Proc. Nati. Acad. Sci. USAVol. 81, pp. 5081-5085, August 1984Biochemistry

Binding of [3H]forskolin to rat brain membranes(adenylate cyclase/receptors/hormone)

K. B. SEAMON*, R. VAILLANCOURTt, M. EDWARDSt, AND J. W. DALYttLaboratory of Bioorganic Chemistry, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,MD 20205; and *National Center for Drugs and Biologics, Food and Drug Administration, Bethesda, MD 20205

Communicated by Bernhard Witkop, May 1, 1984

ABSTRACT [12-3H]Forskolin (27 Ci/mmol) has beenused to study binding sites in rat brain tissue by using bothcentrifugation and filtration assays. The binding isothermmeasured in the presence of 5 mM MgCl2 by using the centrif-ugation assay is described best by a two-site model: Kd, = 15nM, B,,,X (maximal binding) = 270 fmol/mg of protein; Kd2 =1.1 ,uM; Bn,% = 4.2 pmol/mg of protein. Only the high-affini-ty binding sites are detected when the binding is determined byusing a filtration assay; Kd = 26 nM, Bma = 400 fmol/mg ofprotein. Analogs of forskolin that do not activate adenylatecyclase (EC 4.6.1.1) do not compete effectively for [3H]forsko-lin binding sites. Analogs of forskolin that are less potent thanforskolin in activating adenylate cyclase are also less potent incompeting for forskolin binding sites. The presence of 5 mMMgCI2 or MnCl2 was found to enhance binding. In the pres-ence of 1 mM EDTA the amount of high-affinity binding isreduced to 110 fmol/mg of protein with no change in Kd.There is no effect of CaCl2 (20 mM) or NaCl (100 mM) on thebinding. No high-affinity binding can be detected in mem-branes from ram sperm, which contains an adenylate cyclasethat is not activated by forskolin. It is proposed that the high-affinity binding sites for forskolin are associated with the acti-vated complex of catalytic subunit and stimulatory guaninenucleotide binding protein.

The activity of hormone-sensitive adenylate cyclases (EC4.6.1.1) and thus intracellular cyclic AMP is regulated in acomplex manner by both stimulatory and inhibitory hor-mones (1-3). This regulation is initiated by hormone bindingto cell surface receptors. The hormone-receptor complexthen interacts with specific guanine nucleotide binding pro-teins that mediate either stimulatory or inhibitory effects onthe catalytic subunit of the enzyme (3). The stimulatory gua-nine nucleotide binding protein (Ns) is a heterodimer consist-ing of a 35,000-dalton protein (,B) and a 42,000-dalton protein(as) (4-6). It is required for activation of adenylate cyclaseby hormones, NaF, or guanosine 5'-[,B, y-imido]triphosphate(6, 7). The inhibitory guanine nucleotide binding protein (N1)is also a heterodimer, consisting of a 35,000-dalton proteinidentical to the f3 protein of Ns and a 41,000-dalton protein(a,) (8, 9).

Forskolin, a naturally occurring diterpene, can activatemost hormonally responsive adenylate cyclases from mam-malian tissues with an EC50 (50% effective concentration) of5-10 ,tM (10-12). The activation of adenylate cyclase byforskolin does not require the presence of a functional Nssubunit (13, 14), and it has been suggested that forskolin in-teracts directly with the catalytic subunit or acts indirectlythrough an as yet unidentified protein (13). Activation of theNs subunit can markedly potentiate the stimulation of aden-ylate cyclase by forskolin (14). Conversely, forskolin at con-centrations less that 1 ,uM can markedly potentiate hormonal

responses (ref. 12 and references therein). Forskolin is notable to activate adenylate cyclase from ram sperm, and it hasbeen suggested that forskolin may be acting at a subunit oth-er than the catalytic subunit (15, 16). It is clear that a precisedefinition of forskolin's site of action ig lacking.To study and monitor the protein component that binds

forskolin, [12-3H]forskolin (27 Ci/mmol; 1 Ci = 37 GBq) wasprepared with tritium in the C-12 methylene group, adjacentto the carbonyl group. Saturable high-affinity binding sitesfor [3H]forskolin were found to be present in brain mem-branes and appear likely to be associated with activatedstates of the adenylate cyclase complex.

MATERIALS AND METHODSPreparations and Properties of [12-3H]Forskolin. [12-

3H]Forskolin was produced by the base-catalyzed exchangeof protons on the C-12 methylene group, adjacent to the car-bonyl group of forskolin. Forskolin was incubated with thebase 1,5-diazabicyclo[4.3.0]non-5-ene in tetrahydrofuranwith a stoichiometric amount of 3H20 at 450C for 3 hr. Thesite of exchange was independently determined by using2H20 in place of 3H20 and monitoring the reaction by nucle-ar magnetic resonance spectroscopy. Exchange with 3H20afforded [12-3H]forskolin with a specific activity of 27 Ci/mmol. Radiochemical purity of the compound was deter-mined by using reverse-phase high-pressure liquid chroma-tography (DuPont ODS column, 4.6 mm x 25 cm; solventCH3CN/H20, 7:3, vol/vol) with radioactivity monitored bya Radiomatic HS flow detector. Purified [3H]forskolin wasradiochemically pure (>99%) by this procedure. The tritiumon forskolin did not exchange with solvent under basic con-ditions with moderate heat (pH 8.5, 60'C, 3 hr). [3H]Forsko-lin was equipotent with unlabeled forskolin in activating ratbrain adenylate cyclase. [3H]Forskolin is kept as a 500 ,uMstock solution in ethyl acetate. A slight decomposition offorskolin was observed after 4-5 months when it was kept inethanol at room temperature. [3H]Forskolin prepared by theabove procedure is now available from New England Nucle-ar.

Nonspecific binding of [3H]forskolin to tubes was deter-mined as the percentage of total [3H]forskolin that could beextracted from tubes by 0.2 M NaOH after the tubes wereexposed to [3H]forskolin and rinsed three times with cold 50mM Tris HCl, pH 7.5. Polycarbonate and polysulfone tubeshad high nonspecific binding, about 1%. Glass tubes andpolypropylene tubes had the lowest specific binding,<0.05%, and glass tubes were used in all experiments.A solution of [3H]forskolin was prepared by placing an ali-

quot of the stock [3H]forskolin into a glass tube, removingthe ethyl acetate under a stream of N2, and adding 50 mMTris HCl, pH 7.5, to the desired volume. It is crucial to allowat least 20 min for the [3H]forskolin to go completely intosolution. A small percentage of [3H]forskolin will come out

Abbreviations: N, subunit, stimulatory guanine nucleotide bindingprotein; Ni subunit, inhibitory guanine nucleotide binding protein.

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of aqueous solutions in the presence of high concentrationsof unlabeled forskolin (>30 ,uM). This is due to the limitedsolubility of forskolin (<100 ,uM) in aqueous solutions andcan lead to anomalous results when displacement experi-ments are carried out with unlabeled forskolin or with ana-logs with similar solubility characteristics.

Tissue Preparation. One rat forebrain was homogenizedwith a Teflon/glass homogenizer in 10 ml of ice-cold 0.32 Msucrose. The homogenate was spun at 1000 x g for 10 min.The supernatant was removed and spun at 20,000 x g for 10min. The pellet from the second spin was resuspended in 15ml of 50 mM Tris HCl, pH 7.5, with a protein concentrationof about 5 mg/ml. Ram sperm membranes were preparedexactly as described by Stengel et al. (15).

Centrifugation Assay. Membranes (800 ,ug of protein perassay in 0.2 ml of 50 mM Tris HCl, pH 7.5) were added to 10x 75 mm glass tubes. [3H]Forskolin alone or in combinationwith unlabeled forskolin was added (0.2 ml of 50 mMTris HCl, pH 7.5/10 mM MgCl2) to the tubes and allowed toincubate 1 hr at 23°C. The tubes were centrifuged at 20,000 x

g for 15 min at 0°C. The supernatant was aspirated and theassay tubes were washed three times with 3 ml of ice-cold 50mM Tris HCI, pH 7.5. Washing of the tubes was carried outcarefully with a Brandel cell harvester (Gaithersburg, MD)so that the pellet was not disturbed. The pellets were thendissolved in 1 ml of 0.2 M NaOH and radioactivity was de-termined by liquid scintillation counting.

Filtration Assay. Incubations were carried out as describedabove in 13 x 100 mm glass tubes. After the standard incuba-tion the membranes were rapidly filtered with a Brandel cellharvester using GF/C filters. The filters were washed threetimes with 4 ml of ice-cold buffer (50 mM Tris HCl, pH 7.4).GF/C filters had lower blanks (=0.04% of total radioactiv-ity) than GF/B filters.

Binding was maximal in about 30 min at room tempera-ture. Triplicate assays using either the filtration or centrifu-gation assays were remarkably consistent, resulting normal-ly in variations among triplicate assays of less than 10%.Data were analyzed by using the LIGAND program devel-oped by Munson and Rodbard (17).

Adenylate Cyclase. Incubations were in a total volume of250 ,ul containing 50 mM Tris HCl buffer at pH 7.5, 1.0 mM3-isobutyl-1-methylxanthine, 5 mM MgCl2, and 0.1 mMATP. Each assay mixture contained 2 ,uCi of [a32-P]ATP andan ATP-regenerating system of 5 units of creatine kinase and2 mM creatine phosphate. Assays were initiated by the addi-tion of 25 ,l of membranes (-100 Mg of membrane protein),were carried out at 30°C for 10 min, and were terminated bythe addition of 0.5 ml of 10% trichloroacetic acid. Carriercyclic AMP (0.25 ml of 1 mM cyclic AMP containing==20,000 cpm of cyclic [3H]AMP), was added, and radioac-tive cyclic AMP was isolated and analyzed as described bySalomon et al. (18). Assays were carried out in triplicate.The EC50 values for activation by forskolin and forskolin an-

alogs were taken from at least two determinations over aconcentration range from 0.01 to 200 AM of the derivative.The EC50 values for the determinations did not vary more

than 20%.

RESULTS

The binding of [3H]forskolin to membranes was determinedby using two different protocols: In the first, membraneswere incubated with increasing concentrations of [3H]for-skolin (0.007-0.5 AM) and no added unlabeled forskolin. Inthe second, membranes were incubated with a low concen-tration of labeled forskolin (0.01 ,uM) in the presence of in-creasing amounts of unlabeled forskolin (0.05-25 AM). Theconcentrations of bound and free forskolin were calculatedand binding parameters were determined by the LIGAND

program (17), using one-site and two-site models. Theamount of bound forskolin determined by using either proto-col was the same, indicating that [3H]forskolin and unlabeledforskolin bind to an equivalent extent.

Binding data from five different centrifugation experi-ments are shown in Fig. 1. Nonspecific binding of [3H]fors-kolin is defined from the ratio of bound [3H]forskolin to total[3H]forskolin at the highest concentration of forskolin. Thisratio, corresponding to the fractdon of total [3H]forskolinnonspecifically bound, is considered to be constant in allconcentrations of forskolin. Nonspecific binding in the cen-trifugation experiments was largely associated with the frac-tion of radioactivity trapped in the membrane pellet and was2-3% of the total forskolin added. The best parameters de-termined for a one-site model and for a two-site model aregiven in Table 1. The binding curve calculated with the pa-

0.07A

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FIG. 1. Binding of [3H]forskolin to rat brain membranes deter-mined with a centrifugation assay. The membranes (1 mg per assay)were incubated with increasing amounts of [3H]forskolin (0.1-100nM). Binding at higher concentrations of forskolin was determinedwith a constant amount of [3H]forskolin (10 nM) and increasingamounts of unlabeled forskolin (0.01-40 gM). Data frorh five differ-ent experiments (x) were pooled and analyzed by the LIGAND pro-gram. The 95% confidence limits (upper and lower lines) are shownfor each computer-generated fit (middle lines). (A) Binding parame-ters for a one-site model: Kd = 20 nM, Bn.,, (maximal binding) =0.350 pmol/mg. (B) Binding parameters for a two-site model: Kd =11 nM, Bmax, = 0.204 pmol/mg; Kd2 = 0.77 ,uM, BmaX2 = 3.7pmol/mg.

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Table 1. Binding of [3H]forskolin to rat brain membranes

Bmax'pmol/mg

Method Model Kd, nM proteinCentrifugation (14) One-site 27.8 ± 2 0.52 ± 0.03

Two-site 15.4 ± 1.8 0.27 ± 0.031100 ± 500 4.2 ± 1.6

Filtration (5) One-site 25.8 ± 4.4 0.40 ± 0.05Binding assays were carried out at 230C for 1 hr in 50 mM

Tris HCI, pH 7.5/5 mM MgCl2. All data were analyzed by the LIG-AND program and included binding experiments performed with in-creasirg amounts of [3H]forskolin as well as binding experimentswith increasing amounts of unlabeled forskolin. The numbers in pa-rentheses indicate the number of experiments pooled and analyzedby the LIGAND program; ± indicates SEM.

rameters for the single-site model (Fig. 1A) fit the data verywell at concentrations of [3H]forskolin less than 0.1 AM.However, there are large deviations in the fit at higher con-centrations of [3H]forskolin. The curve calculated for a two-

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FIG. 2. Scatchard plots of the binding of [3H]forskolin to ratbrain membranes, determined with a centrifugation assay. Data are

from Fig. 1. Computer-generated fits for a one-site model and a two-site model are shown in A and B, respectively. Binding parametersare given in the legend to Fig. 1. The ratio of bound to total corre-sponding to nonspecific binding has been subtracted from the dataand is 0.026 for the one-site model (A) and 0.023 for the two-sitemodel (B).

site model (Fig. 1B) fit the experimental data at concentra-tions of forskolin greater than 0.1 ,uM much better than thatfor the single-site fit. A Scatchard plot of the data is curvilin-ear, which is also indicative of two classes of binding sites(Fig. 2).

Binding of [3H]forskolin to rat brain membranes was alsodetermined by using a filtration assay. Data from experi-ments with increasing concentrations of [3H]forskolin andfrom competition experiments with a constant amount of[3H]forskolin and increasing concentrations of unlabeledforskolin are plotted in Fig. 3. The data are adequately fit bya one-site model (Table 1). The binding isotherm is not de-scribed significantly better by a two-site model. The disso-ciation constant determined by the filtration assay and thedensity of binding sites are very close to those determinedfor the high-affinity sites by using the centrifugation assay.Unlabeled forskolin (10 AM) displaced about 80% of the to-tal radioactivity bound at 0.1 AM [3H]forskolin (data notshown). It is difficult to define the specific binding of [3H]-forskolin at concentrations greater than 1 AM as displaceablebinding because of the limited solubility of forskolin (<100AM).Various analogs of forskolin were able to compete for

[3H]forskolin binding sites (Fig. 4, Table 2). Ki values forunlabeled forskolin and the forskolin analogs were deter-mined from competition studies. The K1 for unlabeled fors-kolin was 29 ± 8 nM, which is very close to the Kd deter-mined from direct binding studies (vide supra). The most po-tent analog was the 7-ethylcarbonate, which is almostequipotent to forskolin in activating adenylate cyclase. The7-formyl, 7-desacetyl, 6-acetyl-7-desacetyl, 11,-hydroxy,and 14,15-dihydro analogs of forskolin are less potent in acti-vating adenylate cyclase and had Ki values for competitionwith [3H]forskolin that ranged from 176 nM to 622 nM. The7-tosyl analog of forskolin is much less potent in activatingadenylate cyclase and was very weak at competing for[3H]forskolin binding sites. Forskolin analogs that do not ac-tive adenylate cyclase include the 1,6-diketo analog, 1,9-di-deoxyforskolin, and 14,15-oxidoforskolin. These forskolinanalogs did not compete effectively for [3H]forskolin bindingsites in rat brain membranes even at 100 ,M. Ki values de-rived from centrifugation or filtration experiments were thesame for all analogs within experimental error.The binding of [ H]forskolin was decreased in the absence

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FIG. 3. Binding of [3H]forskolin to rat brain membranes, deter-mined with a filtration assay. Membranes (1.2 mg per assay) were

incubated with increasing concentrations of [3H]forskolin (o) or with20 nM [3H]forskolin and increasing concentrations of unlabeled fors-kolin (e). The bound forskolin was determined by using the filtrationassay. The data were fit to a one-site model: Kd = 28 nM, Bmax =0.325 pmol/mg. A Scatchard plot of the data is given in the Inset. F,free forskolin.

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FIG. 4. Inhibition of binding of [3H]forskolin to rat brain mem-branes by analogs of forskolin. Membranes (800 ,g per assay) were

incubated with 10 nM [3H]forskolin in the presence of various con-

centrations of forskolin derivatives: 7-ethylcarbonato-7-desacetyl-forskolin (o), 14,15-dihydroforskolin (e), and 1,9-dideoxyforskolin(o). Binding in the experiment depicted in this figure was deter-mined by using a centrifugation assay. Binding parameters for theanalogs are given in Table 2.

of magnesium (Fig. 5) and in the presence of 1 mM EDTA(data not shown). The Bmax was 270 fmol/mg of protein inthe presence of 5 mM MgCl2 (standard assay condition) and200 fmol/mg of protein in the absence of added MgCl2 (Fig.5) with no change in Kd. The presence of 1 mM EDTA withno added magnesium ions reduced the binding to 110fmol/mg of protein with no change in Kd (data not shown).The maximal increase in binding was achieved at about 5mM magnesium. Manganese (5 mM), like magnesium, in-creased the maximal number of sites with no change in theaffinity for [3H]forskolin (Fig. 5). Calcium had no effect onbinding in concentrations up to 20 mM in the presence of 1mM EDTA (data not shown). Sodium chloride (100 mM) wasnot able to increase the number of binding sites (Fig. 5).There was no detectable high-affinity binding of [3H]fors-

Table 2. Forskolin analogs: Inhibition of binding of [3H]forskolinand activation of adenylate cyclase

Ki forinhibition of EC50 forbinding of activation of

[3H]forskolin,t adenylate cyclase,Analog* nM yM

Forskolin 29 ± 8 4

7-Ethylcarbonatoforskolin 120 ± 11 4

11,-Hydroxyforskolin 158 ± 22 25

7-Formylforskolin 176 ± 19 306-Acetylforskolin 329 ± 49 1014,15-Dihydroforskolin 496 ± 56 307-Desacetylforskolin 622 ± 56 307-Tosylforskolin 5000 ± 200 >251,6-Diketoforskolin >105 >10314,15-Oxidoforskolin >105 >1031,9-Dideoxyforskolin >105 >103*Forskolin has an acetyl moiety at the 7-hydroxy function. The 7-ethylcarbonato, 7-formyl, and 7-tosyl analogs are derivatives offorskolin with the indicated functional group replacing the acetylgroup at the 7 position. The 11P-OH analog is a derivative of fors-kolin with the 11-keto group reduced. The 1,6-diketo analog has the1- and 6-hydroxy functions oxidized.

tInhibition constants were determined from at least two separatedisplacement experiments; ± indicates SEM. K, values were calcu-lated by using a single-site model, with the Kd for forskolin beingthat determined from the filtration experiments.

0

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FIG. 5. Effect of MgCl2, MnCl2, and NaCl on [3H]forskolin bind-ing. Membranes were incubated with increasing concentrations of[3H]forskolin in 0.4 ml of 50 mM Tris HCl, pH 7.5 (o), and with thefollowing additions: 100 mM NaCl (A), 5 mM MgCl2 (o), or 5 mMMnCl2 (o). Bound forskolin was determined by using a filtration as-

say. Data were analyzed with the LIGAND program. Bmax withbuffer alone (o) was 200 fmol/mg of protein; with 0.1 M NaCl (A),190 fmol/mg; 5 mM MgCl2 (o), 293 fmol/mg; and 5 mM MnCl2 (o),330 fmol/mg. There was no significant difference in the Kd (13 nM)under all the above assay conditions.

kolin to ram sperm membranes (1 mg of protein per assay) atconcentrations of [3H]forskolin from 1 nM to 1 AM. Theseexperiments were carried out with the filtration assay. Theratio of bound [3H]forskolin to total [3H]forskolin was con-

stant and was unaffected by the inclusion of 20 ,M unla-beled forskolin in the incubation mixture.

DISCUSSION[3H]Forskolin binds to rat brain membranes at two classes ofsites: The high-affinity binding sites have a Kd of 15 nM anda Bmax of 270 fmol/mg of protein. The low-affinity bindingsites have a Kd of 1.1 ,M and a Bmax of 4 pmol/mg of pro-tein. The 14,15-ditritio derivative of forskolin with the dou-ble bond reduced has also been used as a radioactive ligandto measure forskolin binding sites in rat brain: The K1 valuesfor 14,15-dihydroforskolin and forskolin determined by com-petition experiments were 225 nM and 100 nM, respectively(19). In the present study the Ki values of 14,15-dihydrofor-skolin and forskolin were determined to be 476 nM and 29nM, respectively. The maximal binding determined with the14,15-ditritioforskolin was about 3 pmol/mg of protein (19),which is very close to what is determined for the low-affinitybinding sites by using [3H]forskolin in the present paper. Itmay be that the studies with the 14,15-ditritioforskolin (19)did not detect the high-affinity binding sites that are detectedwith the [3H]forskolin.The forskolin binding sites are sensitive to proteolytic en-

zymes, N-ethylmaleimide, and high temperature and thuswould appear to be associated with a protein(s) (data notshown). The adenylate cyclase in ram sperm membranes isnot activated by forskolin (15, 16) and binding sites for[3H]forskolin were not detected in ram sperm membranes,The ability of forskolin analogs to compete for [3H]forskolinbinding sites in brain membranes correlates well with theirability to activate adenylate cyclase (20). However, it is diffi-cult to equate the affinity of the high-affinity binding sitesfor [3H]forskolin with the potency of forskolin as an activa-tor of adenylate cyclase: Activation of adenylate cyclase byforskolin occurs with an EC50 of about 5-10 ,M, which isabout 100-fold higher than the observed Kd for the high-af-

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finity binding sites. The Kd for the lower-affinity sites (=1,uM) is closer to the EC50 for activation of adenylate cyclase.It is possible that the low-affinity binding sites are associatedwith the protein mediating the direct activation of adenylatecyclase by forskolin. If this protein were the catalytic sub-unit, then the amount of adenylate cyclase in rat brain mem-branes would be estimated as about 4 pmol/mg of protein.However, the amount of low-affinity binding sites for[3H]forskolin is much higher than the amounts usually ob-served for hormone receptors in rat brain (50-600 fmol/mgof protein). It has been assumed that the molar amount ofcatalytic subunit of adenylate cyclase is similar to or lessthan that of hormone receptors.

Forskolin has the ability to markedly potentiate hormoneactivation of adenylate cyclase at concentrations (0.01-0.1,xM) much lower than required for the direct activation byforskolin alone (refs. 10 and 12 and references therein). It ispossible that the high-affinity binding sites identified in thepresent study with [3H]forskolin are those associated withhormone potentiation by forskolin and are distinct fromthose associated with the direct activation of adenylate cy-clase by forskolin. This explanation requires that forskolinhave two sites of action. An alternative explanation for thepresence of the high-affinity binding sites and the hormonalpotentiations observed at low concentrations of forskolin isthat forskolin binds at a single site whose affinity for forsko-lin is enhanced by the binding of hormones to their receptorand the consequent formation of an activated complex ofcatalytic and Ns subunits. This high-affinity binding of fors-kolin would not necessitate the simultaneous binding of fors-kolin and Ns subunit at the catalytic subunit, since it is possi-ble that activated states of the catalytic subunit can persistafter dissociation of either forskolin or the activated Ns sub-unit (14). It is tempting, however, to speculate that a ternarycomplex can exist between forskolin, the catalytic subunit,and the activated Ns subunit and that the binding of eitherforskolin or the activated Ns subunit promotes the binding ofthe other. Any agent that would promote the activation ofthe adenylate cyclase by Ns would then promote the forma-tion of the high-affinity state of the protein. This would in-clude Mg2+, Mn2+, and NaF, all of which increase the bind-ing of [3H]forskolin (Fig. 5 and data not shown). In the filtra-tion assay, this results in an increase in the maximal numberof binding sites with no observable change in Kd for forsko-lin. Only the high-affinity state of the enzyme is detected inthe filtration binding assay. Even if the low-affinity bindingsites are the unactivated catalytic subunit, it would be diffi-cult to detect a decrease in Bmax of 0.1 pmol/mg of proteinsince the maximal amount of low-affinity binding sites isabout 4 pmol/mg. Furthermore, it is at present unknownwhether all or any of the low-affinity binding detected byusing the centrifugation assay measure forskolin sites in-volved in direct activation of adenylate cyclase by forskolin.

It has been shown in many different systems that maximalstimulation of adenylate cyclase by forskolin requires thepresence of the Ns subunit. This has been demonstrated foradenylate cyclase in membranes (21), intact cells (22, 23),and solubilized preparations (14). Activation of adenylatecyclase in S49 wild-type membranes displays kinetics of acti-vation by forskolin consistent with two components, a high-affinity component with an EC50 of about 0.1 ,uM and a low-er-affinity component with an EC50 of about 10 gM (21).Only the low-affinity component is observed in the cyc mu-tant of the S49 lymphoma, which does not contain a func-

tional N, subunit. The high-affinity component is thereforeassociated with the presence of a functional N, subunit.These observations led Green and Clark (21) to postulatethat forskolin's affinity for adenylate cyclase will be en-hanced by an activated N, subunit, in agreement with ourmodel for cooperative binding of forskolin and N,.

Forskolin binding sites in rat brain membranes have beenidentified by using [12-3H]forskolin. These sites are satura-ble, of high affinity, and display structure activity require-ments consistent with forskolin's ability to activate adenyl-ate cyclase. This provides a convenient assay for forskolinanalogs, forskolin antagonists, and endogenous forskolin-like compounds. The high-affinity binding sites may be asso-ciated with a state of the adenylate cyclase enzyme promot-ed by stimulatory hormones and the N, subunit. A final con-firmation of this model will require further binding studies inintact cells and other tissues that have hormonally respon-sive adenylate cyclase and in solubilized preparations ofadenylate cyclase.

We are indebted to Dr. David Rodbard for helpful discussions andsuggestions concerning experimental design and computer analysisof binding data. An accurate description of [3H]forskolin bindingsites would have been difficult without his advice and the use of theLIGAND program.

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