applicable to membrane trafficking phenomena, including the recycling of plasma membrane receptors and also regulated secretory processes.

Glucose DansporterDafficking Subcellular

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Acknowledgments-We are grateful to Drs. Shinobu Satoh, Avril E. Clark, and Izabela J. Kozka for carrying out the GLUT4 trafficking experiments on which the modeling is based. We also thank theMedical Research Council (United Kingdom) and the Juvenile Diabetes Foundation International for grant support. REFERENCES 1. Pearse, B. M. P., and Robinson, M. S. (1990) Ann. Reu. Cell Bid. 6, 151-171 2. Sollner, T., Whiteheart, S. W., Brunner, M., Erdmusat-Brimage, H., Geromanus, S., Tempest, P., and Rothman, J. E.(1993) Nature 362,318-324 3. Bourne, H. R., Sanders, D. A., and McCormick, F. (1990)Nature 348,125-132 4. Waters, M. G., Serafini, T., and Rothman, J. E. (1991)Nature 349, 24S251 5. Cain, C.C., Trimble, W. S., and Lienhard, G. E. (1992) J . Bid. Chem. 267, 11681-11684 6. Laurie, S. M., Cain, C. C., Lienhard, G. E., and Castle, J. D. (1993) J. Bid. Chem. 268, 19110-19117 7. Thoidis, G., Kotliar, N., andPilch, P. F. (1993)J. Biol. Chem. 268, 11691-11696 8. Burgoyne, R. D., and Morgan, A. (1987) FEBS Lett. 245,122-126 9. Padfield, P. J., Balch, W. E., and Johnson, J. D. (1992) Proc. Natl. Acad. Sei. U. S.A. 89,16561660 10. Oberhauser, A. F., Monck, J. R., Balch, W. E., and Fernandez, J. M. (1992) Nature 360,27&273 11. Hess, S. D., Doroshenko, P. A,, and Augustine,G. J. (1993) Science 259, 11691172 12. Goldstein, J. L., Anderson, R.G. W., and Brown, M. S. (1979) Nature 279, 679-685 13. Major, S., Presley, P. F., and Maxfield, F. R. (199315. Cell Biol. 121,1257-1269 14. Hopkins, C. R. (1992) Bends Biochem. Sci. 17, 27-32 15. Dunn, K.W., and Maxfield, F. R. (1992) J. Cell B i d . 117, 301-310 16. lboze, J., and Hollingshead, M. (1991) J. Cell Sei. 115, 635-653 17. Suzuki, Y., and Kono, T. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2542-2545 18. Cushman, S. W., and Wardzala, L. J. (1980) J. Biol. Chem. 255,4758-4762 19. Slot, J. W., Geuze, H. J., Gigengack, S., Lienhard, G. E., and James, D. E. (1991) J. Cell Bid. 113, 123-135 20. Slot, J. W., Geuze, H. J., Gigengack, S., James, D. E., and Lienhard, G. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7815-7819 21. Clark, A. E., and Holman, G. D. (1990) Biochem. J. 269,615-622

22. Holman, G. D., Kozka, I. J., Clark,A. E., Flower, C. J., Saltis, J.,Habberfield, A. D., Simpson, I. A,, and Cushman, S. W. (1990) J. Biol. Chem.265, 18172-18179 23. Clark, A. E.,Holman, G. D., and Kozka, I. J. (1991) Biochem. J. 278,235-241 24. Satoh, S., Nishimura, H., Clark, A. E., Kozka, I. J., Vannucci, S. J., Simpson, I. A,, Quon,M. J., Cushman,S. W., and Holman, G. D. (1993)J. Biol. Chem. 268, 17820-17829 25. Jhun, B. H., Rampal, H., Liu, M., Lachaal, M., and Jung, C. Y.(1992) J. Bid. Chem. 267, 17710-17715 26. Yang, J., Clark, A. E., Harrison, R., Kozka, I. J., and Holman, G. D. (1992) Biochem. J. 281, 809-817 27. Yang, J., and Holman, G. D. (1993) J. Bid. Chem. 268,4600-4603 28. Tanner, L. I., and Lienhard, G. E. (1987) J. Bid. Chem. 262,89754980 29. Quon, M. J. (1994) Am. J. Physiol. 266, E14PE150 30. King, E. L., and Altman, C. (1956) J. Phys. Chem. 60, 1375-1378 31. Kozka, I. J., and Holman, G. D. (1993) Diabetes 42, 1159-1165 32. Simpson, I. A., and Cushman, S. W. (1986)Annu. Reu. Biochem. 55,1059-1089 33. Kamieli, E., Zarnowski, M. J., Hissin, P. J., Simpson, I. A., Salans, L. B., and Cushman, S. W. (1981) J. Biol. Chem. 266,47724777 34. Robinson, L. J., Pang,S., Harris, D. S., Heuser, J., and James,D. E. (1992) J . Cell Biol. 117, 1181-1196 35. Czech, M. P., and Buxton, J. M. (1993) J . Biol. Chem. 268, 9187-9191 36. Quon, M. J., and Campfield, L. A. (1991) J. Theor Biol. 160, 93-107 37. Nishimura, H., Zarnowski, M. J.,and Simpson, I. A. (1993)J. Bid. Chem. 268, 1924619253 38. Gibbs, E. M., Lienhard, G. E., Appleman, J. R., Lane, M. D., and Frost, S. C. (1986) J. Bid. Chem. 261,3944-3951 39. Fesce, R., Grohovaz, F., Valtorta, F., and Meldolesi, J. (1994) "Fends Cell Biol. 4, 1-4 40. Davis, R. J., Faucher, M., Racaniello, L. K, Carruthers, A., and Czech, M. P. (1987) J. B i d . Chem. 262,1312G13134 41. Descamps, O., Bilheimer, D., and Herz, J. (1993)J. Biol. Chem. 268,974-981 42. Yanai, S., Sugiyama, Y., Kim, D. C., Iga, T., Fuwa, T., and Hanano, M. (1991) Am. J. Physiol. 260, C457-C467 43. Joost, H. G., Weber, T. M., Cushman, S. W., and Simpson, I. A. (1987) J. Biol. Chem. 262, 11261-11267 44. Vannucci, S. J., Nishimura, H., Satoh,S., Cushman, S. W., Holman, G. D., and Simpson, I. A. (1992) Biochem. J. 288, 325-330