THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 256, No. 21, Issue of November 10. pp. 11365-11371, 1981
Printed in U.S.A.
Association of a 68,000-dalton Protein with Adrenocorticotropin-
Y1
sensitive Adenylate Cyclase Activity in Adrenocortical Tumor
Cells”
(Received for publication, February 25, 1981, and in revised form, June 29, 1981)
Valerie M. Watt$ and BernardP. Schimmer
From the Banting andBest Department of Medical Research, University of Toronto, Toronto, Ontario, Canada M5G lL6
This report explores the biochemical basis for clonal nocortical tumor cell line, Y1, using a genetic approach (for
review
variation in adrenocorticotropin (ACTH)-sensitive ade- see, Schimmer, 1980).In previous reports, we isolated
nylate cyclase activity the Y1 mouse adrenocortical and described several variant subclones of the Yl cell line in
in
tumor cell line. We demonstrate that the level of a which the adenylate cyclase system was unresponsive to
is
specific protein, designated p68, significantly corre- ACTH (Schimmer, 1969, 1972; Schimmer et al., 1979; Rae et
lated with the abilityof adrenocorticotropin to stimu- al., 1979a). Adenylate cyclase activity in these variants was
late adenylate cyclase activity among 1 subclones ( p stimulated by fluoride, guanyl nucleotides, and cholera toxin
Y
= 0.004; r = 0.65). p68 was characterized by molec- (Rae et al., 1979b; Schimmer, 1972), suggestingthat the ade-
its
ular weight i sodium dodecyl sulfate polyacrylamide nylate cyclase system was partially intact. Furthermore, stud-
n
gels (Mr = 68,000) and byits isoelectric point as deter- ies with synthetic fragments of ACTH suggested that the
minedbytwo-dimensional gel electrophoresis(PI = lesions in these clones werenot restricted to the tight binding
7.2). On two-dimensional gels, the protein migrated as region of the ACTH receptor (Rae et al., 1979b).In this study,
a major spot with satellite spots 0.1 pH unit on either
we have explored the molecular basis for the loss of ACTH-
side.Homogenatesandplasmamembranefractions
from cloneshighlyresponsive to ACTH hadlarge sensitive adenylate cyclase activity in variant Y1 cells, by
amounts of p68. In homogenates from highly respon- comparing the profiles of proteins in ACTH-responsive and
siveclones, p68 represented 10 to 12% of thetotal -insensitive clones. We demonstrate that the presence of one
protein. Homogenates and plasma membrane fractions protein (Mr = 68,000; PI = 7.2) correlates significantly with
from clones insensitive to ACTH were deficient in p68. the integrity of the ACTH-sensitive adenylate cyclase system.
In homogenatesfromtheinsensitiveclones Y6 and We suggest that this protein may play a role in determining
0 5 3 , p68 represented 50.8% of thetotalprotein.A the sensitivity of the adenylate cyclase system to ACTH.
somaticcellhybrid,formedbyfusion of these two
ACTH-insensitive recovered
clones ACTH-sensitive EXPERIMENTAL PROCEDURES
adenylate cyclase activity and concomitantly expressed Materials-Nutrient mixture F10 was purchased from Connaught
appreciable levels of p68. It is suggested thatp68 may Laboratories; sera were from Gibco. [2-3H]ATP (16 to 27 Ci/mmol)
of
regulate the transfer information from the occupied and PCS scintillation fluid mixture were from Amersham/Searle; L-
ACTH receptor to the catalytic subunit of adenylate [%]methionine ( 5 0 0 to 980 Ci/mmol) and 2,5-diphenyloxazole from
cyclase. im
New England Nuclear; SB-5 fl from Kodak. Ammonium persulfate,
Dowex 50 (AG 50W-X8), SDS, and ureawere obtained from Bio-Rad;
RNA polymerase from Boehringer Mannheim, N,N”methylenebis-
acrylamide from Eastman; Ampholines from LKB; dimethylsulfoxide
The action of ACTH’ on adenylate cyclase appears to be from Matheson, Coleman and Bell; Nonidet P-40 from Particle Data
similar to the actions of other polypeptide hormones and Laboratories. GPP(NH)Pwas purchased from ICN Pharmaceuticals;
neurotransmitters on the enzyme (Glynn et al., 1979) and theophylline from Schwarz-Mann; bovine serum albumin, catalase,
obligatory for many of the hormone’s effects on the adrenal and Trizma (2-amino-2-hydroxymethyl-l,3-propanediol) from base
Sigma; ACTHI-, (bovine) from Nutritional Biochemicals and
cortex (Halkerston, 1975; Schimmer, 1980). Components of ACTHI-, (porcine) from Sigma. ACTHI-24 (Organon) was a gift from
the ACTH-stimulated adenylate cyclase system in the adrenal Dr. H. Strade (Organon, Inc., West Orange, NJ).
cortex include a specific hormone receptor on the cell surface, CelLs and Cell Culture-All cell lines were derived from the mouse
guanyl nucleotides and guanyl nucleotide-binding proteins, adrenocortical tumor cell l i e , Y1 (Yasumura et al., 1966). This clone
divalent cations, and a catalytic subunit which faces intracel- is available currently from the American Type Culture Collection
lularly (for review, see Glynn et al., 1979). Nevertheless, the (No. CCL 79), and is designated “Yl-ATCC” in this paper. Several
sites and mechanism of ACTH action on the adrenal adenylate stable, ACTH-responsive subclones were isolated from the Y1-ATCC
line by single cell-plating techniques and include: Yl-BS1 (Schimmer,
cyclase system are not well defined. 1979), Y1-BS8, and Y1-BS11. Other clones used in this study were
We have analyzed the mechanism of hormonal regulation two spontaneous, ACTH-insensitive variants, Y6 and OS3 (Schimmer,
of adenylate cyclase activity in the ACTH-responsive adre- 1969, 1972);two cell limes deficient in hypoxanthine-guanosine phos-
phoribosyltransferase activity, Y1-HGPRT- and OS3-HGPRT-
* This work was supported by a grant from the Medical Research (Schimmer et al., 1977); and Kin and Cyc mutants selected for
Council of Canada. The costs of publication of this article were resistance to 8-bromoadenosine 3’,5”monophosphate (Rae etal.,
defrayed in part by the payment of page charges. This article must l
1979a; Schimmer et al., 1979).A l cell lines were grown as monolayers
therefore be hereby marked “aduertisernent” in accordance with 18 on plastic tissue culture dishes at 37 “C. Cells were cultured in
U.S.C. Section 1734 solely to indicate this fact. Nutrient F10 medium supplemented with 15%heat-inactivated horse
$ Recipient of an Ontario Graduate Scholarship. serum, 2.5% heat-inactivated fetal calf serum, and antibiotics. The
’ The abbreviations used are: ACTH, adrenocorticotropin; methods of cell culture have been detailed elsewhere (Schimmer,
GPP(NH)P, guanyl-5”yl imidodiphosphate; SDS, sodium dodecyl 1979).
sulfate. Cell Fusion-Y6 X OS3 hybrids were prepared using the half-
11365
11366 Cyclase Activity
p68 and ACTH-sensitive Adenylate
selection techniques of Davidson and Ephrussi (1965). Monolayers of TABLE I
OS3-HGPRT- cells (3.3 X IO5) were incubated with chemically inac- Adenylate cyclase activity in ACTH-responsive and -insensitive YI
tivated Sendai virus in Eagle's Basal Medium for 20 min at 4 "C. The adrenal clones
medium was removed, a suspension containing 2 X IO3 Y6 cells in 1 Adenylate cyclase activity was measured in the presence of 10 p~
ml of medium was added, the cell mixture was incubated at 37 "C for )
GPP(NH)P; ACTHI-,,(20 p ~ and NaF (15 mM) were added as
20 min to promote fusion, and floating cells were discarded. After indicated. Values shown are averages of duplicate or triplicate samples
fusion, the cultures were maintained in regular growth medium for 4 from representative experiments and are expressed as picomoles of
days and then propagated in growth medium containing amethopterin cAMP formed per rnin per mg of protein.
(5 pg/ml) for 7 weeks. Colonies were isolated and maintained in
growth mediup with amethopterin for 6 months. Thereafter, the Adenylate cyclase activity
Clone Fraction
clones were cultured and assayed in normal growth medium. Kary- Basal Fluoride
ACTHI.21
otype analysis was performed using the method of Rothfels and
Siminovitch (1958). Y 1-BSl Cell homogenate 16 250 140
Preparation of Plasma Membrane Fractions-Fractions enriched Plasma membrane 150 710 350
in plasma membranes were prepared from monolayers of adrenocor- Y6 Cell homogenate 11 20 150
tical tumor cells by differential centrifugation of cell homogenates Plasma membrane 88 140 310
followed by centrifugation in a discontinuous sucrose gradient (Schim- OS3 Cell homogenate
9 7 170
mer et al., 1979). The specific activity of (Na + K)-ATPase in these Plasma membrane 49 50 150
fractions was increased at least 20-fold.
Adenylate Cyclase Activity-Cell monolayers a t saturation density
were rinsed with 20mM Tris. HCI (pH 7.7), 1 m~ MgC12, and 250 m M was 1.7-foldgreater than theeffect of NaF on enzyme activity
sucrose, scraped in 20 m~ Tris.HC1 (pH 7.7), 1 m MgCI2,and I m
M M (Table I). In plasma membrane fractions from Yl-BSl cells,
dithiothreitol, and homogenized at 0 "C in a Dounce homogenizer. adenylate cyclase activity also was stimulated by ACTH,_24 to
Adenylate cyclase activity was determined by measuring the conver- levels greater thanthe levels achieved with fluoride (Table I).
sion of ["]ATP to [3H]cAMP as described by Schimmer (1972). The In the plasma membrane fraction, but not in total homoge-
reaction mixture contained ["HIATP (approximately 1.3 X lo6 cpm), nates, GPP(NH)P was required for the stimulatory effect of
M M
1 m disodium ATP, 2 m MgCIz, 13 m~ Tris.HCI (pH 7.7), 6 m M
theophylline, and 52 pg of albumin in a totalvolume of 80 pl. ACTH, ACTH. In homogenates and in plasma membrane fractions
NaF, and GPP(NH)Pwere included at theconcentrations indicated. from Y6 and OS3 cells, adenylate cyclase activity was mark-
The reaction was carried out for IO min at 37 "C and was stopped by edly increased by fluoride but was stimulated less than 2-fold
adding 0.1 ml of a recovery mixture (40 m~ disodium ATP, 12.5 m M by 20 PM ACTH1_z4 (Table I). The small stimulation of ade-
CAMP)and then by boiling for 3 min. Labeled cAMP was separated nylate cyclase activity seen in the Y6 clone with high concen-
from other labeled compounds by chromatography on columns of trations of ACTH1-z4appeared to result from a nonspecific,
Dowex 50 and treatment with BaS04.Adenylate cyclase activity was
expressed as picomoles of cAMP formed per min per mg of protein.
polycationic effect of the hormone2 similar to that observed
Polyacrylamide Gel Electrophoresis-Cell monolayers were incu- by Wolff and Cook (1977).
bated with ~-[~~S]methionine pCi/ml) in growth medium for 42
(4.55 Gel Electrophoresis of Proteins from ACTH-responsive
h. The monolayers a t saturation density then were rinsed with 250 and -insensitive Clones-The ability of ACTH to stimulate
M M
m sucrose, scraped in 20 m Tris.HCI (pH 6.8), and homogenized adenylate cyclase activity in Y1-BS1 cells,but not in Y6 and
in a Dounce homogenizer. Samples weremixedwith 460 m 0-
M OS3cells, prompted us to lookfor differences in protein
mercaptoethanol and SDS, at a ratio to protein of at least 5:l (w/w), composition in the plasma membrane fractions and in total
and were boiledfor 10 min. For analysis on SDS-polyacrylamide gels,
glycerol was added to a final concentration of 10%(w/v). For two- homogenates from these clones. Yl-BS1, Y6, and OS3 cells
dimensional gel electrophoresis, the following were added: 7.9 M urea, were incubated with ~-[~~S]methionine, plasma membrane
Nonidet P-40, at a ratio to SDS of 12:l (w/w), 3.4% Ampholines (pH fractions were prepared, and the proteins in total homogenates
ranges 3.5 to 10,5 to 8, and 9 to 11 a t a ratio of 5533:12) and 460 mM and in the plasma membrane fractions were analyzed follow-
0-mercaptoethanol. Electrophoresis was carried out on 7.5% poly- ing electrophoresis in SDS-polyacrylamidegels. In the plasma
acrylamide gels according to the method of Laemmli (1970). Gels membrane fraction from Y1-BS1cells, at least six major
were calibrated using bovine serum albumin (Mr= 67,000), catalase
(M, = 60,000) and RNA polymerase (Mr of a subunit = 39,000, /3 bands, ranging in molecular weight from 30,000 to 108,000,
subunit = 155,000, /3' subunit = 165,000). For two-dimensional gel were visible in stained gels and in fluorographs (Fig. la). In
electrophoresis, proteins were first separated by equilibrium-isoelec- the plasma membrane fractions from the ACTH-insensitive
tric focusing with ampholines consisting of pH ranges 3.5 to 10, 3 to Y6 and OS3 cells, there was a striking reduction in the stained
5,8 to 10, and 9 to 11 at a ratioof 7:1:0.5:1.5as described by Alton and and labeled band at 68,000 daltons (Fig. la). Although the
Lodish (1977). After electrophoresis, the gelswerefixed i 12.5% n protein profiles of these clones weresimilar, differences in the
trichloroacetic acid for 30 min, stained with 0.025% Coomassie Bra-
liant Blue G for approximately 6 h and destained in 7.5%acetic acid. amounts of other bands also were observed (e.g.M , E 30,000;
To prepare fluorographs, gels were impregnated with 2,5-diphenyl- Fig. la). We have concentrated on the 68,000-dalton protein
oxazole (Bonner and Laskey, 1974), dried on Whatmann No. 3MM since the change in the amount of this protein was dramatic,
paper using heat and vacuum, and exposed at -70 'C on SB-5 film. and was observed in both stained and labeled preparations
to
Films were prefogged give a linear relationship between the amount, and in both variants. The difference in the amount of the
of radioactivity and the intensity of the f h images (Laskey and 68,000-dalton protein present in the ACTH-responsive Y1-
Mills, 1975). Protein bands were quantitated by scanning stained gels
or fluorographs with a Joyce-Loebl densitometer and by determining BS1 clone and the insensitive variants also wasvisible in
peak areas using a PDP 11 computer program. stained gels and fluorographs of total cell proteins (Fig. 16).
Protein Determination-Proteins were measured by the method This difference also was observed after labeling cells with L-
of Lowry et al. (1951), using bovine serum albumin as the standard. [35S]methioninefor 1 h or with [3H]leucine for 24 h. Quanti-
tative analysis of fluorographs indicated that in Yl-BSl cells,
RESULTS the band at 68,000 daltons represented 10.6% k 0.7% (n = 7)
Adenylate Cyclase Activity in ACTH-responsive and
-insensitive Adrenal Clones-Homogenates and fractions en- The polycationic nature of ACTHI-Uwas demonstrated previ-
riched in plasma membranes were prepared from clones Y1- ously (Raeand Schimmer, 1974). ACTHI-24 stimulatedadenylate
cyclase in Y6 cells only a t high concentrations of the peptide (2 to 20
BS1, Y6, and OS3. In homogenates prepared from Yl-BS1 p~). at these concentrations was without effect ( e g . Table
cells, ACTHI-,, stimulated adenylate cyclase activity 16-fold 111). Two other polycations, polylysine and polyarginine, also stimu-
over basal levels (Table I). Although NaF also stimulated lated adenylatecyclase in Y6 cells over the same concentration range
adenylate cyclase activity, the stimulatory effect of ACTH1-z4 (J. Tsao and B. P. Schimmer, unpublished observations).
p68 and ACTH-sensitive Adenylate Cyclase Activity 11367
a. stain
Coomassie Fluorograph
b. Coomassie stair Autoradiograph
OS3 .Yl-BSl Y6 OS3 Y6
Y1-BS1 OS3 Y1-BSI YE 3S3 Y1-BS1 Y6
Mr Mr
10-3, ( X 10-3
108 108
83
83
68
68 57
57
41
41
30
30
"
" e "
FIG.1. Gel electrophoresis of proteins from ACTH-responsive and -insensitive clones. Plasma membrane fractions ( 0 ) and total
homogenates ( b )were prepared from cells labeled with methionine. Samples were electrophoresed on 7.5%polyacrylamide gels.
of total radioactive protein. In Y6 and OS3 cells, the band at present in the soluble fraction (track 0. T o determine
68,000 daltons represented 50.8%of the labeled protein. Pro- whether the soluble p68 could associate with plasma mem-
teins from total homogenates of Yl-BSl, Y6, and OS3 cells branes during fractionation, a soluble fraction was prepared
also were analyzed by two-dimensionalgel electrophoresis. In from Y1-BS1 cells labeled with ~-["'SS]methionineand mixed
homogenates from Y1-BS1 cells, 83% of theradioactively with a totalhomogenate of unlabeled Y1-BS1 cells. The
*
labeled 68,000-dalton protein focused at a PI of 7.2 0.1 ( n plasma membrane fraction was isolated from this mixture,
= 4), with satellite spots displaced 0.1 pH unit on either side solubilized, and electrophoresed. A 68,000-dalton band from
(Fig. 2). This protein is designated p68. T h e 68,000-dalton the labeled soluble fraction did associate with the plasma
protein associated with the plasma membrane fraction Y1- of membranes during purification (Fig.3, track D). comparison
A
BS1 cells had the same isoelectric point (Schimmer et al., of the specific activities of p68 in each fraction,however,
1979). In both variant adrenal clones, Y6 and OS3, the amount suggested thatnotmorethan 50% of p68 in the plasma
in
of p68 in plasma membrane fractions and total cell homog- membrane fraction resulted from co-fractionation of the cy-
enates, as analyzed by two-dimensional gel electrophoresis, tosolic p68. Attempts to reconstitute ACTH sensitivity in Y6
was less than in Y1-BSl. Quantitative analysisof two-dimen- cells or modify ACTH-sensitive adenylate cyclase activity in
sional gels of total Y6 homogenates gave the same result as Yl-BS1 cells by adding back soluble fractions containingp68
analysis of one-dimensional tracks, i.e. the amount of p68 in were unsuccessful (data not shown).
Y6 cells was approximately 3%of that in Y1-BS1 cells. Adenylate Cyclase Activity and Levels ofp68 in Y6 X OS3
Distribution ofp68 between Cytosol and Membranes-The Hybrids-Y6 cellsinlimiting numbers and OS3-HGPRT-
abundance of p68 in Yl-BSI cells precluded it from being cellswerefused with Sendai virus and twocolonieswere
exclusively localized in the plasma membrane fraction, an isolated following selective growth in amethopterin. Whereas
organellewhich comprises a small fraction (approximately both Y6 and OS3-HGPRT- cells had nearly diploid chromo-
2.5%) of total cell protein.:' Therefore, the distributionof p68 some numbers, the two clones isolated after fusion and selec-
of
between the cytosol and particulate fractions Yl-BSI cells tion had polyploid karyotypes, indicating theirhybrid nature
was determined. Fluorographs of radioactively labeled pro- (Table 11). Inasmuch as this fusion and selection procedure
teins from total homogenates and cytosolic and particulate favors the isolation of heterologous hybrids (Davidson and
fractions separated on SDS-polyacrylamide are shown in
gels Ephrussi, 1965), these clones are tentatively designated Y6 X
Fig. 3. Although a significant amount of p68 (30%) was asso- OS3-HGPRT- hybrids. The two parental clones and the Y6
ciated with the particulate fraction(track B ) , 70% of p68 was x OS3-HGPRT- hybrid, clone 2, had adenylatecyclase activ-
ities which were stimulated by fluoride but which were insen-
V. M. Watt and B. P. Schimmer, unpublished observations. sitive to ACTHI-,, (Table 11). Inone hybrid, Y6 X OS3-
11368 p68 and ACTH-sensitiveAdenylate Cyclase Activity
A B C D
Mr
(x 10-3)
108
108-
83 83 -
68 68 -
57 57-
41-
41
30-
30
I I I
6 7 8
PH
FIG.3. Gel electrophoresis of proteins in subcellular frac-
FIG.2. Two-dimensional gel electrophoresis of proteins tions from Yl-BS1 cells. Y1-BSl cells, labeled with ~-[%]methio-
from Y1-BS1 cells. A fluorograph from cells labeled with L-[%] M
nine, were homogenized in 20 m Tris.HCI (pH 6.8) containing 0.1
methionine is shown. The outlined area indicates the position of the m~ phenylmethylsulfonyl fluoride and centrifuged a t 100,OOO X g for
major protein with M , = 68,OOO. 60 min. The pellet was washed once with the Same Tris-HCl buffer
and the resultant supernatant fractions were combined. The total
TABLE I1 homogenate (trackA ) , 100,OOO X gpellet (track B), and the combined
Adenylate cyclase activity and karyotype analysis of Y6 X OS3- supernatant fractions (track C) were electrophoresed on 7.5% poly-
HGPRT- hybrids acrylamide gels and analyzed by autoradiography. In a separate
Y6 and OS3-HGPRT- cells were fused with Sendai virus and two experiment, the soluble fraction from radioactively labeled Y1-BSl
colonies were isolated following selective growth in amethopterin. cells was mixed with 10 times the number of unlabeled Y1-BS1 cells
Chromosome numbers for parental lines are modal numbers based on and the plasma membrane fraction was isolated and analyzed by
scores from a t least 16 spreads. Values for hybrids are averages with autoradiography followinggel electrophoresis. Track D shows the
the range for more than 85% of the spreads counted shown in labeled soluble proteins which were recovered in the plasma mem-
parentheses. Cell homogenates were assayed for adenylate cyclase brane fraction.
activity with no additions, ACTHI-2, (20 p ~ or) NaF (15 m). Activ-
ities are expressed as picomoles of cAMP formed per min per mg of adenylate cyclase activity were determined in 18 independent
protein and in hybrid clones are averages of triplicate samples. subclones of the Y1 line and compared with the level of p68
Adenylate cyclase activity in each clone in order to further explore the relationship
Clone Chromosome no.
Basal ACTH,.*, Fluoride between these two variables. Adenylate cyclase activity was
Y6 39 8 21 220 assayed in cell homogenates with no additions or with maxi-
OS3-HGPRT- 42 4 7 200 mally effective concentrations ofACTHI-39 or NaF (Table
Y6 X OS3-HGPRT- 111). Basal levels of adenylate cyclase activity in all clones
Clone 1 70 (54-75) 4 240 240 were low and ranged from 2 to 12 pmol of cAMP formed/min/
Clone 2 73 (62-81) 2 10 190 mg of protein. Fluoride-stimulated adenylate cyclase activity
ranged from 160 to 340 pmol of cAMP formed/min/mg of
HGPRT- clone 1, adenylate cyclase was activated by protein and, in all clones, reflected a substantial increase in
ACTH1-24to the same level achieved with fluoride (Table 11). enzyme activity. The relative effectiveness of ACTH1-s on
Associated with the recovery of ACTH-sensitive adenylate adenylate activity among the different clones varied consid-
cyclase activity in the hybrid, Y6 X OS3-HGPRT- clone 1, erably (Table 111). Clonal variations in responsiveness to
was an increased level of p68 (Fig. 4). The level of p68 in the ACTH were apparent in the absolute levels of ACTH-stimu-
ACTH-responsive hybrid, clone 1, was increased at least 16- lated adenylate cyclase activity, in the fold increase over basal
fold over the level of p68 in the two-unresponsive parental levels and in the ratio of activities produced by ACTH1-m and
clones. The ACTH-insensitive hybrid, clone 2, had a level of fluoride (ACTH/F- activity ratio; Table 111). The expression
p68 simiiar to thatfound in the unresponsive parents (Fig. 4). of the effectiveness of ACTH1-m on adenylate cyclase activity
Clonal Variations in ACTH-sensitive Adenylate Cyclase as an ACTH/F- activity ratio was used as an estimate of the
Activity and the Levels of p68-The effects of ACTH1-39 on relative sensitivity of adenylate cyclase to ACTH. The Y1
p68 and ACTH-sensitive Adenylate Cyclase Activity 11369
A C clones were ranked in order of decreasing responsiveness to
ACTH as determined by the ACTH/F- activity ratio (Table
111). Clones such as Y1-BS1 and several of the Kin mutants
(Kin 7, Kin 8, and Kin 9) had ACTH-stimulated adenylate
I cyclase activities which were greater than or equal to the
activity measured in the presence of fluoride. Clonessuch as
Cyc-101,Y6,OS3, and Y6 X OS3 HGPRT- clone 2 were
insensitive to ACTH. The other clones listed showed inter-
(.
10-3) mediate levels of response to ACTH. Adenylate cyclase activ-
ity in the parent Y1 clone, Y1-ATCC, was stimulated approx-
imately 8-fold byACTH; however, this response was less than
20% of the catalytic activity seen in the presence of fluoride
(Table 111). In previous studies, similar ACTH/F- activity
ratios were observed in homogenates of Y1-ATCC cells (Taun-
108 I ton et al., 1969; Schimmer, 1972). Each Y1 subclone in mono-
" l
r for
layer culture was labeled with ~-[~S]methionine 42 h, and
the amount of p68 was quantitated following SDS-polyacryl-
83 amide gel electrophoresis of total homogenates and autora-
diography. The amount of p68,like ACTH-sensitiveadenylate
cyclase activity, varied among the subclones of the Y1 line
68 M
(Table 111). The addition of0.1 m phenylmethylsulfonyl
fluoride to the homogenizing buffer in order to inhibit protein
degradation in many of the subclones did not alter the level
57 to
of p68. In clones highly responsive ACTH such as Y1-BS1,
Kin-7, and Kin-8, p68 represented approximately 10 to 12% of
the total labeled protein. In clones which were much less
sensitive or insensitive to ACTH, such as Y1-ATCC, Y6, Y6
x OS3-HGPRT- clone 2, Cyc-101and OS3, the amount of p68
41 ranged from 0.3 to 2.5% of total labeled protein. The greatest
variability in the correlations between levels of p68 and re-
sponsiveness to ACTH were observed in clones with inter-
FIG. 4. Gel electrophoresis of Y6 X OS3-HGPRT- hybrids.
30 Cells were labeled with L-["Slmethionine, and homogenates were
electrophoresed on 7.5% polyacrylamide gels and analyzed by auto-
radiography. OS3-HGPRT- (track A); Y6 X OS3-HGPRT- clone 1
(track B ) ; Y6 X OS3-HGPRT- clone 2 (track C). The profde of
proteins obtained from homogenates of the Y6 parent is shown in Fig.
16.
TABLE I11
Clonal variatwns in ACTH-sensitiveadenylate cyclase activity and in theleuekr ofp68
Cell homogenates were assayed for adenylate cyclase activity with no additions, NaF (16 mM), or with maximally effective concentrations
of ACTH1- (2 p for ACTH from Nutritional Biochemicals; 20 p~ for ACTH from Sigma). Adenylate cyclase activity is expressed as
picomoles of CAMP formed per min per mg of protein. The level of p68 was determined by quantitation of autoradiographs from SDS-
wlvacrvlamide eels and is exmessed as the Dercentage of total cell Drotein.
Adenylate cyclase activity
Clone ACTH/F-
Basal ACTH Fluoride
%
Yl-BSI 8 f l 260 f 10 220 f 10 1.22 It 0.07 (24) 10.6 f 0.7 (7)
Kin 7 8f2 240 f 10 220 f 30 1.13 f 0.18 (4) 9.8 (2)
Kin 9 2 180 170 1.08 (1) 11.4 (1)
Kin 8 7 f l 290 f 20 310 f 25 0.96 f 0.08 (4) 9.6 f 0.8 (3)
Y6 X OS3-HGPRT-
Clone 1 7 f2 140 f 10 190 f 15 0.71 f 0.07 (4) 12.1 f 0.5 (3)
Kin 4 2 130 200 0.63 (1) 12.1 (1)
Yl-HGPRT- 6 f l 140 f 15 250 f 20 0.53 f 0.04 (14) 0.8 f 0.1 (4)
Y 1-BS8 7 f l 120 f 6 260 f 10 0.45 f 0.02 (30) 0.8 f 0.1 (3)
120
cyc 102 12 260 0.43 (1) 12.4 f 1.1 (3)
Kin 1 6 f l 85 f 15 220 f 20 0.41 f 0.08 (10) 3.9 f 0.3 (4)
Y1-BSll 6 f 1 300130 f 20 0.40 f 50 1.2 f 0.07 (1)
Cyc 103 4 105 280 0.36 (1) 11.0 (2)
Kin 2 5 f l 60 f 10 230 f 25 0.25 f 0.04 (8) 10.5 k 0.7 (4)
Y 1-ATCC 8f2 65 f 10 0.17
320 f 30 f 0.02 (10) 1.7 f 0.1 (4)
Y6 6 f 1 16 f 6 260 f 70 0.03 f 0.01 (3) 0.3 (2)
Y6 X OS3-HGPRT-
Clone 2 3 7 160 0.02 (1) 0.4 (2)
cyc 101 7 12 340 0.02 (2) 2.5 (2)
OS3 4 f l 3 f l 220 f 45 <0.01 (3) 0.8 f 0.1 (3)
11370 ACTH-sensitive
p68 and Adenylate Cyclase Activity
mediate ACTH/F- activity ratios (Table 111).Clones such as cyclase activity and the level of p68 may have resulted from
Cyc-103 and Kin-2 had low ACTH/F- activity ratios but had variations in one or more of the other components of the
levels of p68equivalent to thelevels found in highly responsive system. In some hepatoma and lymphoma clones, for example,
clones. In contrast, the ACTH-responsive clones Y1-HGPRT- lack of hormone-responsiveadenylate cyclase activity resulted
and Yl-BS8 had levels of p68 comparable to thelevels found from alterations in hormone receptors, guanyl nucleotide-
in ACTH-resistant clones (Table 111).In a l 18 clones of the
l binding proteins, and catalytic subunits (Insel et al., 1976;
Y1 line, the relative amount ofp68 correlated significantly Haga et al., 1977;Wicks et al., 1978). Second, thisstudy
with hormone-sensitive adenylate cyclase activity expressed focused on the level ofp68 in relation to ACTH-sensitive
as the ACTH/F- activity ratio ( p = 0.004; r = 0.65). The adenylate cyclase activity. The precise function of p68, how-
correlation Coefficient wasdetermined using product-moment ever, remains undefined, and it is conceivable that in some
correlation analysis (Ostle and Mensing,1975). Significant clones the activity of p68 has been altered. Therefore, the
correlations also were observed when the relative amount of putative activity ofp68 rather than the absolute level of
p68 was compared with the absolute level of ACTH-sensitive protein may be more relevant to ACTH-sensitive adenylate
adenylate cyclase activity ( p = 0.01) or with the fold stimu- cyclase activity.
lation by ACTH over the basal level ( p = 0.01). Little or no p68 was detected when total proteins from
normal mouse tissues (liver, skeletal muscle, fat, testis, brain,
DISCUSSION kidney, lungs, heart, stomach, and adrenal) were analyzed by
two-dimensional gel electroph~resis.~ Therefore, we consider
We have found a significant correlation between the ability the high levels of p68 found in some Y1 subclones (Table 111)
of ACTH to stimulate adenylate cyclase activity and the to be atypical and to have resulted from overproduction by
amount of a specific protein, p68, among subclones of the Y1 some clones.The overproduction of p68 may beanalogous to
adrenocortical tumor cell line. Clones highly stimulated by the overproduction of receptors for epidermal growth factor
ACTH have large amounts of p68; cloneslacking ACTH- and nerve growth factor in human epitheliod carcinoma and
responsive adenylate cyclase activity have little p68 devia- human melanoma cell lines (Fabricant etal., 1977). In clones
tions from the correlation are found in clones with interme- which contain high levels of p68, the protein was found mainly
diate sensitivity to ACTH (Table 111).An association of p68 in the soluble fraction. Nevertheless, the avidity ofp68 for
with ACTH-sensitive adenylate cyclase activity also was ob- plasma membrane fractions (Fig. 3) is consistent with a role
served in hybrids formed between the two ACTH-insensitive for p68 in regulating adenylate cyclase activity. In other
adrenal clones, Y6 and OS3. One hybrid, clone 1, recovered systems, soluble factors have been reported to influence the
ACTH-sensitive adenylate cyclase activity and, concomi- responsiveness of adenylate cyclase to hormone (eg. MacNeil
tantly, acquired appreciable levels ofp68. In contrast, the et al., 1980).Although the relationship between these soluble
hybrid clone 2 remained insensitive to ACTH and retained factors and p68 is unknown, p68, unlike the soluble factors,
the low levels ofp68 associated with unresponsive clones does not influence adenylate cyclase activity when added to
(Table 11).The mechanism responsible for the reconstitution plasma membranes. Based on our earlier studies on the
of ACTH-sensitive adenylate cyclase activity in the hybrid ACTH-insensitive clones Y6 and OS3 (Schimmer, 1969, 1972;
clone 1 is unknown; however, preliminary evidence suggests Rae et al., 1979b; Schimmer et al., 1979),we suggest that p68
that restoration of ACTH sensitivity through cell fusion is an has a role in the transduction of the signal of ACTH binding
infrequent event and, therefore, probably not due to a simple to activation of adenylate cyclase.
complementation of two different mutations.
The co-expression of ACTH-sensitive adenylate cyclase Acknowledgments-We thank Jennivine Tsao and Sheila Power
activity and p68 may be the result of their coordinate regula- for their excellent technical assistance; Drs. C. J. Ingles, A. Kuksis,B.
tion and need not imply a role for p68 in the modulation of G. Louis, and C. C. Yip for their helpful discussion and advice; and
Drs. D. H. MacLennan and J. Logothetopoulos for their critical
ACTH-sensitive adenylate cyclase activity. The factors influ- reviews of the manuscript.
encing the differential expression of p68 amongY1 clones are
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