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[78] P r o b i n g f l - A d r e n e r g i c R e c e p t o r s By KATHRYN E. MEIER and ARNOLD E. RUOHO

Adrenergic receptors mediate responses to endogenous catecholamines. These receptors are subdivided into two major types according to their relative affinities for catecholamine agonists.l For a-adrenergic receptors, the order of agonist potency is epinephrine > norepinephrine > isoproterenol. For/3-adrenergic receptors, the order is isoproterenol > epinephrine > norepinephrine./3-Adrenergic receptors have been further subdivided into/3~ and/32 subtypes on the basis of their relative affinities for agonists and antagonists. 2 Agonist occupation of/3-adrenergic receptors results in activation of adenylate cyclase, leading to increased intracellular levels of cyclic AMP./3-Adrenergic receptors are intrinsic membrane glycoproteins. The complete amino acid sequences of the mammalian/32-adrenergic receptor 3 and the avian/3-adrenergic receptor4 have been deduced from their cDNA nucleotide sequences. Cellular events involved in the regulation of receptor expression and function can be studied by means of microscopy techniques that allow visualization of the receptors on the cell surface and in intracellular compartments./3-Adrenergic receptors can be sequestered into an intracellular compartment following agonist binding and are down-regulated in response to prolonged exposure to agonist) ,6 However, these conclusions have been reached through studies of radioligand binding to the receptor, rather than by microscopy. Adrenergic ligands are small molecules that cannot be directly visualized, and antibodies to the receptor have been difficult to obtain. Furthermore, the receptors are generally present in low densities on the surface of responsive cells (< 10,000 receptors/cell); this fact has complicated attempts at their localization. In order to visualize R. P. Ahlquist, Am. J. Physiol. 153, 586 (1948). 2 A. M. Lands, A. Arnold, J. P. McAuliff, F. P. Ludvena, and T. G. Brown, Nature (London) 214, 597 (1967). 3 R. A. F. Dixon, B. K. Kobilka, D. J. Strader, J. L. Benovic, H. G. Dohlman, T. Freille, M. A. Bolanowski, C. D. Bennett, E. Rands, R. E. Diehll, R. A. Mumford, E. E. Slater, I. S. Sigal, M. G. Caron, R. J. Lefkowitz, and C. D. Strader, Nature (London) 321, 75 (1986). 4 y . Yarden, H. Rodriguez, S. K.-F. Wong, D. R. Brandt, D. C. May, J. Burnier, R. N. Harkins, E. Y. Chen, J. Ramachandran, A. Ullrich, and E. M. Ross, Proc. Natl. Acad. Sci. U.S.A. 83, 6795 (1986). 5 T. K. Harden, Pharrnacol. Rev. 35, 5 (1983). 6 L. S. Mahan, R. M. McKernan, and P. A. Insel, Annu. Rev. Pharmacol. 27, 215 (1987).

METHODS IN ENZYMOLOGY, VOL. 184

Copyright © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.

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fl-ADRENERGIC RECEPTORS

0 HN'I~NH

O

BN

H OH

0

HN~NH

~

H O H O H O H NvJL N"~ N,.,,~N'h~ N,.,J[N"Y N ~ N",~ O'~h~ O H O H O H O H OH ~I,.~.,~I II

I

BGN

0

HN~NH

~

H 0

BCA

O"~ NL

N~I.S,~I

OHH

OH

0

HN~'NH

~

H

O

O

BCCA

OH~

N~'II'N~S'~'VI~¢~I H

OH H O,,,,..I~Ny

0

HNJI'NH

~

H

O

BDCA

OH OH.

O

H

Ov, L,~ NY

FIc. 1. Structures of biotinylpropranolol and biotinylalprenolol derivatives.

the receptor by the techniques of light and electron microscopy, it is therefore necessary to create receptor-specific ligands that can be linked to a fluorescent or electron-dense marker. We synthesized a series of biotinylated derivatives of propranotol and alprenolol, two fl-adrenergic antagonists that bind with high affinity to the receptor (Fig. 1). 7-9 The goal of this work was to obtain derivatives that 7 K. E. Meier and A. E. R u o h o , J. Supramol. Struct. 9, 243 (1978). 8 K. E. Meier and A. E. R u o h o , Biochim. Biophys. Acta 761, 257 (1983). 9 K. E. Meier, Diss. Abst. Int. B 42, 4036 (1982).

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would bind simultaneously to both the fl-adrenergic receptor and to avidin. The interaction between avidin and biotin is essentially irreversible. The interaction between fl-antagonists and the receptor is of sufficiently high affinity (equilibrium Ko 1-10 nM) to allow excess ligand to be washed from the cell surface prior to fixation. Avidin can be linked to fluorescent, electron-dense, or macromolecular marker molecules. Using biotinylated antagonists, it should therefore be possible to visualize fladrenergic receptors on the cell surface. Success in achieving this goal will be dependent on the receptor density on the cell surface, as well as on the sensitivity of the method used to detect the label. The remainder of this chapter is concerned with the preparation and characterization of biotinylpropranolol and biotinylalprenolol derivatives. Discussion focuses on biotinyldodecanoylcysteaminylalprenolol (BDCA) because this compound possesses the optimal characteristics desired for a bifunctional reagent.

Methodology General Methods for Detection and Quantification of Biotin. The presence of biotin in reaction products is detected using p-dimethylaminocinnamaldehyde as previously described by McCormick and Roth. 10 A solution containing 0.2% (w/v) of the reagent in I% sulfuric acid-ethanol is applied as a spray to thin-layer chromatography plates containing separated reaction products. Biotin-containing compounds appear as pink spots following this treatment. The amount of biotin present in solutions of biotinylated products is determined by a colorimetric avidin titration technique described by Green. 11This procedure involves spectrophotometric titration (500 nm) of a known quantity of biotin-binding sites in an avidin solution by competition with 10 m M hydroxyazobenzene-2'-carboxylic acid. The observation of a sharp end point to the titration curve is indicative of high-affinity binding of the biotinylated compound to avidin. The stabilities of the complexes between biotin derivatives and avidin are assessed as follows. A 50-~1 aliquot of a 1 m M solution of the biotinyl derivative is added to 500/.d of 0.1 m M avidin in 50 m M Tris-HCl (pH 7.4). This solution is incubated for 10 min at 25 °. A 50-gl aliquot of a solution of 10 m M [14C]biotin (50 ~Ci/ml) is then added. The incubation is continued at 4 °, 25°, or 37°. At various times, 100-/.d aliquots of the mixture are removed and diluted with 100/~l of 50 m M Tris-HCl (pH 7.5). The l0 D. B. M c C o r m i c k and J. A. Roth, this series, Vol. 18, p. 383. H N. M. Green, this series Vol. 18, p. 418.

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diluted samples are applied to a 7 × 80 mm column of Sephadex G-50 and then eluted with 50 m M Tris-HCl (pH 7.5). Two 0.9-ml fractions are collected; the 14C content of each fraction is determined by liquid scintillation spectrometry. The avidin-biotin complex elutes in the first fraction (void volume); free biotin is contained in the second fraction. Data are normalized for column recovery by expressing the bound [14C]biotin as a percentage of the total [14C]biotin eluted from each column.

Methods for Assessment of Binding Affinity of Derivatives for fl-Adrenergic Receptor. Several assay systems can be used to determine the affinity of biotinylated antagonists for the fl-adrenergic receptor. The ability of the compounds to inhibit isoproterenol-induced increases in cyclic AMP levels in intact duck erythrocytes or HeLa cells is assessed using a commercially available radioimmunoassay method 7 or a binding protein assay 8 to measure intracellular cyclic AMP. Similar studies have been carried out with duck erythrocyte membranes using [a-32p]ATP as substrate for adenylate cyclase activity. 7 The KD values of the compounds for fl-adrenergic receptor binding are determined using radioligand binding assays with membrane fractions (or solubilized membrane fractions) prepared from avian erythrocytes8 or cultured mammalian cells (see footnote to Table I). 9'12 These determinations are based on the abilities of the derivatives to compete for binding of a radiolabeled antagonist ([3H]dihydroalprenolol or [125I]iodohydroxybenzylpindolol) to the receptor preparation. Synthesis of Biotinyl-N-hydroxysuccinimide. Biotinyl-N-hydroxysuccinimide (BNHS) is prepared by the method of Jasiewicz et a1.13; this reagent is now commercially available under the designation N-hydroxysuccinimidobiotin (Sigma Chemical Co., St. Louis, MO). Synthesis of Biotinylpropranolol Derivatives. Biotinylpropranolol compounds are prepared from an aminopropranolol derivative, 1-(1naphthoxy)-3-N-(ethylamino)propan-2-ol (NEDA), as previously described. 7 NEDA is reacted directly with BNHS to give biotinyl-NEDA (BN). This compound, whose synthesis and characterization has independently been reported by Atlas and co-workers, TM is now commercially available under the designation "biotinylpropranolol analog" (Sigma). A second derivative is obtained by first reacting BNHS with hexaglycine to give biotinylhexaglycine.7 This intermediate is then reacted with NEDA to give biotinylhexaglycyl-NEDA (BGN). 12 K. E. Meier and A. E. Ruoho, Biochem. Biophys. Acta 804, 331 (1984). 13 M. L. Jasiewicz, D. R. Schoenberg, and G. C. Mueller, Exp. Cell Res. 100, 213 (1976). 14 D. Atlas, D. Yaffe, and E. Skutelsky, FEBS Lett. 95, 173 (1978).

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Synthesis o f 1-[2-Hydroxy-3-(2-propyl)amino]propyloxy-2-[2-hydroxy3-[(2-amino)ethyl]thio]propyl Benzene (Cysteaminylalprenolol). Bromoalprenolol is synthesized from N-bromosuccinimide (Sigma) and (-)-alprenolol (+)-tartrate (Sigma) as described by Vauquelin et al., ~5 using a reaction time of 4 hr. The reaction mixture, containing 2 mmol of bromoalprenolol, is adjusted to pH 9.0 with NazCO3. This mixture is extracted 5 times with 200 ml of ether. The ether extract, containing bromoalprenolol, is reduced to 10 ml using a rotary evaporator. This solution is added to 320 mg NaOH (8 mmol) and 454.4 mg cysteamine (4 mmol) in 50 ml of ethanol. The mixture is stirred for 30 min under N2 in the dark. The N2 source is then removed, and the reaction is allowed to proceed for 18 hr at 25 ° with stirring. The mixture is then dried under vacuum. The residue is dissolved in 25 ml of water and is extracted 6 times with 25 ml of ethyl acetate. The ethyl acetate extract is reduced to 50 ml using a rotary evaporator. This solution is extracted 3 times with 50 ml of water that had been adjusted to pH 6.0 with HC1. The aqueous extract is then adjusted to pH 10 with NazCO3. This solution is extracted 6 times with 50 ml of ethyl acetate. These extractions are monitored by thin-layer chromatography on silica gel using chloroform-methanol-acetic acid (1 : 5 : 1, v/v); a ninhydrin spray [0.29% (w/v) in acetone] is used to visualize the amine-containing starting material and product. The final ethyl acetate extract is dried under vacuum. This residue is dissolved in 15 ml of methanol, filtered, and dried under vacuum to a yellow oil (411 mg, 1.2 mmol, 60% yield). The product gives a single component (Rf 0.6) that can be visualized using UV light or ninhydrin spray following thinlayer chromatography on silica gel with the solvent system described above. Synthesis o f Biotinylaminododecanoic Acid. Biotinylaminododecanoic acid is prepared by a modification of a method described by Bayer et al. 16A solution of 12-aminododecanoic acid (258.4 mg, 1.2 mmol, Aldrich, Milwaukee, WI) in 12 ml of 0.2 M NaHCO3 is added to a solution of BNHS (341 mg, 1 mmol) in 10 ml of dimethylformamide (DMF). The reaction is allowed to proceed for 9 hr at 25° with stirring. The reaction mixture is then filtered to remove the resulting white precipitate. The precipitate is washed with water followed by water acidified to pH 2.0 with HCI. The washed precipitate is dried under vacuum to a white powder (380 mg, 86% yield, mp 216°). Synthesis o f Biotinyldodecanoylcysteaminylalprenolol. A solution of biotinyldodecanoic acid (88.2 mg, 0.2 mmol) in 5 ml of DMF is heated to 95°; 1,1'-carbonyldiimidazole (32.4 mg, 0.2 retool, Sigma) is then added, 1~G. Vauquelin,P. Geynet,J. Hanoune,and A. D. Strosberg,Proc. Natl. Acad. Sci. U.S.A. 74, 3710 (1977). 16E. A. Bayer, T. Viswanatha, and M. Wilchek,FEBS Lett. 60, 309 (1975).

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The reaction is allowed to proceed for 5 min at 95 °, followed by 30 min at 25 °. A solution of cysteaminylalprenolol (137 mg, 0.4 mmol) in 10 ml of DMF is then added. The reaction mixture is stirred overnight at 25 °. The solvent is removed under vacuum. The residue is dissolved in 5 ml of chloroform-methanol-acetonitrile-acetic acid (9 : 9 : 9 : 2, v/v). This solution is applied to a silica gel column (60-200 mesh, 3 × 14 cm, Sigma). The column is eluted with the solvent mixture described above with collection of 5-ml fractions. Recovery of the product (BDCA) is monitored by thin-layer chromatography of the column fractions on silica gel, using the same solvent system; dimethylaminocinnamaldehyde spray is used to detect the biotinylated product. Fractions 20-36 are pooled. The solvent is removed under vacuum, leaving a yellow oil (0.052 nmol by avidin titration, 26% yield). The product appears as a single component (visualized with either UV light or dimethylaminocinnamaldehyde spray) in two different thin-layer chromatography systems (silica gel): chloroformmethanol-acetic acid, 1:5: 1, Rf 0.81; chloroform-methanol-acetonitrile-acetic acid 9 : 9 : 9 : 2, Rf 0.81. BDCA can also be detected as a single component (visualized by iodine vapor) using reversed-phase thin-layer chromatography plates with methanol as solvent (gf 0.23). BDCA is stored as a solution in methanol and is stable in this form for at least 1 year. The concentration of the stock solution is determined by avidin titration, as described above.

Synthesis of Biotinylcysteaminylalprenolol and Biotinylcaproylcysteaminylalprenolol. Biotinylcysteaminylalprenolol (BCA) is synthesized by reaction of BNHS with cysteaminylalprenolol. 8,9 Biotinylcaproylcysteaminylalprenolol (BCCA) is synthesized by reaction of biotinyl eaminocaproic acid (prepared by analogy to biotinylaminododecanoic acid, as described above) with cysteaminylalprenolol. 8,9

Comments Our initial studies of two biotinylpropranolol derivatives (BN and BGN, Fig. 1) led to the following conclusions. BN, which did not contain a spacer group between the biotin and propranolol residues, was a potent fl-adrenergic antagonist in the absence of avidin, but it bound to the receptor with very low affinity in the presence of avidin (Table I). A hexaglycyl spacer group was introduced into BGN in order to allow simultaneous binding of the ligand to avidin and to the receptor. This goal was achieved at the expense of substantial loss of affinity of the ligand for the receptor (Table I). This reduced affinity is presumed to be due to the addition of a bulky side chain to the amino portion of the parent compound. A second series of derivatives were synthesized from alprenolol, an-

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TABLE I BINDING CONSTANTSFOR BIOTINYLPROPRANOLOLAND BIOTINYLALPRENOLOLDERIVATIVES Ligand

K D (nM) ~

Propranolol BN Avidin-BN BGN Avidin-BGN Alprenolol BCA Avidin-BCA BCCA Avidin-BCCA BDCA Avidin-BDCA

I1 3.9 >650 47 320 3.5 12 59 13 13 2.0 2.0

The apparent equilibrium dissociation constants for each ligand are determined at 4° using a digitonin-solubilizedduck erythrocyte membrane preparation. The radioligand used is [3H]dihydroalprenolol, and the binding constants are determined by competition for radioligand binding by the indicated antagonists. The avidin complexes of the ligands are prepared with a 10% molar excess of biotinbinding sites over biotinylated ligand. Similar trends in affinity values are obtained using a duck erythrocyte membrane preparation (binding and adenylate cyclase assays), except that the differences between the values for BN versus avidin-BN and BCA versus avidin-BCA are more pronounced.

o t h e r f l - a d r e n e r g i c a n t a g o n i s t t h a t p o s s e s s e s high affinity for t h e r e c e p t o r . I n t h e c a s e o f a l p r e n o l o l , it w a s p o s s i b l e to d e r i v a t i z e a d i f f e r e n t p o r t i o n o f t h e p a r e n t m o l e c u l e t h a n h a d b e e n d e r i v a t i z e d in t h e b i o t i n y l p r o p r a n o lol s e r i e s . T h e b i o t i n y l a l p r e n o l o l d e r i v a t i v e s c o n t a i n e d s p a c e r g r o u p s o f v a r y i n g c h a i n l e n g t h . A l l o f t h e c o m p o u n d s b o u n d w i t h high affinity to t h e / 3 - a d r e n e r g i c r e c e p t o r in t h e a b s e n c e o f a v i d i n , w i t h B D C A p o s s e s s i n g t h e h i g h e s t affinity ( T a b l e I). I n t h e p r e s e n c e o f a v i d i n , B C C A a n d B D C A r e t a i n e d t h e i r affinities f o r t h e r e c e p t o r , w h i l e B C A b o u n d w i t h m u c h l o w e r affinity. T h e s e d a t a , c o n s i s t e n t w i t h t h e d a t a o b t a i n e d f o r t h e b i o -

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/~-ADRENERGIC RECEPTORS

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tinylpropranolol compounds, indicate that it is necessary to introduce a spacer sequence between the two ends of these bifunctional reagents in order to allow simultaneous binding of the compounds to both the /3-adrenergic receptor and to avidin. Similar requirements for a spacer sequence were observed in the studies of Pitha et al., ~7who found that such a sequence is required for binding of fl-adrenergic receptors to alprenolol which had been linked to an affinity column support resin. All of the derivatives described here bind with high affinity to avidin. Their dissociation rates from avidin are slightly higher than that of biotin, e.g., 20% dissociation of BDCA in 20 hr at 25 ° as compared to 3% dissociation of biotin. 9 This rate is not rapid enough to interfere with use of the complex in biological assay systems. The avidin-BDCA complex is always prepared shortly before each use. We have attempted to use BDCA as a receptor visualization reagent with cultured mammalian cell model systems (HeLa epithelial cells, L6 myoblast cells). 9 Using binding and activity assays, we found that BDCA is capable of binding to the/3-adrenergic receptor of intact HeLa cells in the presence of ferritin-avidin conjugates. HeLa and L6 cells were incubated with ferritin-avidin-BDCA, washed, fixed, embedded, and thin sectioned. Examination of the sections revealed that the level of labeling was too low to allow positive identification of/3-adrenergic receptor sites, despite low levels of nonspecific binding of ferritin-avidin to the cell surface in the absence of BDCA. Similar results were obtained with various incubation temperatures, with various incubation times, with avidin linked to horseradish peroxidase, with various types of fixatives, and by prelabeling the cells with BDCA prior to addition of ferritin-avidin. Subsequent analysis of receptor content, showed that, in contrast to earlier reports of high levels of receptor expression, these cells expressed approximately 5000 receptors/cell. 9 With this level of receptor expression, one would expect to visualize between 10 and 30 receptors in a 75-nm thin section of a cell. At this level of labeling, it was not possible to distinguish nonspecific binding of ferritin-avidin from specific binding of the ferritinavidin-BDCA complex. Several new developments in this field may lead to the successful visualization of/3-adrenergic receptor sites on intact cells using the biotin-avidin approach developed here. First, the techniques of molecular biology have made it possible to transfect cells with the nucleotide sequence encoding the receptor. Such cells can theoretically be made to express greatly elevated levels of receptor protein. Second, improved techniques for detection of fluorescent marker molecules have enabled 17 j. Pitha, J. Zjawiony, R. J. Lefkowitz, and M. G. Caron, Proc. Natl. Acad. Sci. U.S.A. 77, 2219 (1980).

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the detection of very low levels of ligand binding to cell surfaces. The application of such approaches to visualization studies utilizing the highaffinity biotinylalprenolol probe described here should result in interesting new information regarding the location, expression, and regulation of/3adrenergic receptors.

[79] P r o b i n g A d e n o s i n e R e c e p t o r s U s i n g B i o t i n y l a t e d Purine Derivatives By KENNETH

A. JACOBSON

Extracellular adenosine acts as a neuromodulator through two subtypes of membrane-bound receptors. Agonist activation of AI- or A2adenosine receptors is generally linked to an inhibitory or stimulatory effect, respectively, on the enzyme adenylate cyclase, which converts intracellular 5'-ATP to cyclic Y,5'-AMP. Many N6-alkyl - and N6-aryl substituted adenosine analogs have been synthesized as potent agonists having nanomolar Ki values at the high-affinity Aj site.1 At each receptor subtype, xanthines act as competitive antagonists. Nonselective antagonists caffeine and theophylline have Ki values in the 10 ~ M range, and at A~ receptors some synthetic 1,3-dialkyl-8-substituted analogs have Ki values close to 1 nM. 2 Adenosine antagonism is the principal mechanism by which caffeine acts as a stimulant in vivo. The avidin-biotin complex has been utilized in studies directed toward the isolation, histochemical localization, and microscopic structural probing of the receptor protein. 3 Both adenosine and xanthine analogs have been coupled to biotin, 4,5 forming bifunctional probes to serve as noncovalent cross-linkers between adenosine receptors and avidin. Structure-activity relationships for adenosine agonists and antagonists were studied initially to identify potential sites for derivatization. Since neither adenosine nor theophylline contains a readily derivatized functional group that is nonessential for biological activity, a "functionalized congei K. A. J a c o b s o n , K. L. Kirk, W. L. Padgett, and J. W. Daly, J. Med. Chem. 28, 1341 (1985). 2 K. A. J a c o b s o n , K. L. Kirk, W. L. Padgett, and J. W. Daly, J. Med. Chem. 28, 1334 (1985). 3 G. L. Stiles, Trends Pharmacol. Sci. 7, 486 (1986). 4 K. A. J a c o b s o n , K. L. Kirk, W. Padgett, and J. W. Daly, FEBS Lett. 184, 30 (1985). 5 K. A. Jacobson, D. U k e n a , W. Padgett, K. L. Kirk, and J. W. Daly, Biochem. Pharmacol. 36, 1697 (1987).

METHODS IN ENZYMOLOGY, VOL. 184

Copyright © 1990by AcademicPress, Inc. All rights of reproduction in any form reserved.

Probing beta-adrenergic receptors.

660 APPLICATIONS [78] [78] P r o b i n g f l - A d r e n e r g i c R e c e p t o r s By KATHRYN E. MEIER and ARNOLD E. RUOHO Adrenergic receptors...
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