relationship between the p-adrenergic receptor and ... · the journal or biological ch~m,~~ry vol...

THE JOURNAL or BIOLOGICAL CH~M,~~RY Vol 252, No 16, Issue of August%, pp. 5161-5715, 1911

Prrnted ,n U.S.A.

Relationship between the P-Adrenergic Receptor and Adenylate Cyclase STUDIES OF LIGAND BINDING AND ENZYME ACTIVITY IN PURIFIED MEMBRANES OF S49 LYMPHOMA CELLS*

(Received for publication, December 27, 1976, and in revised form, February 28, 1977)

ELLIOTT M. Ross,+ MICHAEL E. MAGUIRE,~ THOMAS W. STURGILL, RODNEY L. BILTONEN, AND ALFRED G. GILMAN~

From the Departments of Pharmacology and Biochemistry, University of Virginia School of Medicine, Charlottesville, Virginia 22903

Purified membrane fractions prepared from S49 lymphoma cells have been used to assess binding to the p- adrenergic receptor and activation of adenylate cyclase under identical assay conditions. In this preparation, activation of the enzyme by isoproterenol (or by prostaglandin E,) is dependent on the presence of a purine nucleotide (GTP, ITP, guanyl-5’-yl imidodiphosphate (Gpp(NH)p), etc.) in addition to the 0.5 to 1 mM ATP used as substrate. The affinity of the /3-adrenergic receptor for agonists, but not for antagonists, is decreased by these purine nucleotides. All such nucleotides, when present in maximally effective concentrations, reduce affinity for agonists to the same extent. This change in affinity is accompanied by an elevation of the apparent Hill coefficient for the binding of agonists from 0.5 to 0.9. These changes in binding are reversible by washing when either GTP or Gpp(NH)p is the nucleotide. Variants of the S49 cell that are deficient in adenylate cyclase activity show agonist-specific changes in binding to the /3-adrenergic receptor. Binding of agonists to the receptor in these variants in the presence or absence of a regulatory nucleotide is indistinguishable from binding to receptors from wild type cells assayed in the presence of nucleotide.

Coupling between binding of agonists to the /3-adrenergic receptor and activation of adenylate cyclase is variable and is dependent on the nucleotide present. While the K,, for isoproterenol is nearly 500 nM in the presence of GTP, ITP, guanyl-5’-yl(&y-methylene)diphosphate (Gpp(CH,)p), or Gpp(NH)p. values ofK,,, for isoproterenol in the presence of these nucleotides are approximately 100, 50, 5, and 4 nM, respectively. When Gpp(NH)p or Gpp(CH,)p is used, the value of K,,., for isoproterenol is dependent on time since these nucleotides activate the enzyme irreversibly; isopro-

* This work was supported by United States Public Health Service Grants NS10193 and AM17042. A preliminary report of some ofthese data has been presented (1).

$ Recipient of National Institutes of Health Postdoctoral Fellow- ship CA05279.

# Present address, Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106.

11 Recipient of National Institutes of Health Research Career De- velopment Award NS00026. To whom correspondence concerning this manuscript should be addressed.

terenol enhances the rate of this process but has no effect on the irreversibly activated enzyme. The limiting value of the K,,, for isoproterenol at t = 0 in the presence of Gpp(NH)p is 14 nm-still greatly discrepant from the K,,.

Exposure of cells to cholera toxin also appears to alter the stoichiometry of coupling between receptor and enzyme. While the K,, for isoproterenol in the presence of GTP is identical in membranes from treated or control cells, the K,,, is lowered by a factor of 10 by treatment of the cells with toxin.

These data are discussed in terms of equilibrium and kinetic models of the hormone-sensitive adenylate cyclase system.

Elucidation of the mechanism of interaction between the /3- adrenergic receptor and adenylate cyclase is important to those interested in the metabolism of cyclic AMP,’ the action of adrenergic neurohormones and drugs, or the interaction of integral membrane proteins. A necessary step in the approach to a mechanism has been the recent development of specific assays for the receptor itself (2-4); these allow the determina- tion of the fractional amount of receptor bound either by an agonist or antagonist under the conditions where activation of the enzyme is being measured. Nevertheless, assays of adenylate cyclase activity in membrane fractions have not been as informative as one might hope. Gross differences between cell types and specie: of origin, extreme dependence on the pH, ionic strength, and composition of the medium, and the possible requirement for nonsubstrate nucleotides at one or more regulatory sites have made it very difficult to arrive at definite conclusions about the action of hormones. This situation is obviously confounded when enzyme activation and ligand

I The abbreviations used are: cyclic AMP, adenosine 3’:5’-mono- phosphoric acid; Hepes, 4-(2-hydroxyethylj-l-piperazineethanesul- fonate; HME buffer, 20 rnM sodium Hepes, 2 mM MgCl,, 1 mM sodium EDTA, pH 8.0; IHYP, iodohydroxybenzylpindolol (hydroxybenzylpindolol is (~)-3-indoloxy-l-(2-p-hydroxybenzylpropyl-2-amino)iso- propanol); PGE,, prostaglandin E,; R020-1724, 4-(3-butoxy-4-me- thoxybenzyl)2-imidazolidinone; Gpp(NH)p, guanyl-5’-yl imidodiphosphate Gpp(CH,)p, guanyl-5’-yl(P,y-methylenejdiphosphate; lpp(NH)p, inosinyl imidodiphosphate; kinetic and thermodynamic constants are defined in the text.

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5762 p-Adrenergic Receptor-Adenylate Cyclase Relationship

binding are studied under different conditions. Alternative approaches to the problem have been the disec-

tion of hormone-sensitive adenylate cyclase systems either by genetic techniques (5-7) or by attempts to isolate the individual components of the system. Hypothetically, these include a receptor, a catalytic moiety, and a regulatory purine nucleotide-binding component. Progress in this area is now evident (8-12). On balance, the S49 lymphoma cell appears to possess the most favorable combination of properties that are requisite for a combined genetic, biochemical, and pharmacological resolution of a hormone-sensitive adenylate cyclase system. This clonal murine line is amenable to genetic dissection, since cyclic AMP is cytocidal to the cells (13); it is diploid and grows to relatively high density in simple suspension cultures; and the cells synthesize high concentrations of cyclic AMP in response to both P-adrenergic agonists and prostaglandins, particularly those of the E series (5). We have thus chosen to concentrate on the resolution of the adenylate cyclase system of this clone and on appropriate variant clones derived there- from.

To further this study, it was necessary to develop techniques for preparation of relatively purified plasma membrane fragments. The fraction described has favorable properties for study of the regulation of enzyme activation by hormone and the role of guanine (and other) nucleotides in this process. The current goal has been to relate activation of adenylate cyclase by catecholamines to the binding of ligands to the P-adrenergic receptor. The complex results of these experiments have provoked our attempt to rationalize them with models for interaction between the P-adrenergic receptor and adenylate cyclase. In this communication, as the data on binding and activation of adenylate cyclase are presented, we will attempt to described simultaneously certain components of the models that are invoked by the increasing complexity of the observations. More general discussion will then concentrate on integration of these components and speculation on their biochemical correlates.

EXPERIMENTAL PROCEDURES

Assays - Adenylate cyclase activity was measured in a final volume of 100 ~1 containing 0.5 rn~ I &‘PlATP (10 to 60 cpm/pmol), 10 mM MgCl,, 1 mM sodium EDTA, 0.1 mM R020-1724, 3 mM K, phosphoenolpyruvate, 10 pglml of pyruvate kinase, 0.1 mg/ml of bovine serum albumin, 0.1 mM sodium ascorbate. and 50 mM sodium Hepes, pH 8.0. Unless otherwise noted, reactions were started by the addition of membrane protein (5 to 20 ILR) and were incubated for 20 min at 30” with constant shaking. Reactions were stopped and cyclic I”‘PlAMP was quantified by the method of Salomon et al. (14). The observed rate of reaction for the NaF-stimulated enzyme was a linear function of time and membrane protein concentration.

Binding to the p-adrenergic receptor was assayed with the ligand I ‘“:‘IlIHYP, essentially as described (41. These reaction mixtures (100 ~1) were incubated for 30 min at 30” with shaking, and the components were the same used far the assay of adenylate cyclase, except that ATP was not radioactive and 1 ““IIIHYP was included. Specific binding is defined here as the difference in the amount of 1 ““IIIHYP bound in the presence and absence of 1 FM (-)-propranolol. When purified membrane fractions and the usual concentration of ligand (90 to 120 PM) were used, specific binding constituted approximately 95% of that observed in the absence of propranolol.

Protein was assayed after precipitation with trichloroacetic acid according to Lowry et al. (15); bovine serum albumin was the stan- dard.

Cells and Cell Culture-The catecholamine- and prostaglandin- sensitive (wild type) clone 24.3.2 of the S49 lvmnhoma cell line was obtained from Dr: H. Bourne, University of California, San Fran- cisco. The catecholamine-resistant variant clone 94.15.1 (5) and other similarly derived clones were obtained from Dr. P. Coffino, University of California, San Francisco. Additional adenylate cyclase-deficient variants were selected from the wild type clone by

culture of cells for 6 days in medium containing 3 &ml of cholera toxin, 30 pM R020-1724, and 100 PM I-methylI3-isofbutylxanthine, with two changes of medium, followed by cloning in soft agar (16) containing the same agents. Cells selected in this way retained sensitivity to killing by N”, 0”-dibutyryl cyclic AMP, but they were insensitive to the cytocidal or adenylate cyclase-stimulating effects of isoproterenol or cholera toxin. Lymphoma cells were routinely grown in stationary suspension or in spinner cultures at 37” in Dulbecco’s modified Eagle’s medium (4.5 g/liter of d-glucose) containing 7.5 to 10% heat-inactivated horse serum. Mouse embryo fibrobhasts, used for the feeder layer required for the cloning tech- nique, were purchased from Microbiological Associates and were propagated in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal calf serum, at 37” in an atmosphere of 90% air and 10% co,.

Preparation of Plasma Membranes - Cells were harvested at a density between 2.0 and 3.5 x lO”/ml by low speed centrifugation and were washed twice at room temperature in 137 mM NaCl, 5.36 mM KCl, 1.1 mM KH,PO,, and 1.08 mM Na,HPO,, pH 7.2. Subsequent steps were performed at O-4”. Cells were susnended to 3 x lO’/ml in 156 mM NaCl, 20 mM sodium Hepes, 2 mM MgCl,, 1 mM EDTA (pH 7.4 at O”), and were lysed by rapid decompression after equilibration for 20 min with N, at 400 p:s.i.in a Parr cell disruption bomb. (Parr Instrument Co., Moline, Ill.). The lysate was centrifuged for 5 min at 2000 rpm in an IEC #269 rotor (relative centrifueal force....,, = 900 x g). The supernatant was centrifuged for 20 minat 18,560”rpm in a Sorvall SS-34 rotor (relative centrifugal force,,, = 43,000 x g), and this pellet was homogenized in a type B Dounce homogenizer in 20 mM sodium Hepes, 2 mM M&l,, 1 mM EDTA, pH 8.0 (HME buffer) containing 10% (w/w) sucrose (about 1 ml/lo” ceils). Approximately 5 ml of this suspension was applied to a discontinuous sucrose density gradient containine 40% sucrose (12 ml). 30% sucrose (10 ml). and 20% sucrose (10 mfi, all in HME buffer. The gradients were centrifuged for 80 min at 27,000 rpm in a Spinco SW-27 rotor. The bands at the 20 to 30% and 30 to 40% sucrose interfaces were separately diluted with HME buffer and centrifuged for 40 min at 100,000 x g. The two pellets from this centrifugation, referred to as the plasma membrane fractions, were suspended in HME buffer to a protein concentration of approximately 1 mg/ml. After addition of 1 mM dithiothreitol, preparations were frozen in a bath of dry ice and ethylene glycol monomethyl ether; both adenylate cyclase and p- adrenergic receptor binding activities were stable for several months at -85”. A routine preparation now consists of 5 x 10”’ cells grown in two &liter spinner cultures; this quantity may be processed in the six tubes of the SW-27 rotor.

Materials- 18-“HlAdenosine 3’:5’-mononhosnhate, 18-“Hlauanine, _ . ~- cholera toxin, and enzyme grade sucrose were obtained from Schwarz/Mann, isotopically pure Na”“I from AmershamSearle, and 1 a-:“PlATP from either Amersham/Searle or New England Nuclear. (tl-Hydroxybenzylpindolol, (-)-propranolol, and R020-1724 were gifts of Drs. D. Hauser (Sandoz, Inc., Basel), D. J. Marshall (Ayerst Research Labs), and H. Sheppard (Hoffmann-La Roche), respectively. (-)-Isoproterenol was obtained from Sigma, while nucleotides and pyruvate kinase were purchased from Boehringer Mann- heim. 1 “VIHYP was prepared as described (4).

RESULTS

Preparation ofS49 Lymphoma Plasma Membranes - Puri- fication of S49 lymphoma plasma membranes is described under “Experimental Procedures,” and a typical preparation is summarized in Table I. Lysis of the cells by decompression cavitation in neutral isotonic medium containing Mg2+ pre- vents the destruction of most of the nuclei (17). This homogeni- zation procedure also leads to significantly improved subsequent fractionation and to far less aggregation of membrane fragments, compared to cell disruption in a Dounce homogenizer after swelling in hypotonic solutions. The nuclei are removed by centrifugation at 900 x g, and the bulk of the remaining particulate material and most of the adenylate cyclase activity are then collected and applied to a discontinuous sucrose gradient. Adenylate cyclase activities assayed in the presence of NaF, Gpp(NH)p, isoproterenol plus GTP, or PGE, plus GTP fractionate with each other and with P-adrenergic receptor binding. These specific activities are all increased 3- to lo-fold in the 20 to 30% and 30 to 40% sucrose


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p-Adrenergic Receptor-Adenylate Cyclase Relationship 5763

interface fractions, relative to the activity of the material applied to the gradient, and in the best preparations they are up to 40-fold greater than those of the crude cell lysate. The recovery of each of these activities from the gradient (receptor, adenylate cyclase measured as above), expressed as a fraction of the amount applied, is similar to the recovery of protein. In contrast, adenylate cyclase activity measured in the presence of either isoproterenol or PGE, alone represents a recovery of less than 10% of the activity measured under these conditions that was applied to the gradient. In fact, with the purified membrane fraction and the assay conditions used, neither isoproterenol, PGE,, nor GTP alone increases adenylate cyclase activity (Fig. 1); however, in the presence of GTP or ITP, either isoproterenol or PGE, can stimulate the enzyme markedly, and vice versa. Gpp(NH)p itself can activate adenylate cyclase (Fig. 1); this will be discussed more fully below. The gradient procedure does appear to be effective for separation of plasma membrane fragment from mitochondrial membranes. Analysis of cyanide-sensitive cytochrome c oxidase indicates that 0.8% of this mitochondrial activity is at the 20 to 30% interface, 6% is at the 30 to 40% interface, and the remainder is in the pellet.

We speculate that this loss of response to hormone alone in the plasma membrane fractions is caused by the removal of nucleotides (or sources of free nucleotides, e.g. mitochondria and RNA) which are required for hormonal stimulation of adenylate cyclase. To test this hypothesis qualitatively, 13Hlguanine (0.6 &i/ml, 6 Ci/mmoll was added to the culture 16 h before cells were harvested to monitor distribution of guanine nucleotides (and their metabolic products) among the various subcellular fractions. Table I shows that less than 2% of the tritium in the homogenate is found in the plasma membrane fraction; this amount is essentially all insoluble in trichloroacetic acid. While detailed experiments would be necessary to substantiate this hypothesis further, these data are consistent with the presence of significant quantities of guanine nucleotides in washed crude particulate fractions, and such nucleotides are readily detected by high pressure liquid chromatography. In any case, purification has resulted in a preparation in which a purine nucleotide in addition to 0.5 to 1 mM ATP is requisite for hormonal activation of adenylate cyclase.

Binding of I “V]IHYP to the S49 P-Adrenergic Receptor - As shown previously, binding of the P-adrenergic antagonist [‘2”IlIHYP can be used for the assay of the P-adrenergic receptor, and the degree of binding of other adrenergic ligands can be assessed by study of competition for binding sites. The shape of each of the curves in Fig. 2A for the time-dependent association of l’2”I]IHYP with receptor is consistent with a simple bimolecular reaction. An apparent second order rate constant, K, is calculated to be approximately 4 x 10R liters.molll ‘min-!, and a constant amount of bound ligand is achieved by 30 to 60 min under the usual assay conditions. While most binding studies were performed with 30-min incubations to approximate the time of the adenylate cyclase assay more closely, essential features of the binding data are unaltered if examined affer 60 min.

The dissociation process is more complex, as shown in Fig. 2B; the data are consistent with the existence of two kinetic components of dissociation-a fast process characterized by a t1,2 of approximately 2 min and a slower phase with a t,,z of 3 h. The rate of formation of the rapidly dissociating component is probably very fast. At times after 2.5 min, the absolute amount of this component appears to be independent of time,

suggesting equilibration of this component with free ligand. By contrast, the amount of the slowly ,dissociating component continues to increase until a limiting value is attained.

A Scatchard representation of the binding of I ““IIIHYP to the plasma membrane fraction of S49 lymphoma cells is shown in Fig. 3. It cannot be claimed that the experiment shown is representative. Despite meticulous attention to detail, experiments of this type have yielded a relatively broad range of values for K,, (50 to 140 PM), and the data for individual Scatchard plots have frequently displayed considerable scat- ter. However, there is no perceptible trend toward deviation from linearity; this fact and experiments of the type shown in Fig. 3 are indicative of the existence of a set of thermodynamically equivalent sites. An average K,, = 90 PM and the entire range of values will be utilized in calculations as appropriate. In the various preparations used in this study, the receptor density varied from 100 to 300 fmol/mg of protein.

Kinetic experiments thus yield dam indicative of two components of binding, while equilibrium binding experiments are consistent with the existence of a set of equivalent sites. Similar situations were encountered in studies of lJH]prostaglandin E, binding (181 and in our previous experience with 1 12”IlIHYP (4). If in fact a second independent set of sites exists, they must represent a minor fraction of the total. Thus, such sites are not visible in Scatchard plots as a class with differing affinity, and, if their affinity for l’2”I]IHYP is the same as that of the receptor sites, the data of Fig. 2B indicate that they represent on the order of 10% of the total. In this case, these sites will have essentially no impact on the conclusions of this study.

The data are also consistent with the possibility that both kinetic phases represent dissociation of I ““IIIHYP from the /3- adrenergic receptor. In this case, a monotonic Scatchard plot would indicate that two forms of the receptor are interconvertible. It is necessary to consider the relationships between kinetic and equilibrium constants for such a situation in order to decide if the experimental values obtained are internally consistent. The following model is hypothesized:

kl k2 R + IHYP\ RlIHYP,\ R2IHYP (1)

k-1 k-2

where R, RJHYP, and RJHYP, respectively, represent the free receptor, a rapidly formed complex which can dissociate quickly, and the thermodynamically favored complex which is slowly formed. At equilibrium, the fractional degree of saturation, f, will be specified by the relationships:

f= IR,IHYPI + [R~IHYP]

(2) Rt

f= KapplIHYPl

(3) l+K app [ IHYPI

where K,,,, is the apparent association constant for binding, R, is the total concentration of all forms of the receptor, and bracketed entities are concentrations of the individual species in Equation 1. The apparent association constant is:

K [R~IHYP] + [R~IHYP]

aPP = [RI lIHvP1 (4)

In terms of the association constant for the first step in Equa- tion 1, K,, and the isomerization constant for the second step, L

K aPP

= Kl(1 + K2) (5)


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5764 @Adrenergic Receptor-Adenylate Cyclase Relationship

TABLE I Preparation of S49 lymphona plasma membranes

13HlGuanine” l’*~IlIHYP bindin?

Fraction Protein (mg) Total counts per min

(S recovery) Trichloroacetic acid-insoluble counts per min

(% recovery)

Homogenate

900 x supematant g

43,000 x g pellet, ”

10 to 20% interface

20 to 30% interface

30 to 40% interface

Gradient nellet

517 4.4 x 10" UOO)

339 2.4 x lo6 (55)

68 6.4 x loj (15)

1.2 3 x 104 (0.6)

1.5 3 x 104 (0.6)

5.1 5 x 104 (1.2)

32.2 3.7 x 105

1.5 x 10” (100)

7.5 x lo” (50)

2.4 x 109 (16)

2 x 104 (1.3)

3 x 104 (2.4)

5 x 10" (3.3)

1.4 x lo:'

9.5 (1) 11 (1.2) 28 (2.9)

45 (4.7)

264 (28) 154 (16)

6.5

4910 (100)

3780 (77)

1890 (39)

54 (1)

398 (8.1)

785 (16) 209

(8) (9) (0.7) (4.2) ~___

” Total radioactivity was measured in aliquots of each fraction. Trichloroacetic acid-insoluble radioactivity was determined by adding one- fourth volume of cold 50% trichloroacetic acid; after 1 h each sample was filtered (0.45 pm Millipore), washed with cold 10% trichloroacetic acid, and counted.

b Measured as described under “Experimental Procedures.” Total ligand concentration was 175 PM.

FIG. 1. Activation of adenylate cyclase as a function of concentrations of hormones and nucleotides. Assays were performed as described under “Experimental Procedures.” The data shown are taken from four experiments. A, adenylate cyclase activity as a function of hormone concentration. O,O, isoproterenol; q ,W, prostaglandin E,. Open symbols, no nucleotide present; closed symbols, assayed in the presence of 50 PM GTP. B, activity as a function of nucleotide concentration. O,., GTP, A,A, ITP; W, Gpp(NH)p. Closed symbols, no hormone present; open symbols, assayed in the presence of 1 FM isoproterenol.

Since the data of Fig. 2B indicate that RJHYP represents support this contention.

0 Log [Hormone] (Ml

approximately 10% of the total at equilibrium, K, = 10 and

K =PP = K1(1+K2) '% K1K2 (6)

Kthe first binding step is fast, such that R,IHYP is essentially at equilibrium with respect to IIHYPI and lR1 at all times, and if lIHYP1 is constant, then (see Appendix A)

[R*IHYPl = [R~IHYPI~~~~~ * 0 (7)

where lR,IHYP&,,,, is the equilibrium value of I RJHYPI and

0 = l-expt- (k2 K,. [IHYPI

1 + Kl[IHYPl + km2)t) (8)

As a function of time, the fractional degree of saturation, f, is: 0 + Kl[IHYP]

f= K1tIHypl + KlK21IHYPl 1 + K1lIHYP,

(9) 1 + Kl[IHYP] + K~Kz[IHYP]

While each time course in Fig. 2A is consistent, within experimental error, with a simple bimolecular reaction, the analysis in Equation 9 predicts that in detail the observed kinetics will not be strictly second order. The data of Fig. 2A do show a slight dependence of k on ligand concentration, but a more detailed analysis of the forward reaction would be necessary to

-1 . 0 -0 -7 -6 -5 -4 -3

Log [Nucleotide] (M)

Thus, the approach to true equilibrium in such a scheme is governed by the pseudo-first order rate constant contained in H:

kapP = k2 ~l~l::;:,ypl] + kw2= k[IHYP] (10)

where k is the apparent second order rate constant (4 x 10’ liters. mall’ min’) referred to previously.

This derivation allows a comparison of the apparent equilibrium constant K,,, with the rate constants that were obtained. K,,,,, for the proposed mechanism is = K,K, (Equation 61. In terms of the rate constants, Ka,,p = k,k,lk,k,. Under conditions at which:

k2 [:r::;:HYP,l ’ k-2

then

(11)

Furthermore, under the conditions of our experiment,


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/3-Adrenergic Receptor-Adenylate Cyclase Relationship 5765

Adenylate cyclase 10 rn~ NaF Adenylate cyclase 50 /LLM Gpp(NH)p Adenylate cyclase 1 P’M isoproterenol Adenylate cyclase 10 /LLM rostaglan- + 50 /.m GTP din E, + 50 PM &P

pmol/min/mg C-fold purifica- pmolhin (% re- pmollmi+ng pmol/min/mg

tion) COV2I.y) (-foldtpun~iica- pmollmin (% re- pmol/mir$ng

6foMjx&ica- pmolhnin (% re- (-fold purifica- pmolimin (% re- COW@ COWS) tion) COWQ4

51 (1)

62 (1.1)

122 (2.1)

26 (0.5)

508 (8.9)

938 (16.5)

46

3.0 x 104 (100)

2.1 x 104

(70) 8300

(28) 32 (0.1)

762 (2.5)

3800 (13)

1480

34 (1)

35 (1) 80 (2.3) 26 (0.7)

411 (12) 593 (17)

39

1.8 x lo4

(100) 1.2 x 10’

(67) 5400

(30) 31 (0.2)

617 (3.4)

3000 (17)

1246

62 (1) 59 (1)

114 (1.9) 15 (0.2)

326 (5.3)

794 (13) 44

3.2 x lo4 (100) 2 x 104

(63) 9800 (24)

18 (0.06)

489 (1.5)

4000 (13)

1420

27 (1)

32 (1.2)

65 (2.4) 18 (0.7)

236 (8.7)

466 (17) 26

1.4 x 104 (100)

1.1 x 10”

(79) 4400

(31) 21 (0.2)

354 (2.5)

2400 (17)

837 (0.8) (5.0) (1.1) (6.9) (0.7) (4.4) (1) (6.0)

’ Recovery of adenylate cyclase and binding activity in the 43,000 x g pellet is usually 30 to 50%. Essentially no activity is found in the supernatant, nor, in preliminary experiments, could more activity be accounted for if the pellet and supernatant were recombined.

” The low recovery of protein (60%) from the gradient relative to the 43,000 x g pellet is unusual. Recovery is usually 85 to 95%.

Ii 2.4 i0 Time (mid

FIG. 2. Forward and reverse kinetics of binding 112”IlIHYP to S49 cell plasma membranes. A, forward reaction. Membranes were added to complete assay mixture at 30” containing l”“I]IHYP at the following initial concentrations: 47 PM (01, 93 pM CO), 185 PM (A). Final protein concentration was 0.14 mg/ml, and reaction mixtures were incubated at 30”. This preparation of protein displayed a K,, for l’““I]IHYP of 90 PM and contained approximately 200 fmol/mg of specific lLYzI]IHYP binding sites. At the times shown, quadruplicate loo-p1 aliquots were removed and assayed for bound I”‘IlIHYP. Nonspecific binding, which did not change with time, was subtracted from the total observed. At equilibrium, the fractions of I’2”IlIHYP bound in each case were 0.14 (A), 0.18 (O), and 0.22 (0). B, reverse reaction. Membranes were incubated at 30” in medium containing 150 to 200 PM l’2”I]IHYP for 2.5 min (x), 6 min (a), 10 min (01, or 60

K,[IHYP] < 1. Therefore:

kapp k2kl =- and kapp K aPP (12) [IHYPI km1 [IHYPlk-2

With our kinetic results we find that:

where k is the apparent second order rate constant referred to above. This value should overestimate K,,,. Alternatively, one may attempt to estimate a lower bound for K,,,. The best approximation for k 1 is that obtained from the initial slopes of Fig. 2A; this is in fact a lower estimate for k,. Thus, k,lk-, = 4.10s/0.3 = 1.3.10”, and K,,, = 1.3.10”.K, = 1.3.10”‘. This should be an underestimate that depends on the true value of K,; however, it does agree well with the K,, determined from equilibrium studies.

0% Time (mid

min (01, and then diluted 20-fold with medium free of IHYP but containing 1 PM (-l-propranolol. At the times shown, l-ml aliquots were withdrawn and assayed for bound radioactivity. Data were corrected for nonspecific binding by the use of control incubations containing 1 KM (-)-propranolol in the initial volume. The solid lines represent theoretical curves obtained by assuming two first order dissociation processes. The slower was assumed to have a tI12 of 180 min. Using this t,,*, data obtained at 20 to 60 min were extrapo- lated to zero time to estimate the fraction of the slowly dissociating component. All the data were then fit by optimization of the till of the fast component to best approximate the observations. The estimated values of t,,2 for the rapid process and the fraction of slowly dissociating ligand were 2.0 min, 63% (x); 2.2 min, 71% (A); 2.6 min, 79% (0); and 3 min, 90% (0).

The purpose of this description of a phenomenological reaction scheme for the binding assay is not to deduce the correct sequence of reactions, but is to demonstrate that the data are internally consistent and that good estimates of apparent equilibrium constants can be obtained if certain experimental conditions are maintained (i.e. t SJ /c,,,~~; 0 + 1) and to provide a basis for interpretation of the binding isotherms to be presented.2 It is certainly true that other reaction schemes could be developed for the binding of [“WIHYP to the /3-

2 For example, this model predicts, as we have demonstrated by computer simulation, that if true equilibrium is obtained (t = ~1, neither apparent positive nor negative cooperativity should be observed. On the other hand, if t is not substantially larger than k,,,-’ (which changes with ligand concentration), apparent positive cooperativity (to varying degrees) will be observed. For such reasons, caution must be exercised in the assessment of cooperative interactions based on the shape of binding isotherms in systems such as this.


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5766 /3-Adrenergic Receptor-Adenylate Cyclase Relationship

[~*JI]IHYP Bound (PM)

FIG. 3 (left). Binding of 1 ‘zzIIIHYP to S49 cell plasma membranes. Binding was assayed as described over the range of 11 to 417 PM total I”‘IlIHYP. Incubation time was 60 min; the protein concentration was 0.27 mg/ml. The concentration of free ligand was estimated to be the total concentration minus the concentration of specifically bound ligand. The slope of the Scatchard plot yields a K,, for I ““IIIHYP of 70 PM.

FIG. 4 (rigght). Effects of purine nucleotides on (-)-propranolol

adrenergic receptor. In order to deduce the most appropriate mechanism with confidence, extensive experimental studies would be necessary. Furthermore, it is doubtful whether data of sufficient accuracy could be obtained at this time, and details that might be added to the kinetic scheme would not affect the primary conclusions derived from this study.

Binding of Other Ligands to PAdrenergic Receptor-The binding of unlabeled P-adrenergic agonists and antagonists was measured by competition with 1 ‘2”I]IHYP. Fig. 4 demon- strates the ability of (-)-propranolol, an antagonist, and (-)- isoproterenol, an agonist, to decrease 1 izzI]IHYP bound. The fractional degree of 1 “‘IIIHYP binding is given by

f= x;lIIsYPl 1 + K;'[IHYP] + K;'[H]

(13)

where K, and K,, are the apparent dissociation constants for binding of 1 ““I]IHYP and the competing ligand, respectively. The reported values of K,, for competing ligands (K, in the above equation) are thus concentrations at which 50% of maximal binding occurs, corrected for the competitive effect of I “‘IIIHYP. That is

KH = ln11,2(1 - fI) (14)

where 1 HI,,, = the concentration of competitor at which the binding of I ““IIIHYP is 50% of the maximal observed and f, = fractional degree of saturation by 1 ‘2”I]IHYP in the absence of competing ligand. The derived K,, values are approximately 0.7 nM and 80 nM for (-)-propranolol and (-)-isoproterenol, respectively (Fig. 4 and Table II).

As we have shown previously for other cell clones (191, the affinity of the putative receptor for agonists, but not for antagonists, is decreased by certain purine nucleotides. These observations are extended for this preparation in Fig. 4. There is no effect of purine nucleotides on 1 ““IIIHYP binding or on compe-

Log Cmcentrotm (M)

and (-)-isoproterenol binding to plasma membrane receptors. Spe- cific binding of each ligand was assayed by competition for l’2”IlIHYP binding sites as described under “Experimental Proce- dures.” Data are shown from several experiments in which protein concentrations were approximately 0.2 mglml; 1125111HYP concentrations were 130 PM for experiments with propranolol or 60 to 75 PM for experiments with isoproterenol. The incubation time was 30 min.

tition for binding sites by propranolol,” nor is the number of l’2”IlIHYP binding sites affected by these nucleotides. The apparent K,, for isoproterenol is increased to 485 nM in the presence of maximally effective concentrations of GTP, ITP, and the GTP analogs Gpp(NH)p and Gpp(CH,)p. Similar results were obtained with XTP and Ipp(NH)p. The concentrations of GTP required to cause this decrease in affinity (data not shown) are approximately equal to those required to stimulate adenylate cyclase activity in the presence of isoprotere- no1 (see Fig. 1). Although ATP also decreases the affinity of agonist binding, it is much less potent than GTP or ITP, little or no decrease is observed with the 0.5 mM ATP used in the enzyme and binding assays.

It is to be noted that the competition curve for isoproterenol in the absence of purine nucleotide is rather broad, and these data yield an apparent Hill coefficient of -0.5 (Fig. 5). In the presence of nucleotide, however, the shape of the curve approaches that described by a Hill coefficient of 1 and becomes essentially identical to the shape of curves for competition by antagonists. The explanation of this observation is not clear. For example, the former curve could result from true negatively cooperative interactions between receptor sites or from the existence of two or more sets of independent sites. Perhaps more important are the shifts ofK,, to higher values and of the Hill coefficient to one more consistent with a single set of sites that are brought about by purine nucleotides. The increase in K, indicates a heterotropic negatively cooperative interaction between the purine nucleotide and agonist binding sites.

Reversibility of Effect of Nucleotide on Agonist Binding- Work in various laboratories (21-23) has shown that most, if not all, effects of the nucleotide analog Gpp(NH)p on ade-

3 Although hydroxybenzylpindolol has been shown to be a partial agonist in rat adipocytes (20), IHYP has no agonistic activity in the S49 cell at concentrations up to 1 nhi.


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p-Adrenergic Receptor-Adenylate Cyclase Relationship

TABLE II Effect of nucleotides on concentration dependence of ligand binding

and activation of adenylate cyclase Ligand Nucleotide’ K,? K,,, or K,

n&f

Isoproterenol None 83 (60-100) 30d GTP \ 107 f 27

(N = 5)F ITP 51 ‘- 21

485 (360-580) (N = 5)P GPP(CH,)P 4.6’ Gpp(NH)p 3.9’

Isoproterenol GTP 1.5-200 (Cholera toxin- treated cells)

Propranolol None 1.0 GTP

Gpp(NH)p i

0.67 (0.45-0.85) 1.2

a Saturating concentrations (50 to 100 PM) were used. b Calculated from the concentration required to inhibit [‘251]IHYP

binding by 50% using Equation 14. Values given are for K,,, IHYP = 90 PM. Values in parentheses are calculated for K,,, IHYP = 50 pM and 140 PM.

c K,,., values were calculated from Hill plots of the data of Fig. 8. K, values were calculated from the equation: K, = (I,,.K,,.,)/(A + K,,,) where I,, is the concentration of propranolol to produce 50% inhibition, K,,, is for isoproterenol, and A is the concentration of isoproterenol used (1 PM).

d Km cannot be determined in the absence of nucleotide. The value shown was obtained with minimal (0.1 PM) GTP present (Fig. 81.

e Data are shown k SD. The values for GTP and ITP are significantly different (p < 0.01).

‘The apparent K,,., is dependent on time. Values given were obtained with 20-min incubations. The calculated K,,, for Gpp(NH)p at t = 0 is 14 nM (Table III).

9 The range of values in six experiments is given.

nylate cyclase activity are irreversible; in contrast it has at least been assumed that the effects of GTP are reversible. These findings are consistent with data obtained with S49 cell plasma membranes (see below). In Fig. 6, however, it is shown that the effect of purine nucleotide on affinity of the p-adrenergic receptor for agonist is reversible, regardless of which nucleotide is used. Membranes that were incubated with either Gpp(NH)p, which irreversibly activates the enzyme, or GTP, which does not, were washed and assayed for the binding of (-)-isoproterenol to the p receptor, with or without added GTP. The experiment shows that neither the position of the agonist binding isotherm nor the ability of GTP to alter its position during the binding assay is substantially affected by prior incubation with either GTP or Gpp(NH)p. The results are the same if GTP plus isoproterenol are used in the initial treatment.

Binding of Adrenergic Ligands in Adenylate Cyclase-deficient Variants -The necessity of guanine nucleotides for activation of adenylate cyclase by isoproterenol and their effects on agonist but not antagonist binding suggest that the shift in K,, may reflect molecular interaction of the P-adrenergic receptor and other components of the adenylate cyclase system. Recently, several variant clones of the S49 lymphoma line have been selected that lack measurable adenylate cyclase activity (less than 0.2% of wild type activity in the presence of NaF) but that have retained the binding activity characteris-

5767

f I

FIG. 5. Effect of Gpp(NH)p on the apparent Hill coefficient of (-)- isoproterenol binding. The concentration dependence of (-l-isoproterenol binding to plasma membranes was determined as in Fig. 4, but in the presence of different concentrations of Gpp(NH)p. The data are expressed as Hill plots which were fitted to straight lines by a least squares formula. The lines yielded slopes (apparent Hill coefficients) of 0.52 in the absence of Gpp(NH)p (Al, 0.72 at 10 pM Gpp(NH)p (01, and 0.83 at 300 PM Gpp(NH)p (01. For comparison, data on propranolol binding (V) were redrawn from Fig. 4. They display an apparent Hill coefficient of 0.84.

.

l-N I 0 10-S 10-7 10-6 10-5

[ Isoproterenol], M

FIG. 6. Reversibility of the effects of guanine nucleotide on the binding of (-l-isoproterenol. Plasma membranes at 1.2 mg/ml were incubated for 30 min at 30” in adenylate cyclase assay reagents containing 170 PM Gpp(NH)p tA,Al, 170 PM GTP plus 17 pM c-l- isoproterenol (m,O), or without added nucleotide (0,O). The mixtures were diluted 20-fold with cold HME buffer and centrifuged 30 min at 100,000 x g..The pellets were washed twice and resuspended in HME buffer. The concentration dependence of isoproterenol binding to each fraction was then measured in the presence (O,A,O) or absence (O,A,W of 100 FM GTP. The concentration of [““IIIHYP was 145 PM. Similar results were obtained if the initial incubation contained 170 FM ITP or GTP alone.

tic of the P-adrenergic receptor (6). Since the /3 receptor presumably cannot interact with enzyme in membranes from these variant cells, it was of interest to characterize binding of agonists and antagonists to p receptors in such preparations. Fig. 7 shows the binding of (-)-propranolol and (-)-isoprotere- no1 to membranes of independently selected but phenotypi- tally identical adenylate cyclase-deficient variant clones. In all clones tested, l’~“IlIHYP and competing antagonists bind


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5768 FAdrenergic Receptor-Adenylate Cyclase Relationship

have similar effects on the binding of agonists, a detailed investigation of their effects on enzyme activity was under- taken; results are summarized in Fig. 8 and Table II.

-// -10 -9 -6 -7 -6 -5 -4

MUTANTS IIOp0termo

-// -10 -9 -9 -7 -6 -5 -4

Log Concentration (M) FIG. 7. Binding of an agonist and an antagonist to membranes of

adenylate cyclase-deficient variants. Binding of (-)-isoproterenol and (-)-propranolol were assayed in plasma membranes from wild type cells and from two variant cell lines that are deficient in adenylate cyclase. The data shown in the figure were obtained using a crude membrane fraction described previously (7). Identical data were obtained with one clone of variant cells when purified membranes (see “Experimental Procedures”) were used, and with crude membranes from two other clones of variant cells. Top, membranes from wild type cells; bottom, membranes from adenylate cyclase- deficient variants. 0, 0, propranolol; l , isoproterenol; w, isoprote- renal plus 300 PM Gpp(NH)p. There is no effect of Gpp(NH)p on the competition by propranolol in either wild type or variant clones (not shown).

with the same affinity that is observed with wild type membranes, and binding is again unaltered by the presence of guanine nucleotides. In contrast to the wild type membranes, binding of (-)-isoproterenol is of much lower affinity and is no longer influenced by guanine nucleotides. In addition, the Hill coefficients for the binding of isoproterenol and propranolol are both zO.9. Thus, agonist binding in the variants in the absence of purine nucleotides is indistinguishable from that observed in the wild type cells in their presence. It is unfortu- nately difficult to ascertain at the moment if this is significant or fortuitous. At a descriptive level, it is possible to state that the molecular entities to which nucleotides and agonists bind are thermodynamically uncoupled in these variant cells.

Effects of Purine Nucleotides on Hormonal Activation of Aaknylate Cyclase - Certain purine nucleotides or their analogs are essential for activation of adenylate cyclase. These effecters can be separated into two categories. The naturally occurring nucleotides, GTP and ITP, only activate the enzyme in the presence of /3-adrenergic agonists under the conditions used in this work, and they do so reversibly (not shown). On the other hand, Gpp(NH)p, Gpp(CH,)p, and Ipp(NH)p can activate in the absence of hormone, and they do so in an apparently irreversible manner.“ Since all of these nucleotides

4 We have attempted to reverse the Gpp(NH)p-mediated activation of adenylate cyclase by prolonged washing or by incubation at 30 or 37” with 50 PM GTP, 10 or 100 pM isoproterenol, or both. Incuba- tion with agonist and nucleotide causes a decrease in activity, and the decrease is greater at the higher temperature. However, mem-

Isoproterenol cannot activate adenylate cyclase by itself, although it does bind to the P-adrenergic receptor with K,, = 80 nM. If a concentration of GTP (0.1 FM) is included that is just sufficient to allow measurable stimulation of the enzyme by isoproterenol but is insufficient to cause a major shift in the K,, for the catecholamine, the effect of isoproterenol is characterized by a K,,, = 30 nM -not greatly discrepant from the K,,. When maximal concentrations of GTP (100 PM) are present, however, the K,, for isoproterenol (485 nM) shifts more than does the K,,, (100 nM). The discrepancy is clearer when ITP is the nucleotide. In this case, the K,, for isoproterenol is also 485 nM, but the K,,,, is only 50 nM. The K;,,., values for isoproterenol obtained in the presence of GTP or ITP do not change as a function of time of the adenylate cyclase assay.

Nucleotides thus control the interaction between the p- adrenergic receptor and adenylate cyclase, and the quantitative relationship between ligand binding and enzyme activity is different when ITP, rather than GTP, is used. Furthermore, even when the apparent Hill coefficient for binding of isoproterenol is low (minimal concentrations of GTP), the isotherm for activation of adenylate cyclase by the catecholamine has an apparent Hill coefficient near 1. Insofar as both hormone binding and enzyme activation in the presence of GTP and ITP are reversible, these results can be interpreted in terms of equilibrium models to be discussed later.

Apparent discrepancies between K,,, and K,, for isoprotere- no1 are even more marked when enzyme activity is measured in the presence of Gpp(NH)p or Gpp(CH,)p. However, the effects of the nonhydrolyzable GTP analogs differ from those of GTP and ITP in at least one important aspect: they activate the enzyme irreversibly, and they do so in the absence of a receptor ligand. In the presence of Gpp(NH)p, isoproterenol increases the rate of activation by Gpp(NH)p, but a maximal enzymatic activity is eventually attained that is not influenced by the catecholamine (Fig. 9). Because of the irreversible effect on enzyme activity, a kinetic scheme is required to rationalize the results. This scheme is similar to that recently proposed by Sevilla et al. (24) and by Jacobs and co-workers (25, 26), although these authors did not test their models by quantitatively fitting their data to them.

It is necessary to postulate at least four states of the system. These states are given in the following scheme, where the adenylate cyclase system (0 is unliganded, singly liganded by either hormone (HC), or Gpp(NH)p (GC), or doubly liganded (HGC).

(15)

In this scheme, P denotes the irreversibly activated state, which can be formed from either of the two Gpp(NH)p-liganded species.”

branes treated in this way show no further response to 10 ELM isoproterenol in the presence or absence of GTP. None of these results appears to be inconsistent with simple denaturation. Activa- tion by Gpp(CH,)p and by Ipp(NH)p is stable to washing. Further manipulations have not been attempted.

’ The designation P (priapic) state was chosen, since the enzyme is irreversibly activated and the rate of catalysis is thus persistently elevated.


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P-Adrenergic Receptor-Adenylate Cyclase Relationship 5769

In the experiment, 106 PM Gpp(NH)p is assumed to saturate Assuming that equilibrium is rapidly established and main- the system completely, resulting in the simplified scheme: tained between GC and HGC, solution of the differential equa-

GC. k tions for the system gives (see Appendix B):

(16)

where K is the association constant of isoproterenol for G-C and k and KH are first order rate constants for the formation of the P state from GC and HGC. The velocity as a function of time, u(t), for this scheme is given by

v(t) = fv + fHVH + fpVp (17)

where f, fs , and fp are the fractions of enzyme in the GC , HGC , and P states with velocities V, VH, and VP, respectively:

-1 80

1

60 s

40 i

OJ-

. - GppWH)p ‘)- GpplCH2)P *- ITP cl-0.1 YM GTP .- IOOPM GTP

-10 -9 -8 -7 -6 -5 J

Log ~soproterenoi] (M)

FIG. 8. Effect of purine nucleotides on the activation of adenylate cyclase by (-J-isoproterenol. Enzyme activity was assayed over the range of isoproterenol concentrations shown in the presence of 50 ELM Gpp(NHJp (01, 100 /AM Gpp(CH,)p (01, 100 /AM ITP (A), 0.1 yM GTP (01, or 100 PM GTP (ml. Data are expressed as per cent of maximal stimulation by the agonist during a ZO-min assay. The range of calculated values of the K,, for isoproterenol in the absence or presence of nucleotides (Table II) is shown for comparison.

300,7l A

270

240 1 I 2101

!.A O7 3 6 9 I2 I5 16 21 24 27 30 33 76

Time (tin)

FIG. 9. Effect of (-J-isoproterenol on the time course of adenylate cyclase activation by Gpp(NH)p. Membranes (0.25 mg/mlJ were incubated at 30” in an adenylate cyclase reaction mixture containing 100 PM Gpp(NHJp andO, 1,3, 10,30,100, and 1000 nM (-)-isoprotere- no1 (Curves A to G, respectively). Aliquots (100 $1 were removed at the times shown and assayed for cyclic 132PlAMP. The solid curves represent an optimized fit to the model shown in Equation 16 using the integrated rate formula of Equation 19. Fitting was performed using all the data according to a modified Gauss-Newton nonlinear fitting procedure (27). The values for the parameters of Equation 19 which were obtained from the fit are shown in Table III. B, using

[ 1 V_ + KIHJl$I v(t) = Q Q e-kavt + V,(l-e -ka”t) (18)

where

and Q = 1 + KIH]

kav;j+I(IHIXHI

P state is thus formed in first order fashion with rate constant k,,.. Note that k,,. is simply the average of the rate constants k and k, weighted by the relative probabilities of GC and HGC. Integration gives:

[CAMP ‘1 = 17 KIHIVH G+ Q I 1

k,, (l-e-kavtJ +

1 (191

17P(t-c (l-e -kavt) ) av

The data in Fig. 9A were fit simultaneously by nonlinear least squares methods to the 6 parameter model in Equation 19. The program package used for this fit (27) uses a modified Gauss-Newton method. Note that even the large number of parameters used are reasonably well determined by the fitting procedure because of the large number of points (126) and the mathematical form of the model. The constants obtained from this procedure (Table III) are thus consistent with the experimental observation that isoproterenol enhances the rate of formation of P state. The calculated t,,, for the reaction GC + P is 11 min, whereas in the presence of saturating concentrations of isoproterenol the tljZ for HGC -+ P is 1.8 min. The dissociation constant of isoproterenol for GC (possibly different from that for P state and C) was calculated to be 14 nM. This is markedly different from the K,, (485 nM) measured in

0 3 6 9 12 15 I8 21 24 21 30 33 36 Time (mid

these values, the rate of accumulation of cyclic AMP, i.e. the specific activity of adenylate cylase, was calculated as a function of time for four selected isoproterenol concentrations. These changes in rate are shown as the solid curues (ordinate at left): A, 0 UM; C, 3 nM; E, 30 nM; G, 1000 nM. The apparent K,,,, i.e. the concentration of isoproterenol that caused a half-maximal increase in the rate, was also calculated from these values as a function of time. The dotted curve (ordinate at right) depicts the change in this apparent K,,, as a function of time. The K,,, at t = 0 is 14 nM and the limiting K,,, at 35 min is 0.9 nM.


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5770 PAdrenergic Receptor-Adenylate Cyclase Relationship

TABLE III Calculated parameters for activation of adenylate cyclase by

Gpp(NH)p and isoproterenol

The data of Fig. 9A were fit by a nonlinear least squares method as described by Johnson et al. (27) according to the model described in Equation 16. The error limits are approximately 65% confidence intervals.

K 7.0 +- 1.9 X lo’ M-’

k 0.06 -t 0.01 min’ k,, 0.4 k 0.2 min-’ V 14.8 2 34 pmol/min/mg v,, 66 -t 69 pmollminimg VP 432 I 8 pmollminimg -1" -9 -8 -7 -6 -5

Log [Pr,pronoloi] Ovl)

.k

FIG. 10. Effect of nucleotides on the antagonist activity of C-j- propranolol. Adenylate cyclase activity stimulated by 1 pM (-I- isoproterenol was assayed over the range of propranolol concentrations shown in the presence of 100 PM GTP (0) or 100 C(M Gpp(NH)p (01. The incubation was for 20 min. The activity observed at 10 pM

propranolol in the presence of GTP is equivalent to basal activity, and the activity at 10 FM propranolol in the presence of Gpp(NH)p is equivalent to that seen with the nucleotide alone (in the absence of

the presence of Gpp(NH)p in equilibrium binding experiments. The estimated velocities indicate that P state is nearly lo-fold more active than HGC. The low activity of GC is consistent with the conclusions of Sevilla et al. (24) and is reasonable in view of the minimal effect of GTP on the rate of catalysis. One can calculate an apparent constant for activation of the enzyme by isoproterenol in the presence of isoproterenol) Gpp(NH1p as a function of time (Fig. 9Bl. While this number decreases as a function of time by approximately an order of magnitude and reaches a value consistent with that obtained in Table II, it is important to point out that the K,,, as calculated has no thermodynamic meaning, except in the limit of zero time, since an irreversible process occurs. This limiting value of K,,, is, in terms of the model, the apparent dissociation constant for isoproterenol to the CG form of the adenylate cyclase system. As noted, it is discrepant from the actual K,,.

The ability of propranolol to prevent activation of adenylate cyclase by isoproterenol in the presence of GTP or Gpp(NH)p has also been examined. The data of Fig. 10 show an apparent dependence on nucleotide of antagonist potency in the adenylate cyclase reaction. However, this dependence is apparently due to the difference in the potency of the agonist, rather than to a change in the true affinity of the antagonist. While the concentration of propranolol needed to inhibit 50% of enzyme activity stimulated by 1 pM isoproterenol is 11 nM in the presence of GTP and 370 nM in the presence of Gpp(NH)p, the calculated K, values are both 1 nM and these values are in good agreement with the K,, estimated from binding studies (Table II).

Effect of Cholera Enterotoxin on Agonist Binding and Acti- vation of Adenylate Cyclase -Since cholera enterotoxin activates adenylate cyclase from many sources in a time-dependent and irreversible manner (281, it was of interest to consider its effect on hormone activation and to compare it to Gpp(NH)p. Plasma membranes from S49 lymphoma cells that had been exposed to cholera enterotoxin (1 ~g/ml) for 2 h were examined for binding and adenylate cyclase activities. Isopro- terenol binds with the same affinity to membranes from treated or untreated cells, and the effects of GTP or Gpp(NH1p on the affinity and apparent Hill coefficient are the same as those shown above. The effects of the toxin treatment on adenylate cyclase activity are shown in Fig. 11. Basal activity is markedly stimulated relative to untreated cells (6- to 12-fold in four experiments). In contrast to membranes from untreated cells, GTP can activate the toxin-treated membranes as effectively as can Gpp(NH)p. However, this activation is reversed when membranes are washed, while activation by Gpp(NH)p is irreversible in either treated or control membranes. The extent of hormonal stimulation of the enzyme in

1. I 0 10-O IO-9 10-e 10-7 10-6 IO

[Isoprotefenol]

-5

FIG. 11. Activation of adenylate cyclase by isoproterenol and GTP in membranes from cells treated with cholera toxin. Cells were harvested and washed once in serum-free growth medium by low speed centrifugation and were suspended to 10’ cells/ml in serum-

free medium containing 1 pg/ml of cholera toxin. After 2 h of incubation at 37”, plasma membranes were prepared as described under “Experimental Procedures,” except that membranes at the 20 to 30% and 30 to 40% sucrose interfaces were pooled before the final centrifugation. Adenylate cyclase activity was measured in the presence (0) or absence (0) of 100 PM GTP at varying concentrations of isoproterenol. The specific activity of enzyme in the same membranes was 340 and 410 pmol/min/mg measured in the presence of 10 mM NaF and 50 pM Gpp(NH)p, respectively.

the treated membranes, which is still dependent on the presence of guanine nucleotide, is reduced relative to the elevated basal activity, but the concentration of isoproterenol needed for half-maximal stimulation in the nresence of GTP is shifted to lower values. The change was variable from experiment to experiment, but the range of K,,, observed with membranes from treated cells was 1.5 to 20 nM -values that are much lower than the K,,, of 100 nM obtained with untreated membranes.


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fl-Adrenergic Receptor-Adenylate Cyclase Relationship 5771

DISCUSSION

The hormone-sensitive adenylate cyclase system of mammalian cell membranes is a complex one in terms of both the multiplicity of its components and the mass of data collected during its investigation. We have begun an analysis of the S49 lymphoma adenylate cyclase complex by both genetic (6, 29) and biochemical (30) techniques, but before one can hope to separate and reconsitute the components, one must establish criteria for the behavior to be expected of a reconstructed system. In our attempt to obtain these criteria for S49 cells, we have devised models that may help us understand the manner in which the components of the system interact and the signifi- cance of their interactions.

There are several experimental practices in this study that we believe to be important. The use of clonal wild type and variant S49 cells means that membranes are derived from a homogeneous cell population. Throughout these studies, an enriched plasma membrane preparation has been used. While we have not measured enzyme markers of organelles other than mitochondria, the fraction used is significantly purified with respect to both adenylate cyclase activity and the ,8- adrenergic receptor. Its purity is apparently correlated with a loss of hormone response that can be restored by the addition of certain purine nucleotides. This dependence on nucleotides for activation by hormones was first suggested by Rodbell and co-workers (31) as a general attribute of mammalian adenylate cyclase, and the hypothesis has been supported by other studies (321. Additional important experimental practices in this study have been the maintenance of identical conditions for the comparison of ligand binding and enzyme activation and the use of ATP at a concentration that is insufficient to permit activation by hormone or to cause an alteration of the apparent affinity of the receptor for agonists. Without conditions where addition of purine nucleotide is mandatory to observe hormone-stimulated enzyme activity, it is difficult to assume that endogenous regulatory ligands (nucleotides) are absent.

We have previously shown that the purine nucleotides that are necessary to permit P-adrenergic activation of adenylate cyclase specifically shift the dissociation constants for agonists to higher values (19). The magnitude of this change in K,, is the same for all such nucleotides, and the shift occurs at nucleotide concentrations similar to those that are necessary for activation of the enzyme. The alteration in K,, could be explained by simply assuming competitive binding between the nucleotide and an agonist. It would then be impossible, however, to rationalize the requirement for both hormone and nucleotide for enzyme activation, and there would not be a limiting value for the change in affinity. It is therefore necessary to assume a negative heterotropic interaction between the binding of agonist and of nucleotide.

The simplest such model consistent with the data is

Kl H+G+R#-RH+G

K4

Jl II

K2 (20)

H + RG \- RHG

K3

where R, RG, RH, and RHG, respectively, represent species of the receptor system that are free of ligands, binary complexes with the nucleotide or hormone, and a ternary complex with

both ligands.” Association constants for the reactions are indi- cated. The shift in the binding constant for agonist that is mediated by the nucleotide means that K, > K,; this dictates that K, > K,. This latter relationship has not been verified experimentally because of the difficulty of measuring nucleotide binding to relevant sites on the plasma membrane (8, 33, 34). In terms of this model and the data presented, the ternary complex RHG is the only form that is relevant to the activation of adenylate cyclase. That is, there are interconvertible forms of the receptor, and the species with lower affinity for agonist is that which facilitates activation of the enzyme (35).

At first glance it is not apparent why two ligands should be “used” to regulate enzymatic activity. The utility of such systems may be that both the efficacy and the affinity of a particular ligand can be influenced by the other. For example, in the absence of guanine nucleotide, agonist affinity is high and activation of the enzyme is not possible. If the rate of dissociation of agonist is sufficiently slow, the continued presence of agonist bound to the high affinity state of the receptor could explain the apparent loss of binding sites that is observed when the status of the receptor is subsequently examined with radioactive ligand (36, 371. This “loss” of binding, sites has been correlated with a loss of response of the system, commonly referred to as refractoriness, and response and binding can be restored in the presence of guanine nucleotide (36). It has not been possible to examine this phenomenon directly with a labeled adrenergic agonist. However, Brunton et .al. (18) studied the binding of agonist to a receptor for prostaglandin E, and observed the apparent time-dependent formation of a ligand. receptor complex from which l>H]prostaglandin E, was released very slowly. Such alterations in receptor affinity need to be studied in detail to ascertain if they will quantitatively account for the loss of respon- siveness that is observed in vitro and in uiuo. Models that call for a slow transformation of receptor. agonist complex to a refractory state have also been proposed for the nicotinic cho- linergic receptor (39, 42).

The most important facet of the linked binding of nucleotide and agonist is that hormone binding in the presence of nucleotide is associated with enhancement of catalytic activity. How- ever, the quantitative relationship between binding and activation is not simple; the K,,, for agonist in the presence of a saturating concentration of nucleotide is significantly less than the K,, for its binding. The K,, for agonist binding is independent of the identity of the nucleotide, but the relationship between binding of agonist and activation of enzyme (degree of coupling) does vary with the nature of the nucleotide. While the K,, for isoproterenol is approximately 500 nM in the presence of any active nucleotide, values of K,,, for this agonist range from 14 nM (at t = 0 in the presence of Gpp(NH)pl to approximately 100 nM in the presence of GTP. This variability of Kg,,., is increased still further by treatment of the cells with cholera toxin, where K,,, values for isoprotere- no1 as low as 1.5 nM have been observed (in the presence of GTP).

This discrepancy between K,,, and K,, may be simply ration- alized by assuming that either the binding of hormone to a single receptor site can activate multiple catalytic moieties or that the number of receptors is much greater than the number of adenylate cyclase molecules. For the latter situation, the

fi The use of this simple model is not meant to imolv that both nucleotide and hormone bind to the same molecular entity. It is quite possible, for example, that separate binding species exist for each and that the interaction of such species is controlled by the binding of ligands.


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5772 p-Adrenergic Receptor-Adenylate Cyclase Relationship

activation of adenylate cyclase can be described in equilibrium terms by expansion of the reaction scheme previously given in Equation 20 to include interaction of RHG with C:

KQ RHG + C \ - RHCC , (21)

where C is inactive adenylate cyclase and RHGC is an active, doubly liganded receptor’enzyme complex. (While this reaction is written as binding equilibrium, steady state processes such as nucleotide hydrolysis may be involved and are men- tioned below.) The fractional degree of activity, fill.,, is assumed to be proportional to the fraction of enzyme in the RHGC form:

[QHW] K"[C] [RHG]

f(RHr,C) = [Cl + [QHGC] = [Cl + KOICIIRHG] = (22)

KOIRHC,l

1 + KQIRHGl

where K, is the apparent association constant for the reaction written in Equation 21. Thus, in the absence of nucleotide, IRHGI = .O and no activation can occur. In the presence of saturating nucleotide, all of the receptors are distributed among the species RG. RHG, and RHGC. Since IRHGJ =

&I RGII HI,

f(RHGC) = KOK3[Rc,l [HI (23)

1 + KOKJIRGI [HI

where Kc, is defined by Equation 20. The model assumes that 1 R,,,,;,,] b 1 C ,,,, ;!,I. Furthermore, the

observation that prompted the model, that K,, FJ K;,,., . suggests that K, must be large, since a relatively small amount of hormone need bind to RG over the range of 1 H] that is relevant to the fractional activation of enzyme. We may assume therefore that 1 RG] is relatively constant. Equation 23 now has the form of a simple hyperbolic activation function, i.e.

f(~~cx) = fact = [Ll /Kact

1 + ILl/Kact

where K,,., = (K,JK,,lRG1)~‘. The presence of saturating nucleotide and the assumptions above imply that Kc,,., = (KQK,[R,,,,])-‘. The variation in K,,, for the different nucleotides is interpreted in this case to mean that K, is a function of the specific nucleotide, since K:, appears to be independent of nucleotide. While this model is related to the popular concept of “spare receptors,” it requires that individual receptor molecules be equivalent, in that all must potentially be able to interact with enzyme.

An alternative explanation for the discrepancy between Kc,,., and K,, can be found in the concept of a domain of RC units. A simple form of this model has been described by Biltonen (35). According to this scheme, the cyclase system exists in domains that contain multiple receptors and adenylate cyclase moieties. (For the sake of calculation, the number of receptors is envisioned to be equal to the number of adenylate cyclase molecules, but this is not crucial to the concept.) The function (or form) of the domain is regulated by nucleotides. Thus, binding to a single receptor in the presence of any nucleotide activates all the enzyme within the domain; the size of the domain (Nl is equal to the number of RC units that it contains. This feature of the model explains discrepancies between K;,,., and K,,. Further, the identity of the nucleotide is envisioned to determine N, consistent with the variability of

the difference between K,,, and K,). If binding is independent within the domain, its size can be calculated (351, since K,,, = K,,(2”’ - 1) (See Appendix C). Values for N = 10 are required by the data of this report.

Neither of these models suggests a detailed molecular mechanism for the coupling between agonist binding and activation of adenylate cyclase. However, it is tempting to suppose a correlation between the coupling role of nucleotides and their hydrolysis by a hypothetical nucleoside triphosphatase (43). If GTP is considered to be the physiological substrate, then a decreased ability of the system to hydrolyze the other nucleotides (ITP, XTP) could be correlated with a decreased K;,,., for agonist,. In terms of the first model, K, in Equation 21 could reflect a nucleoside triphosphatase activity associated with the reaction RHGC e RHG + C; alternatively the average size of domains could be related to the stability of the nucleotide ligand to hydrolysis.’

We have calculated that there are only about 250 P-adrenergic receptors per S49 cell (6). and these studies were carried out with plasma membrane fragments with an area that is probably less than 0.1%’ of that of the total plasma membrane (17). If there is random distribution of receptors and adenylate cyclase and if the numbers of these two components are approximately equal, it is very unlikely that a receptor and an enzyme molecule would be found in the same fragment. Fur- thermore, the models proposed require either about a lo-fold excess of receptors over adenylate cyclase or a domain of 10 or more receptor-cyclase pairs to explain the discrepancies between K;,,., and K,,. These models therefore demand some functional and physical complex of multiple RC units in the intact cell. Nonrandom distribution of receptors has previously been hypothesized for the case of insulin (44) and for the cholera toxin ganglioside G,, , complex (45. 46); such speciali- zation is epitomized by receptor localization in many subsyn- aptic membranes. Affinity partitioning of receptor-containing membrane fragments would presumably yield a significant purification of receptor, whether or not such multiple RC units exist (47).

We clearly lack understanding of the persistent activation of adenylate cyclase caused by the nonhydrolyzable purine nucleotides. The kinetic model described (Equation 19) is consistent with the data and seems to be in basic agreement with the schemes proposed by Sevilla et al. (24) and by Cuatrecasas and co-workers (25, 26). It is noteworthy that this model predicts the time-dependent shift in the apparent K;,,., for agonist. While the initial K;,,., of 14 nM is thought to reflect the true “equilibrium” K,,,., in the presence of Gpp(NH)p, it is not intuitively clear what. if any, biochemical meaning the pro- gressive decrease in the apparent K;,,., may have. The decline in K;,,., with time is a mathematical consequence of the irreversible reaction. Any process represented by this model will exhibit a K;,,., that is extremely dependent on time and the value of Kg,,., obtained in the limit oft = 0 is the only one that can be directly compared with agonist binding affinity.

Kinetic analysis of the persistently activated state of adenylate cyclase obviously still leaves the mechanism of its formation open to question. However, the resistance of Gpp(NH)pH, Ipp(NH)p, and Gpp(CHJp to hydrolysis suggests

’ In this light, cholera toxin might be envisioned as a nucleoside triphosphatase inhibitor since the efficiency of coupling is increased and the requirement for nucleotides is maintained; furthermore GTP is itself capable of activating the enzyme dramatically in such membranes. However, the reversibility of enzyme activation by GTP (in contrast to that by Gpp(NH)p) does not argue for this speculation.

” In separate experiments, 100 PM lS:‘HlGpp(NH)p was added to


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/3-Adrenergic Receptor-Adenylate Cyclase Relationship 5773

that the inability of a nucleoside triphosphatase to remove the y-phosphate group from these nucleotides may be crucial. The apparent reversibility of the shift in agonist affinity caused by Gpp(NH)p was unexpected. If one assumes that there is a single class of binding sites for Gpp(NH)p, the reversibility of the effect of Gpp(NH)p on the binding of agonists suggests that the binding of Gpp(NH)p is also reversible. If this is true, the persistently activated state of adenylate cyclase is due to an alteration of the enzyme itself, rather than to pseudo-irreversible binding of the nucleotide ligand.

Neither of the equilibrium models, in their present forms, attempts to rationalize the fact that the Hill coefficient for the binding of agonist in the absence of nucleotide is less than one. This phenomenon could suggest that more than one form of receptor site exist in the absence of nucleotide or that each receptor entity consists of two or more interacting agonist binding sites. The fact that this apparent “negative cooperativity” for agonist binding is not observed in the presence of nucleotide can be explained if states of the receptor that have differing affinity for agonist are interconvertible only in the presence of guanine nucleotide. Speculation of this type has appeal in view of the observations on refractoriness to agonists discussed above. It is probable that the apparent “negative cooperativity” that has been observed here is different from that reported by Limbird and Lelkowitz (481, since the data of the previous study involved antagonists and the Hill coeff- cients were unaffected by guanine nucleotides.

We are intrigued by the observations on the binding of adrenergic agonists to receptors of the S49 cell variants that are deficient in adenylate cyclase activity. Essentially identical binding data have recently been obtained with another variant clone that contains both p-adrenergic receptors and adenylate cyclase but in which there is no stimulation of the enzyme by adrenergic agonists (29). While it makes intuitive sense that both types of variants could show similar changes, the models presented above do not obviously lead one to predict the observed alterations. Binding of agonists in both types of variants is characterized by reduced affinity, increased apparent Hill coefficient, and lack of effect of purine nucleotide. Thus, binding of agonist in the presence or absence of nucleotide in the variant cells is indistinguishable from the binding of agonists in the presence of purine nucleotides in membranes from wild type cells. Two types of hypotheses seem most plausible to various of us. First, there is no compelling reason to assume more than coincidence in the similarity between binding to variant membranes and to wild type membranes in the presence of nucleotide. However, the character- istics of binding and the lack of effect of guanine nucleotide in the variant tempt us to speculate that the variant membranes are already altered in a manner similar to that which occurs in wild type cells in the presence of guanine nucleotide. For example, if agonist and nucleotide ligands regulate an equilibrium between states of a macromolecule or between different macromolecules in the wild type membrane, it is possible that the equilibrium involved has already been perturbed in variant membranes. A common denominator to cause such a hypothetical perturbation in both types of variants could be an

reaction mixtures from which 1 ““IIIHYP and I”*P]ATP were omitted; the ATP regenerating system was either present or absent. The reactions were terminated after 30 min by the addition of 0.1 ml of cold ethanol and chilling. Ethanol extracts were chromatographed on polyethyleneimine-cellulose plates in 0.8 M KP,, pH 3.4, and the plates were scanned for radioactivity. Degradation products were not detected.

inability of the regulatory components of the system to associ- ate functionally with the catalytic moiety.

The simultaneous absence of high affinity agonist binding sites and of agonist-induced “loss” of binding sites for labeled ligand (371 in the adenylate cyclase-deficient variants seems noteworthy, and this correlation provides evidence for the relevance of such a class of high affinity sites to the phenomenon of refractoriness. This observation leads us to predict that the uncoupled variant will display similar behavior when the binding of 1 “:I]IHYP is examined after exposure to adrenergic agonists.

Acknowledgments-We are grateful to Pamela Van Ars- dale, Hannah Anderson, and Catherine Beeson for excellent technical assistance and to Wendy Deaner and Carol Reden- baugh for preparation of the manuscript and artwork.

APPENDIX A

Kinetic analysis ofthe sequential binding model ofEquation 1

A simple and useful solution of the kinetics of the sequential binding model in Equation 1 is possible when certain simplify- ing assumptions are made. In this model (Equation A-l).

fast slow k, k,

R+ IHYP = R,IHYP k-1 kT2

RJHYP (A-1)

K, = k,/k-,; K, = k,/k-,

a ligand binds to a receptor R, which then undergoes an apparent isomerization that is relatively slow compared to the first binding step. The assumptions are that: (a) the first step is sufficiently fast relative to the slow step that it is essentially at equilibrium with respect to R. IHYP, and RJHYP at t = 0 and all times t thereafter and (6) the free ligand concentration is essentially constant and equal to the initial concentration. This model is of course not new or unique, nor is the result of its solution (39. 40). Briefly,

“R!;“] : k,[RIHYP] - km,[R,IHYP] (A-2)

R,,t = IRI + IRJHYP] + lR,IHYp]

Because of assumptions (a) and (b),

[R,IHYP] = K=YP1m&,,, [RJHYP]) 1 + K,[IHYP]

(A-3)

Substituting for IR,IHYP] in Equation (A-2) and arranging in normal form,

“R’z”’ + a[RJHYP] = b (A-4)

where

a = k, K,[IHYP] + h andb = kxK,[IHYP] ’ 1 + K,[IHYPj ’ -~~ Rot 1 + K,[IHYP]

The general solution of Equation (A-4) is

IRJHYPI = C,e”’ + C,

where C, and C, are arbitrary constants. Given the boundary conditions:

at t = 0, [R,IHYP] = 0

at t = I 4WHYPl

dt

~~~ = 0 and[R,IHYP] = [R,IHYP],,,,,,, = $ ,

it follows that


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5774 FAdrenergic Receptor-Adenylate Cyclase Relationship

Thus,

[R,IHYP] = [&IHYP],,,,,B = i~;;~~$IHy, (A-5)

where 0 = (1 - e-‘I’). ]R,IHYP] is given by Equation (A-3), and IRI = R,, - ((RJHYP] + IRJHYP]).

The fraction of sites filled, f, as a function of time given in Equation 9 is obtained by substitution of the expressions in Equations (A-5) and (A-3) and the expression for [RI into Equation 2, followed by rearrangement.

APPENDIX B

Kinetic model for the formation of P state

The model proposed for the formation of P state given in Equation 16 is

.,hkp K II HGC/k,

It is assumed that: (a) the binding reaction is sufficiently fast to remain essentially at equilibrium with respect to H, CC, and HGC, despite P state formation and (b) the concentration of free hormone is essentially constant and equal to the initial concentration.

C,,, = [GC] + [HGC] + [P]

C’m, = C,,, ~ [P] = [GC] + [HGC]

At any time t because of assumptions (a) and (b),

and

[GC] = i&a C’,,,

The rate of formation of P state is given by

4Pl dt = k[GC] + k,iHGC]

Substituting and arranging in normal form,

G’l -;r + a[P] = a c,,,

where

k hK[~ a = ~-~~- + ~~ ~~~ 1+K[w 1+K[m

The general solution of Equation (B-2) is

[P] = C,e?” + c,

(B-1)

(B-21

where C, and CL are arbitrary constants. Given the boundary conditions:

at t = 0, [P] = 0

att= x,[P] = C,,,

it follows that -C, = C, = C,,,,. Thus:

(a) [PI = (1 ~ e-“‘)C,,, = fiC,,, (B-31

(b)

(cl (B-31

The velocity as a function of time is as in Equation 17.

The data in the experiment described in Fig. 9A were expressed as the amount of cyclic AMP accumulated, rather than enzyme velocity; one integration is therefore required. The equation [CAMP] = JO’ u(t)dt, is evaluated using Equations 17 and (B-3) to yield Equation 19.

APPENDIX C

Relationship between domain size, binding constants. and activation constants

This appendix is an abbreviation of a more complete discussion by Biltonen (35). Consider a domain, d, of receptor-cyclase pairs of size N; d = (RC), If we assume that each receptor site is independent, with an intrinsic binding constant. K. the fractional degree of saturation, f. by a ligand. x. is

f = [xllKp 1 + [x]lK,,

However, if the domain is fully activated whenever one or more receptor sites within the domain is occupied. the fractional degree of activation, fiTpI. is given by the relation & = 1 - p wherep is the probability that all receptor sites within the domain are unoccupied. Since the probability that any particular site is unoccupied is (1 + [x]/K,,)~ ’

Thus

p = (1 + [x]‘K,,l ’

fa<, = 1 ~ 11 7 [x]lK,,~ ’

Defining K;,,, concentration of ligand when fi,r, = l/z, it follows that

(1 + K,,,lK,,) ’ = ‘/z

or

Thus

(1 + K,,.,/K,,i’ = 2

K,,,/K,, = 2’ ’ ~ 1

or

K,,, = K,,(2’ ’ - 1).

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E M Ross, M E Maguire, T W Sturgill, R L Biltonen and A G GilmanRelationship between the beta-adrenergic receptor and adenylate cyclase.

1977, 252:5761-5775.J. Biol. Chem.

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