Hypothyroidism Modulates Beta Adrenergic Receptor-

Adenylate Cyclase Interactions in Rat Reticulocytes

GARYL. STILES, JEFFREY M. STADEL, ANDREDELEAN, and ROBERTJ. LEFKOWITZ,Howard Hughes Medical Institute Laboratory, Division of Cardiology,Department of Medicine and Biochemistry, Duke University Medical Center,Durham, North Carolina 27710

A B S T RA C T We have investigated alterations inbeta adrenergic receptor binding sites of rat reticulo-cytes occurring in animals rendered hypothyroid bythyroidectomy. Beta adrenergic receptor interactionswere assessed by measuring the number of (-)[3H]-dihydroalprenolol binding sites and the ability of anagonist to compete for occupancy of the receptors. Thenumber of receptors was significantly reduced in cellsfrom the hypothyroid animals. In addition, there weresignificant agonist-specific alterations in binding. Us-ing computer assisted curve fitting techniques, it wasfound that the ability of (-)isoproterenol to stabilizea high affinity guanine nucleotide sensitive "coupled"form of the receptor was impaired. Reticulocytes fromhypothyroid animals have, in addition, a reduction inthe concentration of the nucleotide regulatory proteinas assessed by the number of 42,000 Mr substrates forcholera toxin catalyzed ADPribosylation. These alter-ations are associated with reductions in catecholamineand NaF stimulated adenylate cyclase activity. Dimin-ished coupling of beta adrenergic receptors with otherregulatory components of the adenylate cyclase systemrepresents a mechanism by which altered thyroid statesmodulate beta adrenergic receptor function and betaadrenergic responsiveness of tissues.

INTRODUCTION

Thyroid hormone levels appear to modulate tissueresponsiveness to beta adrenergic catecholamines,

A preliminary report of this work was presented at theAmerican Society of Clinical Investigation National MeetingApril 1981, San Francisco, Calif.

Dr. Andre De Lean is a recipient of a Centennial Fellow-ship from the Medical Research Council of Canada. Dr.Robert J. Lefkowitz is an investigator of the Howard HughesMedical Institute. Address reprint requests to Dr. Robert J.Lefkowitz, Duke University Medical Center, Durham, N.C.27710.

Received for publication 8 May 1981 and in revised form8 July 1981.

1450

with increased responsiveness often observed inhyper- and decreased responsiveness in hypothyroidism(1, 2). Thyroid hormones regulate the number of betaadrenergic receptors in several tissues including ratheart (3), skeletal muscle (4), and submaxillary gland(5) as well as in turkey erythrocytes (1).

Although changes in receptor number may be par-tially responsible for the altered sensitivity, otherchanges such as alteration in agonist-receptor affinitymight also be important. Radioligand binding tech-niques now permit the assessment not only of thenumber of beta adrenergic receptors but also the statusof their coupling with the adenylate cyclase system.This is most effectively done by quantitative analysisof ligand binding data, using computer assisted curvemodeling procedures (6). Such procedures have in-dicated that in beta adrenergic systems the competi-tion curves of agonists with radiolabeled antagonistsare shallow whereas antagonist competition curves areof normal steepness. This agonist specific heterogeneityof binding appears to be due to the ability of agonists,but not antagonists, to distinguish separate affinitystates of the beta adrenergic receptor (R)l havingrespectively high (KH) and low (KL) affinity for theagonist. The high affinity state of the receptors appearsto represent a "coupled" agonist stabilized complex ofhormone, receptor, and nucleotide regulatory protein(N), which is an intermediate required for hormonepromoted activation of the enzyme (7). Guaninenucleotides, which are required for beta adrenergicactivation of adenylate cyclase, appear to mediate atransition from the high to the low affinity state of thereceptor and thus shift agonist competition curves tothe right. The ratio KL/KH for an agonist correlateswith the efficacy (intrinsic activity) for activation of

'Abbreviations used in this paper: EC,0, ,50% displace-ment; [3H]DHA, [3H]dihydroalprenolol; Gpp(NH)p, guanyl-5'-yl-imidodiphosphate; N, nucleotide regulatory protein; R,beta adrenergic receptor; T4, thyroxine.

J. Clin. Invest. ( The American Society for Clinical Investigation, Inc. 0021-9738/81/1211450/06 $1.00Volume 68 December 1981 1450-1455

adenylate cyclase (6). The ratio KL/KH, reflecting theability of an agonist to stabilize the RN complex is,therefore, a measure of the coupling of the receptorwith the nucleotide regulatory protein.

The binding of agonists but not antagonists to thebeta adrenergic receptor of rat reticulocyte membraneshas been directly shown to induce the coupling of Rwith N (7). It has also been demonstrated that matura-tion of rat reticulocytes to erythrocytes is accom-panied by a gradual uncoupling of the receptor fromadenylate cyclase. This results in a loss of agonist pro-moted high affinity state as documented by quantitativeanalysis of agonist binding properties (8). This modelsystem is now used to investigate whether decreasedR-N coupling might provide a partial explanation fordecreased beta adrenergic responsiveness in hypo-thyroidism.

METHODSHypothyroidism was produced by thyroidectomy in male

Sprague-Dawley rats (175-225 g) obtained from CharlesRiver Laboratories, Newfield, N. J. Changes in thyroidhormone levels were determined 8 wk after thyroidectomy bymeasuring thyroxine (T4) levels as previously described (9).Control serum T4 (n = 3) was 6.9±0.3 gg/dl (mean±SEM)while hypothyroid serum T4 (n = 6) was 1.8+0.2 (P < 0.001).

Reticulocytosis was induced by injecting animals with 7.5,9, and 12 mg (i.p.) of phenylhydrazine on 3 consecutive dfollowed by sacrifice on day 7. "Hypothyroid" reticulocyteswere prepared by injecting the same quantities of phenylhy-drazine 8 wk following thyroidectomy. Percent reticulocyteswas determined after staining with new methylene blue.There were 88±+5% reticulocytes in the control group and87±4% in the hypothyroid group.

Reticulocyte membranes were prepared as previouslydescribed (8). Receptor binding assay was performed by using(-)[3H]dihydroalprenolol ([3H]DHA) at concentrations of2.0-3.5 nMin competition assays in a volume of 1.0 ml. Proteinconcentrations were 0.25-0.5 mg/ml. Incubations were for 10min at 37°C with unlabeled ligands as indicated. Data arepresented as specific binding, defined as binding competedfor by 1 mM(-)isoproterenol, which was >90% of totalbinding.

Adenylate cyclase activity was assayed as previously de-scribed (8). Final concentrations were 32 mMTris-HCI pH 7.65,10 mMMgCl2, 0.2 mMEDTA, 0.04 mMIBMX (3-isobutyl-1-methyl-xanthine) (at 25°C), ATP 0.12 mM, and incuba-tions were for 10 min at 370 in the presence of 0.1 mMguano-sine 5'-triphosphate (GTP). Proteins were measured by themethod of Lovry using bovine serum albumin as standard(10). All data not derived from computer curve fitting wereexpressed as mean±SEMand comparisons were made by ananalysis of variance.

Cholera toxin catalyzed ADP-ribosylation of membranesprepared from reticulocytes obtained from euthyroid andhypothyroid rats. Preparation of reticulocyte membranesand subsequent exposure of these membranes to activatedcholera toxin and [32P]NAD' was carried out as previouslydescribed and validated (7, 8). Equal quantities of each of thereticulocyte membrane preparations (control and hypothyroidat -30 mg/ml) were exposed for 20 min at 25°C to 50 ,ug/ml ofcholera toxin (preactivated for 10 min at 370 with 20 mMdithiothreitol), 5 ,uM [32P]NAD' (5-15 Ci/mM), 750 ,uM GTP,

and a GTP regeneration system that included 2.8 mMphosphoenol pyruvate, 5.2 ,ug/ml pyruvate kinase, and 10 ,g/ml myokinase. As a control experiment reticulocyte mem-branes from both euthyroid and hypothyroid rats wereincubated under similar conditions in the absence of choleratoxin. At the end of the incubation period each preparation wasdiluted fivefold with 75 mMTris-HCl (pH 7.5), 12.5 mMMgCl2, 1.5 mMEDTA and assayed for adenylate cyclaseactivity in the presence of GTP(0.1 mM)and NaF (10 mM). Itwas demonstrated that the incorporation of [32P]ADP riboseinto the 42,000Mr protein was linearly related to proteinsubstrate concentration. The 32P incorporation was alsodirectly related to the extent of cholera intoxication (Fig. 1).The extent of intoxication was determined for both euthyroidand hypothyroid membranes by comparing the GTPand NaF-stimulated adenylate cyclase activity after incubation ofmembranes in the presence and absence of cholera toxin (7).Full (100%) intoxication in the rat reticulocyte system (7, 8)means that adenylate cyclase activity in the presence of GTPisequivalent to that observed in the presence of NaF. The extentof intoxication was 92+7% for euthyroid and 82+8% forhypothyroid membranes in six experiments.

Polyacrylamide gel electrophoresis. The membranes,after incubation in the presence and absence of choleratoxinl, were washed five times in a buffer containing 75 mMTris-HCl (pH 7.5), 2 mMEDTA/Trasylol (5 U/ml) beforebeing dissolved in gel sample buffer (8). Polyacrylamidegel electrophoresis was carried out (8) with equal amountsof protein (control and hypothyroid membrane preparation)placed on each gel lane. [32P]ADP-ribosylated proteins weredetected by autoradiography for 1-3 d usinlg Kodak XR-5 film(Eastman-Kodak Co., Rochester, N. Y.) in the presence orabsence of intensifying screens. [32P]ADP-ribosylated pro-teins were quantitated by slicing each gel lane into 1.5-2-mmslices and then allowing the slices to incubate overnightin 5 ml of scintillation fluid containing Soluene-350 (PackardInstrument Co. Inc., Downers Grove, Ill.) before countingin a Packard Tricarb liquid scintillation spectrometer. The[32P]ADP ribose counts peak representing the 42,000 Mrprotein (1-2 slices in width) were corrected to counts perminute per milligram protein and compared on a percentbasis. In the six experiments a mean of 0.67±0.06 mgproteinwas placed in each gel lane. However, in each individualexperiment there was never a protein difference of >0.03 mgbetween gel lanes.

Data analysis. Comipetitive binding assay data wereanalyzed by a nonlinear least squares curve fitting procedurebased on the law of mass action and applied to comiiplexligand-receptor systems. The method provided estimiiates ofthe affinity of ligands for different states of the receptorsand for the proportion of these states (percent RH and percentRL). Fitted estimates are provided for the data, assuming eitherone or two affinity states. Statistical analysis comparing"goodness of fit" between one and two affinity state modelswas determined as previously described (6) and the morecomplex model was retainied only when it significantlyimproved the fit.

Statistical analysis used in comiparing curves and individualparamneters was previously described (6). Each curve wasformed by averaging individual experiments (control = 3,hypothyroid = 4) anid each of the mean curves was initiallyanalyzed independently allowing each parameter estimate toreach its optimiluIml value. Then, the curves were analyzedsimultaneously and parameters were constrained to a commonvalue. If the effect of sharing parameters was not to sig-nificantly worsen the goodness of fit, then the parameterswere considered indistinguishable. Significance was definedas P <0.05.

Hypothyroid Modulation of Agonist-Receptor Interactions 1451

120

1oo0

-

0

O-

0cx0

0N

80o

601

0 10 20 30 40Cholera Toxin (.ug/ml)

c._

5.28 S0C

4.32 2000I

3.36 ¢0c

2.40 .,00

1.44 Cfio0c

0.48 r?cmI.)1-

0

o

x

50 60

FIGURE 1 The concentration-dependent effects of cholera toxin on adenylate cyclase activity and[32PJADP ribose incorporation into the 42,000-M, protein. Intoxication was performed as describedin Methods. 100% intoxication of the enzyme adenylate cyclase in this system means that GTP(0.1 mM)-stimulated activity is equivalent to the activity elicited by NaF (10 mM). The 32Pincorporated into the 42,000 M, protein was measured by cutting the gel lane into slices andcounting the appropriate region (as determined by a marker protein) in a scintillation counter(Methods). Arrow indicates concentration of cholera toxin used in each experiment. Results are

the means of duplicate determinations.

The dissociation constant (KD) and maximum bindingcapacity for the antagonist [3H]DHA were determined byperforming saturation experiments over a range of 0.5-10nM. The data were analyzed according to the same general-ized model and using a one-state fit. The EC50 (50% displace-ment) and the steepness factors of competition curves were

determined by fitting the data to a four parameter logisticequation (11).

RESULTS

The reticulocytes from hypothyroid animals were

found to have significantly fewer beta adrenergicreceptors 625+42 fmol/mg (n = 3) than did controlreticulocytes 1,351+215 fmol/mg (n = 5) (P < 0.03).Similar decreases in receptor number have been ob-served in other tissues (1). There was no change in theKD for [3H]DHA (control 1.2+0.9 nM and hypothyroid1.0+0.5 nM).

Isoproterenol competition curves always modeledbetter to a two state fit (KH and KL) than a one statefit while isoproterenol curves in the presence of guanyl-5'-yl-imidodiphosphate (Gpp(NH)p) were always ade-quately described by a one state model (KL) which was

not improved by a two state fit. The KL for these twocurves (i.e., with and without Gpp(NH)p) were notsignificantly different, consistent with the intercon-vertibility of these states.

An important finding was that the isoproterenolcompetition curve for the hypothyroid reticulocytewas shifted to the right as shown in Fig. 2 with a sig-nificant increase in the EC56 but no change in the

slope factor (Table I). The isoproterenol plus Gpp(NH)pcurve demonstrated no significant change in eitherEC50 or the slope factor.

m

x

2

z

0m

I

9 8 7 6 5 4 3

- LOGIO [(-)ISOPROTERENOL] (M)

FIGURE 2 Computerized curve fitting of binding data fromdisplacement of [3H]DHA by (-)isoproterenol in reticulo-cytes from control and hypothyroid rats in the presence andabsence of Gpp(NH)p. The reticulocyte membranes were

incubated with 2.0-3.5 nM [3H]DHA in competition withthe indicated concentration of (-)isoproterenol in thepresence and absence of 0.1 mMGpp(NH)p. The datapoints represent the mean of three experiments (control)or four experiments (hypothyroid) each performed in du-plicate. Binding in the absence of competitor was -900fmol/mg in the control membranes. Data were normalizedfor purposes of graphic representation.

1452 G. L. Stiles, J. M. Stadel, A. De Lean, and R. J. Lefkowitz

0

I IIII

40[

201

r% I

TABLE IEffects of Hypothyroidism on (-)Isoproterenol Binding

in the Rat Reticulocyte Using Computer-derivedParameters from Displacement

Curves with [3H]DHA

Hypo-Control thyroidism Signifi-(n = 3) (n = 4) cance

p

Slope factor withoutGpp(NH)p 0.63+0.02 0.70+0.02 NS

Slope factor withGpp(NH)p 0.94±0.03 1.01±0.04 NS

EC50 (nM) withoutGpp(NH)p 180+20 380±30 <0.001

EC50 (nM) withGpp(NH)p 2,800+200 3,700+400 NS

KH (nM) 26+2 63+5 <0.001KL (nM) 1,380+40 1,730+50 <0.001KL/KH 53+8 28+4 <0.001

RH% 71±+5 66+7 NS

For each set of displacement curves (Fig. 1) the computerprograms provided estimates of the EC50 (50% displacement),the slope factors, percentage of total receptors in the highaffinity state (RH%), the high (KH) and low (KL) affinity state

dissociation constants. The results are expressed as mean

+SEE (standard error of the estimate). The statistical sig-nificance was determined as described in Methods. n, thenumber of individual experiments.

The KH and KL of the isoproterenol competitioncurves were both altered significantly in the hypo-thyroid cells but the fold change in KH (2.4-fold) was

much greater than that in the KL (1.2-fold). There was a

concomitant significant decrease in the KLIKH ratiowhile no significant change was seen in the percent RH(Table I).

In several other situations where KLIKH of an agonistfor beta adrenergic receptors is reduced, such as afterdesensitization or with partial agonists, the biologicalcorrelate is a decreased ability of agonists to stimulateadenylate cyclase (6). Accordingly, we investigated thestatus of the adenylate cyclase system in the reticulo-cyte membranes. As can be seen from the representa-tive experiment in Fig. 3, there was a 50% fall in maxi-mumisoproterenol (0.1 mM) and F- (10 mM)-stimu-lated enzyme activity in cells from the hypothyroidanimals. In five experiments basal activity did notchange significantly between control 5.7±1.4 vs. hypo-thyroid 3.1±0.9 pmol/min per mg (P = 0.28) cells. Bycontrast, maximum isoproterenol-stimulated activity51.9+5.7 vs. 20.3+3.8 pmol/min per mg (P < 0.002),and fluoride-stimulated activity 56.0+5.7 vs. 25.2±2.6pmol/min per mg (P < 0.001) were significantly re-

duced. Although the EC50 for isoproterenol stimulationwas not significantly altered 1.1+0.7 ,uM (control) vs.

_!- E 6,> "I_ c-u ._

c t ta,n a'O a,

u> E 4

0 a

Basal 9 8 7 6 5

-log,0 [(-) Isoproterenol] (M )

4

FIGURE 3 Isoproterenol and NaF stimulation of rat reticulo-cyte adenylate cyclase. The reticulocyte membranes were

incubated with the indicated concentrations of isoproterenolor NaF (10 mM) for 10 min at 370 in the presence of 0.1 mMGTPand a standard adenylate cyclase assay mixture (Meth-ods). The data points represent the mean of duplicate deter-minations from a representative experiment (n = 5).

0.7±0.6 uM (hypothyroid) (P = 0.71), the day-to-dayvariability in this parameter was substantial andsmall changes may not have been evident.

Impaired formation of the high affinity state of thereceptor as well as diminished catecholamine- andfluoride-sensitive adenylate cyclase are all consistentwith an alteration of the nucleotide regulatory pro-

tein. Accordingly, in six additional experiments we

quantitated the concentration of 42,000 Mr substratesfor cholera toxin catalyzed ADP-ribosylation in themembranes, since this technique appears to label themajor subunit of the regulatory protein (7, 8). Resultsof a representative experiment are shown in Fig. 4 as

assessed by polyacrylamide gel electrophoresis. Theapparent molecular size of the cholera toxin sub-strate (-42,000 Mr) was not different in hypothyroidvs. control reticulocyte membranes. However, thequantity was diminished by 40% in the experimentshown. In this series of six experiments each performedwith a separate set of animals, we measured and com-

pared isoproterenol (0.1 mM) and NaF (10 mM)-stimu-lated adenylate cyclase activity and the quantity of32P radioactivity incorporated into the 42,000 Mr pro-

tein. We found that in the hypothyroid membranesthe isoproterenol-stimulated activity was decreasedby 44 + 12%, the NaF-stimulated activity was decreasedby 36±11% and the 32P radioactivity incorporatedwas decreased by 40±10% compared to controlmembranes. The percent decrease in isoproterenoland F--stimulated activity is in good agreement withthe percent decrease in 32P incorporation into the42,000 Mr protein. This seems reasonable since bothisoproterenol- and fluoride-stimulated activities are

Hypothyroid Modulation of Agonist-Receptor Interactions

0 Control NaF70-0 Hypothyroid

0

1-040 C?/~~-

30 // o

'640to /~~~~~~~~~~~

50 II tNo/ .~~~~~

10 ///~~~~~~~~~~>"H

1453

DISCUSSION.. ...._......I A. f

CI C ' H I H+C!r

CT T CT. UT":0

FIGURE 4 Autoradiogram of [32P]ADP - ribosylated mem-branes fractionated by SDS - polyacrylamide gel electro-phoresis. Reticulocyte membranes from euthyroid and hypo-thyroid animals were prepared and underwent [32P]ADP- ribosylation and electrophoresis as described in Methods.0,1125-ovalbumin, provides a mobility marker for a Mr = 43,000protein. H, hypothyroid membrane preparation incubatedwith 5 ,uM [32P]NAD+ in the presence (H + CT) or absence(H - CT) of cholera toxin (50 ,g/ml). C, euthyroid membranesprepared in the same manner and incubated in the presence(C + CT) or absence of (C - CT) cholera toxin. The amountof protein placed in the control lane was 0.77 mgand in hypo-thyroid lane was 0.78 mg.

dependent on the presence of the N protein forstimulation to occur (12).

It can be observed that there is also a reduction inthe intensity of two higher molecular weight bands inthe hypothyroid sample in the experiment shown inFig. 4. In fact these two were the only bands whoselabeling was decreased other than the N protein in thisseries of experiments. However the labeling of thesetwo additional proteins was quite variable and low(<5% of the specifically incorporated 32p) and theirfunction is unknown. Since thyroid hormones areknown to produce many of their effects by modulatingthe rate of synthesis of a variety of proteins it is notsurprising that a few proteins other than the betaadrenergic receptor and N protein might also be af-fected by the hypothyroid state.

The present study suggests that thyroid hormonemodulates beta adrenergic function in several ways andindicates that simple enumeration of receptor numberis not sufficient to document these interrelationships.Wehave documented several alterations in the betaadrenergic receptor-adenylate cyclase system of ratreticulocyte membranes derived from hypothyroidanimals. These include the following: (a) the number ofbeta adrenergic receptors is decreased. (b) There is ahighly significant decrease in the ability of agonists tostabilize a high affinity state of the beta adrenergicreceptor. (c) Maximal isoproterenol and NaF-stimu-lated adenylate cyclase are decreased. (d) The amountof ADP-ribosylated cholera toxin substrates (N) isdecreased.

The alteration in high affinity state formation isapparent as a twofold decrease in the ratio of thedissociation constants of the agonist for the high andlow affinity states of the receptor (KLJKH). This is mainlydue to a twofold change in KH. Because the high affinitystate of the receptor appears to consist of a complex ofHRand N (7), these results suggest alterations in theagonist promoted R-N interaction. This could be due tochanges occurring at several sites. Our data demonstratethat the concentrations of both receptor and N proteinare reduced in membranes from hypothyroid animals.Thus, the impaired ability to form this complex in thehypothyroid membranes may be a consequence of thedecreased concentration of both of these components.In addition to the decrease in receptor number, thisimpaired R-N interaction may well contribute to thedecreased sensitivity to catecholamines in hypo-thyroidism. The changes observed in agonist affinity forthe beta adrenergic receptors contrast with the lack ofchange in antagonist [3H]DHA affinity in these cells.Because antagonists do not induce a high affinitynucleotide-sensitive form of the receptor, this resultseems reasonable.

Our data do not exclude other possible factors thatmight contribute to the altered catecholamine sensi-tivity of the adenylate cyclase, such as structuralchanges in R or N (apparent molecular weight isunchanged) or some change in the membrane en-vironment in which R and N interact. Wealso cannotexclude changes in the catalytic unit at this point butthese would not be expected to perturb agonistbinding.

Since N is involved in mediating the stimulatory ef-fects of both catecholamines and F- on adenylatecyclase (12), the alteration observed in N could explainthe decreased KL/KH in the binding assay as well asthe decreased isoproterenol- and F--stimulated adenyl-ate cyclase activity.

1454 G. L. Stiles, J. M. Stadel, A. De Lean, and R. J. Lefkowitz

Our findings in this hypothyroid model are quitesimilar to those found by Brodde et al. (2) in a rat atrialmodel. They found decreased numbers of beta adrener-gic receptors and a significant decrease in maximalcatecholamine- and NaF-stimulated adenylate cyclasein atrial membranes from hypothyroid rats. They, how-ever, did not assess agonist binding or the quantityof cholera toxin substrates.

In contrast, Malbon (13, 15) has found very differentresults in membranes from fat cells of hypothyroidrats. In this system there is no change in beta adrenergicreceptor number (15), and a decrease in catecholaminebut not NaF-stimulated adenylate cyclase is observed(14). In addition, agonist binding is apparently alteredas manifested by a rightward shift in an agonist com-petition curve with [3H]DHA and a complete loss inthe ability of guanine nucleotides to affect agonistbinding. Cholera toxin substrates appear to be slightlyincreased (20-30%) in fat cell membranes from hypo-thyroid animals (14). Because the number of receptorsand N protein as well as maximal adenylate cyclaseactivity are all undiminished in the hypothyroid fatsystem, it becomes necessary to postulate alterationsin as yet undisclosed components of the system toexplain the altered sensitivity. In contrast, in the re-ticulocyte system, alterations in known components ofthe sytstem (R and N) may explain the altered sen-sitivity. As noted above, the alterations observed inrat heart (2) strikingly resemble these in reticulocytesystem.

This study demonstrates the hormonal regulation oftwo components of the adenylate cyclase system (R andN) and this regulation of N highlights the fact thatsimple enumeration of R is insufficient for full evalua-tion of beta adrenergic receptor interactions in patho-physiological conditions. Modification of beta adrener-gic receptor - N site interactions may be a novel mech-anism of thyroid hormone control of catecholamineresponsiveness. Such coupling changes can be quanti-tatively assessed by careful analysis of agonist bind-ing properties as well as quantification of receptornumber and N protein.

ACKNOWLEDGMENTThis work was supported by National Institutes of Health

grant HLO7101.

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The influence of hyperthyroidism and hypothyroidism

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4. Sharma, V. K., and S. P. Banerjee. 1978. Beta-adrenergicreceptors in rat skeletal muscle. Effects of thyroidectomy.Biochim. Biophys. Acta. 534(4): 538-542.

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7. Limbird, L. E., D. M. Gill, and R. J. Lefkowitz. 1980.Agonist promoted coupling of beta-adrenergic receptorwith the guanine nucleotide regulatory protein. Proc.Natl. Acad. Sci. U. S. A. 77: 775-779.

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9. Williams, R. S., and R. J. Lefkowitz. 1979. Thyroidhormone regulation of alpha-adrenergic receptors: stud-ies in rat myocardium.J. Cardiovasc. Pharm. 1: 181-189.

10. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193: 265-275.

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12. Ross, E. M., A. C. Howlett, K. L. M. Ferguson, andA. G. Gilman. 1978. Reconstitution of hormone-sensitiveadenylate cyclase activity with resolved components ofthe enzyme. J. Biol. Chem. 253(18): 6401-6412.

13. Malbon, C. C. 1980. The effects of thyroid status on themodulation of fat cell beta-adrenergic receptor agonistaffinity by guanine nucleotides. Mol. Pharmacol. 18:193-198.

14. Malbon, C. C., and D. M. Gill. 1979. ADP-ribosylationof membrane proteins and activation of adenylatecyclase by cholera toxin in fat cell ghosts from euthyroidand hypothyroid rats. Biochim. Biophys. Acta. 586:518-527.

15. Malbon, C. C., F. J. Moreno, R. J. Cabelli, and J. N.Fain. 1978. Fat cell adenylate cyclase and beta-adrener-gic receptors in altered thyroid states. J. Biol. Chem.253: 671-678.

Hypothyroid Modulation of Agonist-Receptor Interactions 1455