Eur. J. Biochem. 208,693-698 (1992) 0 FEBS 1992

Plasma-membrane-independent pool of the a subunit of the stimulatory guanine-nucleotide-binding regulatory protein in a low-density-membrane fraction of S49 lymphoma cells Petr SVOBODA’, Petr KVAPIL*, Paul A. INSEL3 and Lennart A. RANSNAS’

’ Institute of Physiology, Czech Academy of Sciences, Prague, Czechoslovakia ’ Department of Clinical Chemistry, Sahlgren’s Hospital, University of Goteborg, Sweden

Department of Pharmacology, University of California, San Diego, La Jolla, USA

(Received March 13/June 29, 1992) - EJB 92 0349

We report that compartmentalisation of the stimulatory guanine-nucleotide-binding regulatory protein (G,) exists in S49 lymphoma cells. In addition to the previously reported cytosolic form of the a subunit of G, (Gp) [Ransnas, L. A., Svoboda P., Jasper, J. R. & Insel, P. A. (1989) Proc. Natl Acad. Sci. USA 86, 7900-79031, three membrane-bound forms of G,a were identified through rate- zonal centrifugation in sucrose density gradients, Gpspecific anti-peptide serum and an adenylate cyclase complementation assay. The sedimentation profile of the first pool of G,a in the high-density portion of the gradient (1.13 - 1.16 g/cm3) is identical with that of P-adrenergic-receptor binding, Ma/K-ATPase and adenylate cyclase activity, and may therefore be identified as plasma-membrane fragments. The second pool, which was recovered in the middle portion of the gradient (1.09 - 1.1 1 g/ cm3), contains a much lower total amount of G,a and correlates with the endoplasmic reticulum (microsomal) enzyme markers, NADPH - cytochrome-c reductase and glucose-6-phosphatase.

The identity of the third pool of G,a located at the top of the gradient (1.06-1.08 g/cm3), is unknown. The Golgi apparatus marker, UDPgalactose: N-acetylglucosamine glycosyltransferase, was partially recovered in this area; however, this enzyme was also present in the high-density portion of the gradient. Complete absence of specific adenylate cyclase and Na/K-ATPase activity indicates that this low-density (light) membrane form of G,a is distinct from any plasma-membrane fragments. Furthermore, sedimentation at 100000 x g proves its particulate (membrane) character. The light membrane form of G,a subunit is functionally active in an adenylate cyclase complementation assay using cyc- membranes devoid of G,K.

Overall, our data indicates that a substantial portion of G,a is localized in membrane pools other than plasma membrane.

The stimulatory guanine-nucleotide-binding regulatory protein (G,) represents a signal-transducing protein between stimulatory hormone receptors (e.g. j-adrenergic, glucagon, vasopressin, adrenocorticotropic hormone, luteinizing hor- mone) and the adenylate-cyclase/cAMP system (Stryer and Bourne, 1986; Gilman, 1987; Pfeufer and Helmreich, 1988; Birnbaumer et al., 1990). Agonists acting through stimulatory receptors activate the a subunit of G, (G,a), stimulate adenylate cyclase and increase CAMP.

Correspondence to P. Svoboda, Institute of Physiology CSAV,

Fax: +42 2 4719517. Abbreviations. G protein, guanine-nucleotide-binding protein; Gs.

stimulatory G protein; G,a, a subunit of G,; Gi, inhibitory G protein; Cia, a subunit of Gi; GTP[S], guanosine 5’-[y-thio]triphosphate.

Enzymes. Adenylate cyclase (EC 4.6.1 .l); Na/K-ATPase, sodium/ potassium-activated, ouabain-dependent ATPase (EC 3.6.1.3); glu- cose-6-phosphatase, glucose-6-phosphate phosphohydrolase (EC 3.1.3.9); galactosyltransferase, UDPga1actose:N-acetylglucosamine glycosyltransferase (EC 2.4.1.3.8); succinate dehydrogenase (EC 1.3.99.1); NADH dehydrogenase (EC 1.6.99.3); NADPH- cytochrome-c reductase (EC 1.6.2.4).

Videnskd 1083, CS-142 20 Praha 4, Czechoslovakia

The G,a was identified in plasma-membrane fractions (Birnbaumer et al., 1986; Haga et al., 1977; Bockaert et al., 1987), hepatic microsomes (Codina et al., 1988) and canine sarcoplasmic reticulum (Scherer et al., 1987). The cytosolic fraction was found to contain significant amounts of G,a protein (Ransnas et al., 1989). The lack of data defining the content and functional activity of G,a simultaneously in all subcellular membrane compartments caused us to examine these issues in S49 lymphoma cells by fractionation on sucrose density gradients.

MATERIALS AND METHODS Subcellular fractionation of S49 lymphoma cells

The S49 lymphoma cells were grown in suspension culture in Dulbecco’s modified Eagle’s medium containing 10% (by vol.) heat-inactivated horse serum. They were harvested at a concentration of 0.8 - 1.2 x lo6 cells/ml by centrifugation for 10 min at 900 x g and were washed twice in ice-cold 137 mM NaCl, 5 mM KCl, 1 mM KH2P04 and 1 mM Na2HP04, pH 7.4. As in a standard fractionation procedure, the 16 tis-

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sue-culture flasks, each with 200 ml cell suspension, roughly 3 x lo9 cells, were divided into four groups with four flasks in each group. The contents of the four flasks were combined, sedimented and washed as described above. The final pellets, representing the four independent and identical samples, were resuspended separately in 150 mM NaCl, 20 mM Hepes, pH 7.6,2 mM MgC12 and 1 mM EDTA. They were then equil- ibrated for 30 min at 3.45 MPa of nitrogen in a Parr cavitation chamber. After rapid decompression, the cell lysates were centrifuged at 600 x g for 5 min, and the resulting supernatants (homogenate, 6 - 8 ml) were applied to the top of four discon- tinuous sucrose density gradients. In this way, the amount of material fractionated in a single gradient corresponded to 640- 960 x lo6 cells. Two gradients were used: types I and 11. Type I was essentially the same as that described by Clark et al. (1985) and contained 5 ml 19%, 23%, 27%, 31%, 35% and 43% (by mass) sucrose, 20 mM Hepes, pH 8.0, and 1 mM EDTA. Type I1 consisted of 3 ml15%, 3 ml17%, 10 ml19%, 5 ml 319'0, 5 ml 35% and 5 ml 43% (by mass) sucrose, pre- pared in 20 mM Tris/HCl, pH 8.0, 2 mM MgClz and 1 mM EDTA. The gradients were centrifuged for 60min at 25000 rpm (type I) or 27000 rpm (type 11) in a Beckman SW 28 rotor at 4"C, and were fractionated manually from the meniscus. The first 5 - 7 ml represented the overlayed medium (fraction 0). The rest of the gradient was collected in 2-ml fractions: fraction 1 was composed of a 1-ml overlay plus 1 ml sucrose of lowest density: 19% (I) or 15% (11). The fractions were diluted with 5 ml 20 mM Tris/HCI and 1 mM EDTA, pH 8.0, and centrifuged for 60 min at 40000 rpm in a Beckman Ti50 rotor. The final sediments were resuspended by pipetting and were stored at 2- 10 mg membrane protein/ ml in 20 mM Tris/HCl, pH 7.4, 25 mM NaCl and 1 mM EDTA at - 80°C.

Assay of G,

G,a was determined in a competitive ELISA utilizing anti- peptide serum directed against amino acids 28-42 of G,a (Ransnas and Insel, 1988a, 1989). These antibodies selectively detected G,a but not other proteins present in S49 lymphoma cells (Ransnas and Insel, 1988b). G, was also determined by an in vitro complementation assay, utilizing the mutant S49 lymphoma cell line, cyc-, which is devoid of G,a (Ross and Gilman, 1977a,b; Sternweis and Gilman, 1979). The mem- brane sediments of the sucrose-gradient fractions were extract- ed with 1% cholate (OOC, 60 min, vortexing each 5 min) and were centrifuged for 1 h at 100000 x g. The 100000 x g super- natants were reconstituted (10 min, 30°C) with cyc- mutant membranes and fluoride buffer (10 pM AIC13, 6 mM NaF and 6 mM MgC12), or guanosine-5'-[y-thio]triphosphate(GTP[S] ; 50 pM)-stimulated adenylate cyclase activity was taken as a relative measurement of G, functional activity. The residual adenylate cyclase (fluoride or forskolin stimulated) activity of cholate extracts after 100000 x g centrifugation was zero. The basal adenylate cyclase, measured with or without NaF, in cyc- mutant membranes alone was less than 5% of the G, reconstituted level and was substracted from the total enzyme activity.

fi-adrenergic receptors

The content of P-adrenergic receptors in density-gradient fractions, 20 - 100 pg protein/sample, was determined by an equilibrium-binding study using specific 251-labelled cyanopindolol, as described by Insel et al. (1983).

Marker-enzyme activities

Adenylate cyclase activity was determined in the cell homogenate and gradient fractions (membrane sediments after 100000 x g centrifugation) by the method of Salomon et al. (1974), which involves the separation of [32P]cAMP from [E-~~PIATP on sequential columns of Dowex 50 and alumina.

The Na/K-ATPase was measured as a marker for plasma membranes according to Svoboda et al. (1988).

Glucose-6-phosphatase was used as a marker for the endo- plasmic reticulum according to Swanson (1950) and Canfield and Arion (1988). Inorganic phosphate was assayed according to Ames (1966).

Galactosyltransferase was utilized as a marker for the Golgi apparatus by modification of the original method from Bretz and Staubli (1972), as described by Waldo et al. (1983).

Mitochondria1 enzyme markers, succinate and NADH de- hydrogenases, were measured as described by King (1967) using cytochrome c or ferricyanine as electron acceptors. The absorption coefficients used were those determined by van Gelder and Slater (1962). The electron-transport inhibitors, antimycin A, rotenone and amytal, were added, as rec- ommended by Slater (1967).

Endoplasmic-reticulum marker-enzyme activities, NADH and NADPH - cytochrome-c reductase (rotenone insensi- tive), were measured according to Tolbert (1974) and Ernster at al. (1962).

Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard.

RESULTS

Fractionation of the S49 lymphoma cell homogenate (Parr bomb) was started by low-speed centrifugation ( 5 min, 600 x g) to remove most of the nuclei and non-homogenized cells. A possible loss of components associated only loosely with cell organelles was avoided by application of the 600 x g supernatant (8 ml) directly to the top of discontinuous density gradient type I, consisting of 19%, 23%, 27%, 31%, 35% and 43% sucrose, and centrifuged as described in Materials and Methods. The gradient fractions (7 ml overlay, 2 ml each of fractions 1 - 16) were collected from top to bottom of the gradient, the first fraction represented the interphase between the overlayed medium and 19% sucrose.

Two major membrane bands could be distinguished by macroscopic examination and protein determination (Fig. 1A). The first protein band appeared at the low-density end of the gradient (close to the meniscus, fractions 2 - 4); the second band was located just above the bottom of the centrifuge tube in fractions 13 - 15. All gradient fractions between these two dominant bands contained little protein and there was no sign of any minor local peak in this region. Thus, based on protein distribution, three main areas of the density gradient could be distinguished: low-density (light) band, middle portion and high-density (heavy) band. As much as 81 -94% protein originally present in supernatant 600 x g (applied on the gradient) was recovered in overlay and frac- tions 1 - 16. The total particulate membrane protein rep- resented 60% of this amount, the rest was detected in the 100000 x g supernatant of the overlay and fraction 1 (soluble, cytosolic proteins).

The measurement of Na/K-ATPase, as a marker of plasma membranes (Fig. lB), showed that the peak activity of this enzyme was in fractions 8 - 10. The specific, as well as the total, Na/K-ATPase activity gradually increased over fraction

695

0 4 a 12 16

0 4 a 1 2 16

ftaction

Fig. 1. Distribution of Golgi-apparatus (A) and plasma-membrane (B) marker enzymes on gradient type I. Distribution of protein (B), galactosyl transferase (A), i.e. Golgi-apparatus enzyme, Na/K- ATPase (O) , adenylate cyclase (0) and of b-adrenergic receptors (0) was measured on gradient type I. 8 ml cell homogenate (600 x g supernatant; see Materials and Methods) originating from 8 x lo8 cells was applied to a I!?%, 23%, 27%, 31%, 35% and 43% sucrose- density gradient and fractionated as described in Materials and Methods. The sum of all membrane sediments prepared from fractions 1 - 16 represented about 50-60% of the protein originally applied to the gradient (6-10 mg) in the supernatant 600 x g (homogenate). The residual amount (30- 40%) was detected in the 100000 x g super- natant of overlay fraction 0 which corresponded to soluble, cytosolic proteins. The total protein recovered was 81 -94%. The amount of protein, marker-enzyme activities and of b-adrenergic receptors (specific '251-labelled-cyanopindolol-binding sites) were determined in gradient fractions 0- 16 after dilution and sedimentation at 100000 x g (see Materials and Methods). The data are expressed as percentage of maximum protein content, specific '251-labelled- cyanopindolol binding or enzyme activity in each fraction and are representative of seven similar experiments. 100% values: protein in fraction 3 = 0.68 mg; galactosyl transferase in fraction 14 = 28.6 nmol . h-' . mg protein-' ; Na/K-ATPase in fraction 9 = 16.5 pmol Pi . h-' . mg protein-'; adenylate cyclase stimulated by fluoride buffer in fraction 9 = 892 pmol CAMP. min-' . mg protein - ' ; specific '251-labelled-cyanopindolol binding in fraction 9 = 341.9 fmol/mg.

1 - 9, and decreased over fractions 9 - 16. Thus, a relatively sharp and symmetric peak of Na/K-ATPase was observed in the middle of the gradient. A very similar distribution was obtained for the other plasma-membrane markers, fluoride- buffer-stimulated adenylate cyclase and P-adrenergic recep- tors (Fig. 1B). The highest adenylate cyclase activity was mea- sured in fractions 9-12. The peak values of '251-labelled cyanopindolol-binding sites were also reached in the middle of the density gradient, in fractions 9 and 10. I t may therefore be concluded that the lower/middle portion of the type-I gradi- ent, just above the heavy-protein band, represented by gradi- ent fractions 9 - 11, contains pure plasma-membrane frag- ments.

E 6

x E

-3

dp

E

E -3

x m E

dp

100

75

50

251

I

0

A

0 4 a 12 16

I

0 4 8 12 16

fraction

Fig. 2. Distribution of endoplasmic-reticulum/microsomal (A) and mito- chondrial (B) marker-enzyme activities on gradient type I. The homogenate of S49 lymphoma cells was applied to a 19%, 23%, 27%, 31 YO, 35% and 43% sucrose-density gradient and fractionated as described in Materials and Methods and the legend to Fig. 1. (A) 100% values : endoplasmic reticulum, glucose-6-phosphatase ( B) in fraction 7 = 0.647 Fmol Pi . h- ' . mg protein- ' ; NADPH - cytochrome-c reductase (rotenone insensitive; 0) in fraction 7 = 56.1 nmol . min-' . mg protein-'. (B) 100% values: mitochondria, succinate-cytochrome-c reductase (0 ) in fraction 15 = 174.9 nmol . min-' . mg protein- '; NADH-cytochrome-c re- ductase (0) in fraction 13 = 249.4 nmol . min-' . mg protein- '. The data are representative of three independent fractionation procedures measured in triplicate.

The endoplasmic-reticulum/microsomal markers, NADPH - cytochrome-c reductase (rotenone insensitive) and glucose-6-phosphatase were distributed mainly in fractions 5 - 7 (Fig. 2A). These fractions represent the intermediate re- gion between the low-density protein band and the low-protein area in the middle of the gradient.

Measurement of the mitochondrial marker, succinate de- hydrogenase, using cytochrome c or ferricyanine as electron acceptors, proved that the heavy protein band in fractions 13 - 15 represents mitochondrial membranes (Fig 2B). This conclusion was also supported by measurement of NADH - cytochrome-c reductase (Fig. 2B) and ouabain-independent, Mg-ATPase (data not shown), which increased to very high values in fractions 13-15 and was completely inhibited by oligomycin, a specific inhibitor of mitochondrial ATPase.

Distribution of G,a in the gradient fractions

The measurement of the G,a content (anti-peptide serum) and functional activity (reconstitution assay with cyc- mutant membranes) in gradient-type-I fractions (Fig. 3) showed that membrane bound G,a was distributed in at least three distinct pools, differing in density of membrane fragments. The distri- bution of total and specific content of G,a was identical.

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loot K I

i \ I

0 4 a 12 16

f r a c t i o n

Fig. 3. Distribution of G,a (0) content and G, reconstitutive activity (0) stimulated by fluoride on gradient type I. The content of G,a (ELISA, based on anti-peptide serum against amino acids 28 -42, see Materials and Methods) and G,activity (reconstitution assay with cyc- mutant membranes) was measured in 1% cholate extracts of membrane sediments of fractions 0 - 16 after fractionation of S49 lymphoma cell homogenate on 19%, 23%, 27%, 31%, 35% and 43% sucrose-density gradient and centrifugation at 100000 x g (see Materials and Methods). 100% values: G,GI content of fraction 11 = 21.2 pmol/mg protein; G, reconstitutive activity stimulated by fluo- ride in fraction 11 = 356pmol cAMP.min-"mg protein-'. The data are representative of five G,a-content, and three G,-reconstitu- tive-activity, independent experiments.

The gradient profile of the G,a obtained with the help of immunological detection was identical to that measured by a reconstitution assay (Fig. 3). The first pool of G,a was present in the same region of the gradient (fractions 9 - 11) as fluoride- stimulated adenylate cyclase and Na/K-ATPase activities, and represented about 60 - 70% of the total membrane-associated G,a present in S49 lymphoma cells. This peak of G,a could be unequivocally identified as the plasma-membrane-associated pool (compare Figs 1 and 3) since the P-adrenergic receptors, as well as those for Na/K-ATPase and adenylate cyclase, showed the highest concentrations and activity in the same region of the gradient.

A small peak of Gsa, 10-20% of the total membrane pool, was recovered in a similar manner as the activity of the endoplasmic-reticulum markers NADPH - cytochrome-c reductase (rotenone insensitive) and glucose-6-phosphatase in fractions 5 - 7 (compare Figs 2 and 3).

The third peak of G,a was detected in the topmost, low- density fractions 1-3. This pool of G,a constituted about 20 - 30% of total membrane-bound G,a and was located in or slightly above the light protein band (compare Figs 1 and 3). This low-density form of G,a could not be identified with any marker-enzyme activity, except the Golgi-apparatus marker, galactosyl transferase, showing a biphasic distri- bution (Fig. 1A). Low adenylate cyclase and Na/K-ATPase activity, as well as the minimum number of P-adrenergic recep- tors in a low-density protein band (fractions 2-4), made the plasma-membrane origin of the low-density form of G,a very unlikely. However, some minor contamination with plasma- membrane fragments could not be excluded.

In order to answer this question, namely, whether some minor portion of the light, low-density protein band separated on a type-I gradient might be derived from plasma mem- branes, the S49 lymphoma cell homogenate (supernatant 600 x g) was fractionated on a specially designed gradient, type 11, consisting of 15%, 17%, 19%, 31% 35% and 43%

E 1

X

E

-3

m

*-

0 4 8 12 16

6 e E X m E

.A

*

85 1 M B

0 4 8 12 16

fraction

Fig. 4. Distribution of Na/K-ATPase and adenylate cyclase (A) and G, reconstitutive activity (B) on gradient type 11. (A) Fluoride-stimulated adenylate cyclase (O) , forskolin( 50 pM)-stimulated adenylak cyclase (0) and Na/K-ATPase (0) activities were measured in gradient-type- I1 membrane fractions which were isolated as described in Materials and Methods. The data are representative of five similar experiments. 100% values: adenylate cyclase stimulated by fluoride in fractions 11 = 1063.2pmol cAMP.min-' fraction-' (799.7 pmol CAMP. min-' . mg protein-'); adenylate cyclase stimulated by forskolin in fraction 11 = 7645 pmol cAMP . min - ' . fraction - I (5750 pmol CAMP. min-' mg protein-'); Na/K-ATPase activity in fraction 9 = 25.8 pmol P i . h-' . fraction-' (19.4 pmol Pi -h-' . mg protein- '). (B) Fluoride( H)- and GTP[S](100 pM; 0)-stimulated G, reconstitutive activity was determined in membrane sediments of gradient-type-I1 fractions 1 - 16 (1% cholate extracts). The data are representative of four similar experiments. 100% values: fluoride- stimulated G, reconstitutive activity in fraction 9 = 420 pmol CAMP. min-' . fraction-' (436.6 pmol CAMP. min-' mg pro- tein-'); GTP[S]-stimulated G, reconstitutive activity in fraction 11 = 201 pmolcAMP.min-' .fraction-'(151.4pmol cAMP.min-'.mg protein- ').

sucrose (see Materials and Methods and legend to Fig.4). Fractionation on gradient type I1 achieved complete separa- tion of the light protein band, fractions 2 - 6, from plasma- membrane fragments (fractions 9 - 11, see Fig. 4).

In gradient type 11, the low-density portion was extended when compared with gradient type I and consisted of 3 ml 15%, 3 ml17Yo and 10 ml19% sucrose. Time andg force was also increased. Simultaneously, the high-density portion of the gradient, which prevents the plasma membranes from entering the high-density (mitochondrial) protein band, was the same as in gradient type I. This change resulted in no adenylate cyclase (both fluoride and forskolin stimulated) and Na/K-ATPase activities in the low-density portion of the gradient, fractions 1-7 (Fig. 4A). None of the membrane fractions representing the light, low-density protein band exhibited more than 4% of the plasma-membrane level of marker enzymes (700 - 900 pmol cAMP . min- . mg-

697

for fluoride-stimulated adenylate cyclase; 5 - 7 nmol CAMP. min-' . mg-' for forskolin stimulated adenylate cyclase; and 21 -27 pmol P i . h-' . mg-' for Na/K-ATPase). The overall protein distribution in gradient type I1 was similar to that seen in type I (data not shown). Thus, the plasma- membrane-derived structures represented only a small frac- tion of low-density membranes in gradient type I. The protein recovery on gradient type I1 was 72 - 91 YO.

The separation of plasma-membrane fragments from the low-density membranes on gradient type I1 (Fig. 4A) excluded any possible cross-contamination between the two types of subcellular particle, so that any functional entity demon- strated in the light vesicles may be regarded as being indepen- dent of the plasma membrane. This statement clearly applies to G,a. The relatively high functional activity of G,a was measured in fractions 4 and 5 (Fig. 4B) in a reconstitution assay of fluoride- and GTP[S]-stimulated adenylate cyclase in cyc- mutant membranes. Concomitantly, there was no basal, fluoride- or forskolin-stimulated adenylate cyclase in these fractions (Fig.4A). Therefore, it may be concluded that a hitherto-unknown, plasma-membrane-independent pool of G, was identified in the light membrane fragments of S49 lymphoma cells.

DISCUSSION As the function of G proteins is to allow transfer of infor-

mation between hormone-activated receptor and effector sys- tems and that each of these entities is either a membrane- spanning protein or is in integral association with the plasma membrane, then it should be anticipated that the G proteins should also be in intimate contact with the plasma membrane. In accordance with this idea, membrane-bound G,tl was found to be located in plasma membranes (Ross et al., 1977), although the presence of G,a in cardiac sarcoplasmic re- ticulum (Sherer et al., 1987) or liver microsomes (Codina at al., 1988) has also been reported.

Examination of the location of G protein tl subunits, by either immunoblotting subcellular fractions from various cells (Rotrosen et al., 1988; Bokoch et al., 1988; Ali et al., 1989) or by use of immunocytochemistry (Gabrion et al., 1989), has indicated a more varied location that might be anticipated from their known function. As well as being associated with plasma membranes, G proteins have been detected in various microsomal fractions and indeed in cytoplasm (Ransnas and Insel, 1988b). The j-adrenergic (isoproterenol) stimulation of intact S49 lymphoma cells (Ransnas et al., 1989), was found to redistribute a significant amount of membrane-bound G, into the cytosol defined as supernatant 100000 x g fraction. These data were confirmed by Takahashi et al. (1991) who studied GTP[S]-, GTP- and thrombin-induced release of in- hibitory-G-protein subunit (Cia) from membranes of mouse mastocytoma P-815 cells. The release was specific for GTP and GTP[S] and was markedly (50%) augmented by the cytosol fraction and the released Cia existed in the cytosol as a soluble complex with unidentified component(s).

While it could be suggested that the G proteins detected in the cytoplasm might reflect protein moving towards mem- brane fractions following synthesis, experiments using isolated membrane fractions (Milligan et al., 1988; McArdle et al., 1988; Milligan and Unson, 1989; Ransnas and Insel, 1988b; Ransnas et al., 1991, 1992), have demonstrated that the a subunits of the G proteins can be released slowly from the membrane following activation of G protein with poorly hy- drolysed analogues of GTP and/or hormone.

In addition to the decrease of membrane-bound G-protein tl subunit, caused by an increased agonist or GTP[S] stimu- lation, which is accompanied by an increase of free tl subunit in cytosol (Ransnas et al., 1989; Takahashi et al., 1991; Yamazaki et al., 1983), the decrease of membrane bound G protein a subunit is not accompanied by an increase in tl subunit in the cytosol (McKenzie and Milligan, 1990; Green et al., 1990). This effect, described as down-regulation of G protein, was independent of the generation of CAMP, was not produced by the regulation of G,tl mRNA levels and did not require de novo protein synthesis.

In the view of the above data showing, at a qualitative level, rather heterogeneous distribution of G,a among various subcellular fractions, the present study was undertaken with the aim of characterizing the overall profile of the intracellular membrane distribution of G,tl at a quantitative level. In order to obtain a simple and clear-cut answer, this complex problem was approached in the most simple way, i.e. under control conditions, in the absence of hormone stimulation.

Plasma membranes represent the primary site where recep- tor-coupled G,a would be expected to be localized. However, if one does not intend to exclude, a priori, the existence of Gsa in some other subcellular structures, the majority of the available plasma-membrane isolation techniques can not be used as a basis for subcellular fractionation. Plasma-mem- brane purification is achieved together with discarding or losing of other cell constituents and also a certain portion of plasma membranes. Therefore, we tried to find an approach which would fractionate the cell homogenate by a limited number of steps, allowing quantitative recovery, and would simultaneously prepare plasma-membrane fragments of high purity.

One-step sucrose-density-gradient centrifugation, as used by Clark at al. (1985), served as a good tool for this purpose. Direct application of the 600 x g supernatant onto the top of the gradient, without preliminary concentration of the mem- branes by high-speed centrifugation, prevented the possible loss of light microsomes and/or redistribution of cytosol con- stituents (cf. Ross et al., 1977). Furthermore, by using this procedure, aggregation and cross-contamination of various microsomal membrane elements (Dallner, 1974, 1978) could be avoided.

The results obtained using S49 lymphoma cells in this work demonstrated the presence of G,tl in three distinct membrane pools; plasma membranes, endoplasmic reticulum and low- density membranes. This heterogeneous distribution might reflect some less-understood aspects of G,a metabolism and function, such as protein synthesis, desensitization, intern- alization and/or down-regulation of the components of the G,-mediated signal-transmission pathway. The down-regu- lation of P-adrenergic receptors is known to be associated with endocytosis (internalization) of hormone receptors which appear in the light vesicular fraction distinct from plasma membranes (Harden et al., 1980; Strader et al., 1987; Bouvier et al., 1988). Desensitization of the cell to chronic stimulation by hormone could theoretically be also achieved by down- regulation or another type of inactivation of the more distal components of the adenylate cyclase cascade, i.e. the G pro- teins. The fate of G, under these conditions is unknown, and the data presented in this work, namely the presence of G,tl in the light membrane fraction differing from plasma mem- branes, may have direct relevance to such a phenomena. Haraguchi and Rodbell (1990) described a shift of both G, and Gi from plasma membranes to a low-density microsomal fraction, rich in endosomes and Golgi bodies, after

isoprenaline stimulation of white fat cells. Surprisingly, the plasma-membrane markers 5’-nucleotidase and adenylate cyclase were also elevated. Thus, in this case, the endosomes seem to be derived from plasma membranes.

This work was supported by grants from the Swedish Medical Research Council (B89-04X-08640-01A), NIH (GM31987) and Grant Agency of CSAV (71120).

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